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    The Mystery of Intron Evolution 09/03/2003
    Eukaryotes, the organisms whose cells contain a nucleus, encompass an astonishing diversity of organisms: all plants and animals and a number of one-celled protists. The genomes of eukaryotes contain a mystery: sections of DNA, called introns, that do not code for genes, and are exquisitely removed before translation by a complicated molecular machine called the spliceosome (see 09/12/2002 headline) and its helpers. The spliceosome, a large RNA-protein complex, is conserved throughout the eukaryotic world, from grizzly bears to earthworms to orchids. Coding regions of the eukaryotic DNA, called exons, are meticulously spliced together after the introns are removed. Why are these introns there? What is their function, if any? Why does the cell go to so much work to remove them? Evolutionary biologists want to know.
    In the Sept. 2 issue of Current Biology1, a team of British and American scientists delved into published genomes to compare intron counts and positions, in hopes of determining the evolutionary history of introns. To their surprise, they found “Remarkable Interkingdom Conservation of Intron Positions and Massive, Lineage-Specific Intron Loss and Gain in Eukaryotic Evolution,” as the title of their paper summarizes. They compared eight very different eukaryotes: the malaria parasite and a cousin, two kinds of yeast (fungi), Arabidopsis (a flowering plant favored in genetics research), fruit flies, worms, and humans. Sifting through the published genomes, they found 684 orthologous sets but no clear evolutionary pattern. Surprises include:

    • More introns share identical positions within two or more species than would be expected by chance.
    • Intron phylogeny does not match traditional Darwinian phylogeny.
    • Humans share more introns with the herb, a plant, rather than with animals like fruit flies and worms.
    • Yeast and malaria also both share more introns with plants than with fruit flies and worms.
    • The number of introns in different species varies widely. Humans and plants have many, but yeast and fruit flies have fewer.
    • The majority of plant and animal introns appear to have been gained recently. Humans, for instance, have longer introns than mice (see 12/06/2002 headline).
    • Recently, introns were found in several protists (see 02/26/2002 headline), which “might be the deepest branches in eukaryotic phylogeny.”

    Their surprise is evident in their summary of the findings :

    “The matrix of shared introns in all pairs of analyzed eukaryotic genomes revealed a striking, unexpected pattern (Table 2). The number of conserved introns did not drop monotonically with the increase of the evolutionary distance between the compared organisms. On the contrary, human genes shared the greatest number of introns not with any of the three animals but with the plant Arabidopsis; in the conserved regions (the more accurate results given the uncertainties in alignment in other parts of genes), 24% of the analyzed human introns were shared with Arabidopsis (27% of the Arabidopsis introns) compared to 12%–17% of the introns shared by humans with the fly, mosquito, and the worm (Table 2). The difference becomes even more dramatic when the numbers of introns conserved in Arabidopsis and each of the three animal species are compared: approximately three times more plant introns have a counterpart in humans than in the fly or the worm (Table 2). Although S. pombe [yeast] and Plasmodium [malaria] have few introns compared to plants or animals, the same asymmetry was observed for these organisms: the numbers of introns shared with Arabidopsis and humans are close and are 2–3 times greater than the number of introns shared with the insects or the worm (Table 2).”

    How does an evolutionist make sense of these unexpected findings? The team applied phylogenetic tree-building to the data, and quickly realized that intron presence or absence does not fit Darwinian expectations. A method called Dollo parsimony, for instance, assumes that each derived character state (e.g., intron presence) originated only once on the tree. This led to a very unDarwinian tree, with humans clustered together with plants, and yeast clustered with malaria. They tried other phylogenetic approaches that generated more of a web of branches going nowhere than a tree progressing toward higher organisms (see their Figure 2). “These observations show that intron locations are not suitable markers for phylogenetic analysis at long evolutionary distances,” they confessed.
    “Having shown that evolution of introns in eukaryotic genes did not follow the species tree,“ they inverted the analysis and started by assuming the species tree to deduce the evolution of introns. This led to a conclusion that the “last common ancestor [sic] of the eukaryotic species with sequenced genomes comes out particularly intron rich,” they note. In other words, introns and the spliceosome machinery to juggle them were already present in the earliest eukaryote. For some reason, certain groups lost introns, and others had a net gain, while not a few introns survived nearly two billion years of evolution intact and in their original positions.
    Why would some species conserve introns, and others get rid of them? The authors consider possibilities. “Why have so many ancestral [sic] introns survived almost 2 billion years of evolution?” they ask. “One intriguing possibility is that conserved introns are functionally important, but there is currently little evidence in support of this hypothesis.” On the other hand, maybe losing introns, even if functionless, is hazardous; like trying to remove a bullet, maybe it is sometimes better to leave it in than to risk more damage by removing it. Perhaps their presence influences gene regulation and expression (see 07/22/2002 headline). If so, removing them could be lethal. But then, why would some organisms, like fruit flies, succeed in removing so many of them without harm? No clear reason is given why natural selection would favor removing introns on some groups and adding them in others.
    Without a clear phylogeny emerging from the data, they conclude with some speculations. “It even seems possible,” they imagine, “that invasion [sic] of protein-coding genes by ancestors of introns was part of the dramatic and still mysterious series of events that led to the origin of the eukaryotic cell.” The bottom line is that this “remarkable conservation” across species – and even kingdoms – leaves much to be learned: “The lineage-specific trends of intron loss and gain might reflect more general tendencies for genome compaction and genome expansion, the underlying causality of which remains to be understood.”

    Update: see Sept. 12 headline.
    1Rogozin, Wolf, Sorokin, Mirkin and Koonin, “Remarkable Interkingdom Conservation of Intron Positions and Massive, Lineage-Specific Intron Loss and Gain in Eukaryotic Evolution,” Current Biology Vol 13, 1512-1517, 2 September 2003

    Left-Handed Amino Acids Explained? 09/06/2003
    Another theory has surfaced to explain the origin of left-handed amino acids in proteins. Reported in Science News1, R. Graham Cooks and colleagues at Purdue University studied all 20 biological amino acids, and found that one – serine – formed stable clusters of all left- or all right-handed forms. The third lightest (after glycine and alanine), possessing an uncharged polar side chain, serine not only clustered in single-handed forms, but attracted other amino acids of the same hand. Sugars of the opposite hand were also attracted to the eight-molecule serine rings.

    “Serine clusters’ high stability and selectivity have convinced the researchers that left-handed serine must have forced its chemical siblings to follow its lead [sic]. What caused serine’s left form to become dominant in the first place remains an open question. Some scientists say that ancient minerals may have favored one form over the other (SN: 5/5/01, p. 276). Others point to the effects of radiation hitting primordial Earth. Or, says Cooks, it could have happened by chance.”

    As to this chance event, Cooks speculates in the Purdue News press release that “If somehow polarized light, for example, or a swirling motion in water were present at a critical moment, some of the right-handed clusters could have become left-handed. This could have cascaded into other prebiotic reactions and set the pace for a billion years of evolution” [sic]. He calls serine the “bouncer at life’s dance club.” His team’s paper was published online Aug. 4 in the German chemistry journal Angewandte Chemie.2
    1Science News Week of Sept. 6, 2003 (164:10): Alexandra Goho, “Amino acid lends a heavy hand.”
    2Takats, Z., S.C. Nanita, and R.G. Cooks, “Serine octamer reactions: Indicators of prebiotic relevance,” Angewandte Chemie International Edition, Volume 42, Issue 30, Pages 3521 - 3523 (published online Aug. 4).

    Cells Fight Mutations 09/12/2003
    A team of cancer researchers identified 33 genes in yeast “potentially associated with the suppression of the accumulation of mutations.” Their paper was published online Sept. 12 in the Proceedings of the National Academy of Sciences1, where they begin by stating,

    “Maintaining the stability of the genome is critical to cell survival and normal cell growth. Inherited or acquired deficiencies in genome maintenance systems contribute significantly to the onset of cancer as evidenced by the observation that a number of the DNA-repair and checkpoint genes are mutated in cancer susceptibility syndromes and sporadic cancers. This raises the possibility that other genetic defects causing genome instability and mutator phenotypes could contribute to carcinogenesis.”

    Their blind screening technique found all the known mutation suppressors, but also ten more previously-unknown “nonessential” genes involved in mutation suppression.

    “Of the confirmed genes, five encode components of the oxidative-stress response, and six are genes of unknown function. We believe the data presented here define a nearly complete collection of nonessential genes involved in suppression of mutations in the CAN1 forward-mutation assay [a sample gene used for evaluating effects of mutations] and define several previously unappreciated mutation-suppression pathways.”

    Some of the activities these genes perform are repairing base excisions, fixing oxidated guanine (G) bases, and replacing incorrectly-inserted uracil (U) bases with cytosine (C). Another one suppresses genome rearrangements.
    Huang, Rio, Nicolas and Kolodner, “A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.2035018100.

    Intron Update 09/12/2003
    (See Sept. 3 headline about introns). Scientists have found a possible reason why genes that contain introns are expressed more effectively than those without. Writing in PNAS1, a team from Howard Hughes Medical Institute suspects that the exon junction complex (EJC) that forms at each junction by the splicing process may give messenger RNA (mRNA) a tethering point with “position-specific memory of the splicing event.” EJC components may attach at the junctions to perform expediting functions.
    Of five known protein components of the EJC, some are known to be involved in nonsense-mediated decay, positioning of the mRNA in the cytoplasm, or transport through the nuclear pore complex (see March 4 headline). Other functions of the EJC might include stabilizing the mRNA, enhancing transcription in the ribosome, and translational utilization.
    They found that gene expression was enhanced by the EJC components but not necessarily by the act of splicing itself; that is apparently why some intronless genes can be expressed satisfactorily, especially if EJC proteins can be recruited through other means. Splicing may assist in the efficient formation and localization of EJC components. The stimulatory effect of splicing and the EJC can enhance gene expression more than 30-fold.
    1Wiegand, Lu, and Cullen, “Exon junction complexes mediate the enhancing effect of splicing on mRNA expression,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.1934877100.

    Plasma Blob: It’s Alive? 09/17/2003
    New Scientist claims, “Plasma blobs hint at new form of life.” In an experiment worthy of Frankenstein, a Romanian scientist inserted two high-voltage electrodes into a plasma of argon, and created cell-like spheres of plasma that could replicate by splitting in two and “communicate information” by emitting electromagnetic energy, making atoms in other spheres vibrate.
    Mircea Sanduloviciu and team (Cusa University, Romania) claim this shows that “cell-like self-organization can occur in a few microseconds” instead of millions of years. Sanduloviciu speculates these could have been the first cells on earth, formed in electrical storms. “The emergence of such spheres seems likely to be a prerequisite for biochemical evolution,” he said. Others think that is a stretch, but are intrigued by the implications that life might take on forms much different than the DNA and protein-based life we know on earth.

    Your Motors Perform Cooperative Interactions 09/18/2003
    The motor that powers all life, often called a “splendid molecular machine,” looks even more splendid due to research by Caltech and French scientists. It has parts that help each other out. ATP synthase, which we have reported on frequently before (see 11/15/2002 headline), is a true rotary motor, and probably the most abundant enzyme on earth. Its job is to generate ATP, the energy currency of life. “Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology,” they state in their PNAS paper.1 They found that the structure of the six-lobed F1 upper unit, where ATP is catalyzed from ADP and phosphate, actually is tuned to enhance the productivity of the system.
    Most living things have the F0-F1-ATPase model, composed of an upper and lower mechanism joined by a camshaft and some other parts, although there are variations in some bacteria. The six lobes of the F1 motor, arranged in pairs like orange slices around the camshaft, form three catalytic sites where ATP is synthesized. The researchers did thermodynamic and kinetic modeling of these structures and found that the shapes of the sites change as the camshaft rotates in a way that enhances productivity. Each pair of lobes cycles through three stages as the shaft turns: (1) insertion of ADP + P, (2) catalysis of ATP, and (3) ejection of the product. Each stage is not only finely tuned for its job, but actually stimulates the adjoining pair of lobes to do its job better: e.g., at stage one, the shape of the lobes causes the reaction in stage two of the adjoining lobes to accelerate. The reaction in stage two speeds up the ejection of product in stage three, and so on. Overall, this enhances the productivity of the system by a factor of 300 or more than would occur if a pair of lobes had to work alone. This and the rotation of the camshaft enhances productivity by a factor of 500,000.
    They mention some other interesting facts about ATP synthase in passing. Each motor (and your body has quadrillions of them) can hydrolyze from 40 to 600 ATP per second. With three ATP per revolution, that translates to 12,000 RPM at top speed. Without these enzymes, it would take 500,000,000 times as long for ATP to hydrolyze in solution. Though they studied primarily the hydrolysis cycle, the synthesis reaction, driven by an electric current (proton flow) in the lower F0 subunit, is similarly accelerated because of the efficient mechanical arrangement of the parts.
    1Gao, Yang, Marcus and Karplus, “A model for the cooperative free energy transduction and kinetics of ATP hydrolysis by F1-ATPase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.1334188100, published online 09/18/2003.

    Don’t Mutate This Gene, Or Else 10/01/2003
    Hit the power grid, and you shut down hospitals, businesses and homes. When traffic controllers go on strike, trucks pile up on the highways, and merchants, from florists to toolmakers, cannot get their goods. A whole city or economy can grind to a halt from one failure in a key component. Similarly, mutations to certain genes can have what are called pleiotropic effects, causing harm to very different organs and functions scattered all over the body. In the Sept. 30 issue of Current Biology1, three New York geneticists describe the failures that ensue from a mutation in one protein in the IFT family: “kidney cysts, photoreceptor degeneration, skeletal abnormalities, and spermatogenesis defects. ... A targeted Tg737 knockout [to a particular IFT protein] is embryonic lethal, with early embryonic defects that include randomized left/right asymmetry, a consequence of missing cilia on the embryonic ventral node.” IFT proteins might also be essential for coordinating signals from cilia to other parts of the cell.
    Like most scientific papers, this one is focused on just one narrow aspect of one protein in one organism, the fruit fly. As background information, however, they describe what IFT does and how it works. IFT stands for intraflagellar transport. It is a family of proteins involved in building the insides of flagella and cilia. (These are the whip-like appendages common in most living things, from the outboard motors on bacteria, to the sweepers lining your respiratory tract, to the paddling tails on sperm cells). If you could shrink yourself down to a few microns and watch, here’s what you might see going on inside the shaft of a cilium or flagellum under construction :

    “The eukaryotic cilium or flagellum is a distinct subcellular compartment, with its own characteristic microtubular cytoskeleton, the axoneme, and a membrane that, though continuous with the plasma membrane, can localize distinct sets of proteins. This distinction is maintained by a specific mechanism of intraflagellar transport (IFT). IFT was first observed in the single-celled alga Chlamydomonas as a bidirectional movement of uniformly sized particles along the flagellum, in the space between the axoneme and the flagellar membrane. Biochemical characterization of the particles revealed over 16 constituent proteins associated in A and B subcomplexes. Particle movement toward the plus ends of the axonemal microtubules at the tip of the flagellum is driven by kinesin II, and mutants lacking kinesin II subunits or complex B proteins do not extend cilia beyond the transition zone of the basal body. In mutants that express a temperature-sensitive kinesin, flagella shrink after a shift to the restrictive temperature, and this shrinkage indicates that IFT is needed to maintain and regulate flagellar length. IFT particles and kinesin are returned to the cell body by a nonaxonemal dynein, and mutants with defects in this process typically have swollen cilia that accumulate IFT particles. Some IFT proteins are concentrated in the cytoplasm close to the basal bodies as well as in the cilia proper, and the transition fibers that connect the basal body to the cell membrane are a possible site for the docking and exchange of IFT particles, motors, and cargo.”

    In other words, there is a specialized molecular highway down the shaft of a flagellum, between the membranes, with little molecular trucks (dynein and kinesin) that transport cargo (the protein particles) to and from the tips of the growing end. Intraflagellar transport might be termed the Transportation Department for these organelles. Since everything from eyes, sperm, and lungs depend on cilia or flagella, you can imagine what happens when a mutation shuts down the highway department and brings construction of these essential organelles to a halt.
    A related paper in the same issue2 discusses what happens when another one of the IFT proteins, Kinesin II-mediated anterograde intraflagellar transport, mutates and prevents the kinesin truck from moving down the highway. It makes their cilia sluggish and uncoordinated, and causes auditory defects.
    An analysis by George Witman (U. of Mass. Medical School) of these papers was published in the subsequent (Oct. 14) issue of Current Biology.3
    1Han, Kwok and Kernan, “Intraflagellar Transport Is Required in Drosophila to Differentiate Sensory Cilia but Not Sperm,” Current Biology Vol 13, 1679-1686, 30 September 2003, pp. 1679-1686.
    2Sarpal et al., “Drosophila KAP Interacts with the Kinesin II Motor Subunit KLP64D to Assemble Chordotonal Sensory Cilia, but Not Sperm Tails,” Current Biology Vol 13, 1687-1696, 30 September 2003, pp. 1679-1686.
    3George B. Witman, “Cell Motility: Deaf Drosophila keep the beat,” Current Biology Vol 13, R796-R798, 14 October 2003.

    Mitochondrial Ribosome Structure Casts Doubts on Endosymbiont Theory 10/07/2003
    Have you heard the story that early cells swallowed other ones and made them their slaves? That is supposedly where mitochondria came from, but an article in the Oct. 3 issue of Cell reports that there are some big differences between the mitochondrial ribosomes of eukaryotes and those of bacteria, the presumed captives.
    Manjuli Sharma et al.1 determined the structure of the eukaryotic mitochondrial ribosome (mitoribosome) for the first time. These ribosomes (sites of protein synthesis) differ from those in the cytosol, because they produce 13 specialized proteins dedicated primarily to the production of ATP.

    “According to several genomic analyses, mitochondria are believed to have arisen from an early endosymbiotic event between a eubacterium and its host cell .... Therefore, it has generally been expected that the mitoribosome will display greater structural and functional similarities to a bacterial ribosome than to a eukaryotic cytoplasmic ribosome.”

    They found, “However, the RNA and protein composition of the mitoribosome differs significantly from that of bacterial ribosomes.” Whereas the small subunit has 950 nucleotides and 29 proteins, the bacterial counterpart has 1542 and 21, respectively. The large subunit has 1560 nucleotides and 48 proteins, but the bacterial counterpart has 120 + 2904 nucleotides in two units, and 33 proteins. “Thus, the protein-to-RNA ratio is completely reversed in the mitoribosome (69% protein and 31% RNA) relative to bacterial ribosomes (33% protein and 67% RNA),” they note. Even among the roughly half of the proteins in the eukaryotic mitoribosome that have homologs in bacteria, they are usually significantly larger. And the whole ribosome, though larger, is more porous than the bacterial one.
    The rest of the paper describes the functional units of the mitoribosome. They found exquisite entrance tunnels for the transfer RNA and messenger RNA, and precision exit tunnels for the nascent polypeptides. They feel their analysis “provides new insights into the structural and functional evolution of the mitoribosome.” But the paper also describes large differences between the mitoribosomes and the ribosomes in the rest of the cell:

    “Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.”

    It appears, therefore, that these three classes of ribosomes are quite different from each other. This is probably due to the different jobs they have to do, as expressed in the title of their paper: the component proteins of the mitoribosome suggest they have “an expanded functional role” over their counterparts.
    1Sharma et al., “Structure of the Mammalian Mitochondrial Ribosome Reveals an Expanded Functional Role for Its Component Proteins,” Cell Vol 115, 97-108, 3 October 2003.

    A Self-Regulating Recycling System Found in the Cell 10/07/2003
    Cells are not watertight sacks; they import and export things. But they are not leaky sacks either: everything coming and going is authenticated by sophisticated mechanisms. Small packages, like water molecules or individual proteins, have specially-designed channels embedded in the cell membrane that check their credentials and make them run an electronic gauntlet (see 03/12/02 headline, for instance).2 Larger packages, however, have a surprising method of making their entrance: they dive in and get wrapped in geodesic spheres. The cell membrane neatly reseals itself around the point of entry, which occurs only where specialized receptors allow it. This is called endocytosis (for cargo on the way in) and exocytosis (on the way out).
    The geodesic spheres are made up of a three-armed protein called clathrin. The clathrin molecules envelop the cargo, forming a crystalline polyhedron around it. (You absolutely have to see this cool animation by Allison Bruce of Harvard, showing clathrin forming a spherical vesicle; incredible.) Once the cargo in its crystalline cage has been safely ferried to its destination, the clathrin molecules disassemble and are available for re-use. (This process, and much more, is beautifully illustrated in the award-winning animated short film Voyage Inside the Cell). Exocytosis is the process in reverse, when the cell needs to export cargo to the outside: for example, when a nerve cell needs to send neurotransmitters to another neuron. A host of helper enzymes are involved in making both processes work.
    “Clathrin-mediated endocytosis is one of the primary mechanisms by which eukaryotic cells internalize nutrients, antigens, and growth factors and recycle receptors and vesicles,” begin a team of Pennsylvania scientists in a paper in the Oct. 3 issue of Cell.1 But it should be obvious that the amount of cargo coming in must balance that going out, or else the cell will burst or shrivel. “A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function,” they write, speaking especially of neurons that execute these processes continuously in the central nervous system and the brain. How can the cell maintain this balance?
    These scientists discovered an automatic regulatory process that ensures the materials are recycled properly. A protein called endophilin, a key regulator of the endocytosis process, has two states: open and closed. In the open state, it attaches to the interior side of voltage-gated calcium channels (these are membrane turnstiles that allow only doubly-ionized calcium to pass through). Here, it somehow recruits other protein machines needed for the endocytosis operation. When the calcium concentration reaches 1 micromolar, the endophilin switches into the closed position. Then, it detaches from the calcium gate, “which would presumably allow the liberated endophilin and dynamin [another helper enzyme] to become actively involved in endocytosis immediately after SV [synaptic vesicle] exocytosis.” A similar self-regulating system had been known for exocytosis, but this is the first time a mechanism has been found to regulate endocytosis: “By coupling tightly to both the exocytotic and endocytic machineries,” they conclude, “voltage-gated Ca2+ channels are thus uniquely positioned to coordinate the SV recycling process.” Their model, however, is just a rough picture of a much more elaborate process scientists are just beginning to understand.
    1Yuan Chen et al., “Formation of an endophilin-Ca2+ Channel Complex Is Critical for Clathrin-Mediated Synaptic Vesicle Endocytosis,” Cell Vol 115, 37-48, 3 October 2003.
    2Two American scientists just received the Nobel Prize in chemistry October 8 for their work that revealed the structure and function of the water and ion channels in the cell membrane. See story in FoxNews.

    Life Found in the Genome Desert 10/16/2003
    Southwestern deserts are often filled with living things, if you look closely enough. Similarly, the “deserts” in the human genome, only sparsely populated with protein-coding genes, are turning up some surprising functions. Four California-based geneticists published a paper in Science Oct. 17 that found long-range enhancers in these regions:

    “Approximately 25% of the genome consists of gene-poor regions greater than 500 kb [kilobases], termed gene deserts. These segments have been minimally explored, and their functional significance remains elusive. One category of functional sequences postulated to lie in gene deserts is gene regulatory elements that have the ability to modulate gene expression over very long distances.”

    They found evidence that this is true, and scientists had better pay attention:

    “The demonstration that several of the enhancers characterized in this study reside in gene deserts highlights that these regions can indeed serve as reservoirs for sequence elements containing important functions. Moreover, our observations have implications for studies aiming to decipher the regulatory architecture of the human genome, as well as those exploring the functional impact of sequence variation. The size of genomic regions believed to be functionally linked to a particular gene may need to be expanded to take into account the possibility of essential regulatory sequences acting over near-megabase distances.”
    1Nobrega, Ovcharenko, Afzal, and Rubin, “Scanning Human Gene Deserts for Long-Range Enhancers,” Science 23 June 2003; accepted 8 September 2003, 10.1126/science.1088328.

    Ribosome Does Fast Forward Scanning 10/23/2003
    Remember cassette players that allowed you to scan ahead to the next song? Ribosomes in the cell are like tape readers that can translate one message, written in DNA, to another message, written in proteins. The “tape” that the ribosome reads is a string of messenger RNA, freshly delivered from the DNA code in the nucleus by other molecular machines. Once the tape is inserted into the ribosome, it reads the message and ties amino acids together to form a protein chain. If you watched the film Unlocking the Mystery of Life, you saw a computer animation of the process in slow motion. Now it appears that the ribosome has a scan function.
    Scientists have known about a puzzling phenomenon that occasionally occurs within the ribosome. For unknown reasons, the ribosome can disengage its reading head from the tape and fast-forward to another spot, then continue reading and translating at the next open reading frame (ORF). This is called “translational bypassing.” They know that this is signalled automatically by codes embedded in the messenger-RNA “tape”: a take-off code and a landing code, among others. What they didn’t know is whether the reading head continues to scan the code while disengaged. By analogy with cassette players, is it “fast forward” or “scan”? Apparently, it’s the latter.
    A team of scientists at the University of Washington figured this out by rigging two landing codes into the tape. They found that the ribosome always took the first one. This can only mean that the ribosome is able to scan the message while disengaged and detect the presence of the landing site. Their paper is published in the Oct. 23 online preprints of PNAS.1 Although it is still unclear why the ribosome would want to jump ahead on the recording, other researchers, like Raymond F. Gesteland, believe it is part of a “bag of tricks” the cell has to regulate gene expression or correct errors.
    1Gallant et al., “Evidence that the bypassing ribosome travels through the coding gap,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.2233745100, Published online before print October 23, 2003.

    Time to Junk the Term “Junk DNA” 11/06/2003
    “Heirlooms in the Attic” is the way Mark Johnston and Gary Stormo describe, in the Nov. 7 issue of Science1, the potential discoveries awaiting scientists in the realms of non-coding DNA, disparagingly referred to as “junk DNA.” Genome research has understandably focused on genes that code for proteins, but what are these vast stretches of non-coding DNA? Are they junk or treasures?
    Hints that interesting gems are waiting to be uncovered include the fact that these areas are highly conserved,2 even more so, in some cases, than the genes. According to evolutionary thinking, if something is conserved, it must be important. Scientists are only beginning to uncover hidden functions in the non-coding regions. Do they regulate gene expression? Do they provide scaffolding or attachment points? “Uncovering the part that CNGs [conserved non-genic sequences] play in the cell will certainly require experimentation,” they say, “and that activity is likely to occupy many people for quite some time.” –
    Early in the Human Genome Project, people argued about what to sequence. Some advocated determining just the sequence of the protein-coding regions, because the vast majority of the genome is “junk” DNA. This would, they argued, be cost effective because most of the important information is in protein-coding DNA. Given what we’ve learned about the jewels in the genome’s attic, aren’t we glad they sequenced it all?
    See also Oct 16 headline.
    1Mark Johnston and Gary D. Stormo, “Evolution: Heirlooms in the Attic,” Science Volume 302, Number 5647, Issue of 7 Nov 2003, pp. 997-999; 10.1126/science.1092271.
    2Dermitzakis et al., “Evolutionary Discrimination of Mammalian Conserved Non-Genic Sequences (CNGs),” Science. Originally published in Science Express as 10.1126/science.1087047 on October 2, 2003; Science, Vol. 302, Issue 5647, 1033-1035, November 7, 2003; 10.1126/science.1087047

    Intracellular Railroad Has Park-and-Ride System 12/04/2003
    Cells are like miniaturized cities, with elaborate transportation systems ferrying their cargo to and fro (see Feb. 25 headline). Just like a city may have railroads, busses, cars and monorails, the cell has multiple kinds of transport motors: dyneins, kinesins, and myosins. Scientists have learned that most of the roadways are like one-way monorails: actin filaments and microtubules, upon which the vehicles travel in one direction. But what if a passenger needs to jump from one system to another? ' No problem; the cell has mastered the art of ridesharing with its own park-and-ride system.
    In the Dec. 2 issue of Current Biology1, this is described by Marcus Maniak in a Dispatch entitled “A park-and-ride system for melanosomes.” Melanosomes are organelles (somes) that carry melanin, the pigment chemical that allows some organisms, including fish and amphibians, to change their skin color to match their surroundings. For this to work, the melanosomes need to hitch rides either to the exterior of the cell or the interior. He pulls together several recent findings to describe how this all works:

    “Together these findings suggested how melanosomes might move on actin filaments and showed that this type of motility is required for the even distribution of melanosomes within the cell. From these main observations, it became clear that, during aggregation, a cytoplasmic dynein motor carries melanosomes on the radially arranged microtubules towards the cell center (Figure 1B), while during dispersion a kinesin transports the granules to the periphery (Figure 1C), where they engage via a myosin V molecule with short actin filaments to be distributed further (Figure 1D). This switching of transport systems is a kind of miniature edition of modern urban traffic, where millions of workers leave the city centers in the evening on trains and board their cars at park-and-ride stations to complete their daily journey within the green peripheral belt.”

    As if that were not amazing enough, it appears that the drivers “talk” to each other with a communication system:

    “Although the work of Rodionov et al. has moved the field a large step further, there are obviously several issues that remain to be investigated. Exciting new findings addressing the coupling of motor molecules to the melanosome surface in other experimental animals open the possibility to speculate how the motors may talk to each other on a molecular level. At least for Xenopus there is now clear evidence that both dynein and kinesin couple to melanosomes via the dynactin complex. Moreover, both motors compete for the same protein component; this could allow one motor to gain access to the microtubule while the other is prevented from engaging successfully.”

    He describes how this “tug-of-war” competition is actually a kind of way for the motors to negotiate the right-of-way. Additional factors that attach to the vehicles or trackways may assist in making sure the rules of the road are obeyed. “Thus,” he concludes, “further exciting results are on the way to complete the picture of how melanosomes switch from one transport system to the other.”
    1Marcus Maniak, “Dispatch: Organelle Transport: A Park-and-Ride System for Melanosomes,” Current Biology Vol 13, R917-R919, 2 December 2003.

    Elaborate Quality Control Governs the Cell’s Protein-Folding Factory 12/20/2003
    If it weren’t for quality control in our cells, we’d be dead. That’s the gist of an amazing Insight article in the Dec. 18 issue of Nature.1 “Aberrant proteins are extremely harmful to cells,” the authors begin. How harmful? Here is a short list of diseases that can result from improperly folded proteins or failures in the quality control systems that direct their formation: Creutzfeldt-Jakob disease, Alzheimer’s disease, and other degenerative diseases, scurvy, cystic fibrosis and more. In fact, serious defects in protein assembly are probably never seen, because they could prevent an organism from getting past the first cell division in the embryo. The only way a cell can live and grow is with the assistance of a host of traffic controllers, regulators, monitors, ushers, transporters, inspectors, security guards and emergency technicians maintaining the complex processes of protein assembly. Success must be ensured constantly, 24 x 7, that despite a flurry of activity, must maintain a state of dynamic equilibrium, called homeostasis.
    Each cell in the body is like a city of interrelated factories made up of protein machines and structures operating under strict regulations, built on coded instructions. One of the most important factories is the protein folding system, which ensures that newly-sequenced proteins coming out of ribosomes are folded into their correct (native) shapes. Proteins are made up of amino acids, usually hundreds of them, that are first sequentially assembled in ribosomes, based on templates sent from the DNA code. Then, they are folded into specific, complex three-dimensional shapes that perform numerous and diverse functions in the cell (see 06/13/02 and 05/31/02 headlines.) Protein folding is assisted by enzymes whimsically called chaperones (see 05/05/03 headline) but is also checked and rechecked by numerous other quality control systems (see 09/09/02 headline).
    In the current paper in Nature, the authors have unveiled more of the complexity in the quality controls governing protein folding. Some of the folding occurs in networked subway tunnels that run throughout the cell, called the endoplasmic reticulum (ER). Before getting into the ER, some proteins already begin their folding with assistance from certain chaperones. The authors explain, “In mammalian cells, proteins are translocated into the ER ... where they start to fold co-translationally [i.e., while they are en route into the ER]. Folding is completed post-translationally, and, generally, individual subunits have folded before assembly and oligomerization [the joining together of multiple chains] take place. Sequential interactions with distinct chaperones are required for each of these steps.” The job is completed inside the ER, and the finished protein “tool” is then sent on its way to work. But that is just the tip of a huge iceberg made up of a multitude of processes – hardware and software – that work together to ensure success.
    In the following examples from the article, entitled “Quality control in the endoplasmic reticulum protein factory,” don’t worry about unfamiliar technical terms. Just try to keep track of how many different players are involved in the team of factory workers dedicated to one job: folding a single protein. And keep in mind that each team player is itself a protein, built with the same quality control. You can almost envision little factory workers, each skilled at their specific tasks, alert and knowing just what to do, but it’s all done with chemicals! Be patient in these extended quotes, because the awe is in the details.

    1. Redox regulation: A sensitive chemical balance is maintained between reducing and oxidizing (redox) conditions along the protein’s pathway through the folding factory. “The redox gradient between the ER and the cytosol seems to be important for intercompartmental signalling, particularly in the integrated response to oxidative stress, in which adaptive responses emanating from different compartments are coordinated. And redox reactions with opposite electron fluxes must take place in the ER to mediate formation, isomerization and reduction of disulphides. The wealth of redox assistants allows these fluxes to be separate, and channels electron transport through specific protein–protein interactions.”
    2. Location specificity: “Although most folding factors in the ER are ubiquitously expressed throughout the body, some are tissue-type specific or cell-type specific, and probably fulfil a particular synthetic task.... For example, efficient collagen production requires the expression of hsp47, whereas a tissue-specific protein-disulphide-isomerase-like protein, PDIp, is produced in the pancreas and probably permits the massive secretion of digestive enzymes.”
    3. Bridge builders: Junctions called disulphide bridges are common in protein folds, and these, although weak, are carefully maintained by a host of enzymes called oxidoreductases: “The impressive number of oxidoreductases in the ER suggests that catalysis and regulation of disulphide-bond formation is crucial for folding. Energywise, in most cases, the contribution of a disulphide bond is hardly more than that of a single hydrogen bond [i.e., quite weak], yet, without disulphide bonds, native conformations are not obtained. Disulphide bonds cannot force a folding protein into a given conformation: in the sampling of conformations during folding in the ER, native and non-native disulphide cross-links are transiently formed [i.e., correct and incorrect links form and break easily, and must be guided]. Continuous activity of oxidoreductases probably ensures that these covalent links remain flexible until folding is completed.”
    4. Correct fold recognition: Even though a string of amino acids could conceivably fold in large number of ways, like a Rubik’s cube, somehow the chaperones are able to tell a correct (native) fold from an incorrect one. “Besides providing a unique folding environment, the ER has a crucial quality-control role. How does it discriminate between native and non-native proteins? The answer to this question depends primarily on ER chaperones. When folding or assembly intermediates expose hydrophobic [i.e., water-avoiding] surfaces, unpaired cysteines or immature glycans, ER-resident chaperones or oxidoreductases interact with them, and as a consequence they are retained in the ER or retrieved from the Golgi complex [see 11/12/01 headline]. By forming multimolecular complexes, folding factors in the ER may provide matrices that couple retention to folding and assembly. Immature proteins may also form aggregates that are excluded from vesicles exiting from the ER” [i.e., such that they are not ejected before they are ready].
    5. Fail-safe inspection: A protein needs to pass multiple layers of monitoring: “All proteins are subjected to a ‘primary’ quality control that monitors their architectural design through ubiquitous folding sensors (Table 1). ‘Secondary’ quality-control mechanisms rely instead on cell-specific factors and facilitate export of individual proteins or classes of proteins.... the ER is the main test bench where molecules destined for the extracellular space are scrutinized for their potential toxicity.”
    6. Feedback regulation: The ER not only does quality control, but sends messages back to the nucleus to regulate the production of more chaperones: “The reasons for having a quality-control system in the ER are easy to understand where protein folding and function are concerned, especially in multicellular organisms where development relies on the fidelity of protein secretion. Quality control can also regulate the transport or the activity of certain proteins during differentiation or in response to stress or metabolic requirements.”
    7. Waste control: When a protein cannot be folded after repeated attempts, more assistants are on hand to ensure proper dismantling and recycling: “Mutations or unbalanced subunit synthesis make folding or assembly — and hence exit from the ER — impossible. To maintain homeostasis [dynamic equilibrium], terminally misfolded molecules are ‘retrotranslocated’ or ‘dislocated’ across the ER membrane to be degraded by cytosolic proteasomes” [organelles equipped to break up badly-folded proteins and recycle their parts].
    8. Time limits: Somehow, the cell knows when a protein has had enough time to shape up or ship out: “A fascinating problem is how molecules that have not been given the time to fold (and therefore are unfolded) are discriminated from those that have failed to fold after many attempts (misfolded), and must therefore be disposed of. One way of timing glycoprotein quality control involves the sequential processing of N-glycans and in particular mannose trimming in the ER. It remains to be seen how substrates are eventually targeted to the retrotranslocation channels, how these are opened, and to what extent proteins must be unfolded to negotiate dislocation.”
    9. Workforce regulation: Like a company’s human resources department that responds to managers’ calls for more workers, the cell keeps track of how many chaperones are available, and sends out “help wanted” ads to the nucleus. “To maintain the efficiency of quality-control mechanisms in diverse physiological conditions, living cells have evolved [sic] regulatory circuits that monitor the levels of available chaperones. This is true for both the cytosol and the ER, and compartment-specific responses clearly exist that selectively restore optimal levels of the desired folding factors.”
    10. Emergency squads: The authors provide two examples of rapid-response traffic control teams: “The accumulation of aberrant proteins in the cytosol triggers the heat-shock response, resulting in de novo synthesis of hsp70 and other cytosolic chaperones. But if aberrant proteins accumulate in the ER, cells activate a different response, the unfolded protein response (UPR), which leads to the coordinated synthesis of ER-resident chaperones and enzymes.”
    11. Failure consequences: The authors give an example of what can go wrong when the system gets swamped, starved, or sent defective parts: “Physiologically, ER stress (a condition in which the folding machinery in the ER cannot cope with its protein load) can be caused by synthesis of mutated or orphan proteins, absence of cofactors (an example being scurvy, in which collagen cannot fold because of the lack of vitamin C), or a drastic increase in otherwise normal cargo proteins.”
    12. Unified response to varied inputs: A variety of signals can lead to the same Unfolded Protein Response (UPR) pathway: “How do the diverse unfolded or misfolded proteins that accumulate in the ER provoke the same pathway? The unifying concept is that BiP and other primary quality-control factors maintain the stress sensors in the ER in the inactive state, so that chaperone insufficiency triggers UPR whatever the nature of the cargo.”
    13. Meltdown regulation: What happens when the damage is so great, that further operation of the factory would be dangerous? Three independent controls make sure an orderly shutdown occurs (apoptosis, or cell death: see 04/09/02 headline). “The mammalian ER sensors, Ire1, PERK and ATF6, guarantee a tripartite response with synergic strategies. By phosphorylating eIF2alpha, PERK transiently attenuates translation, limiting protein load. ATF6 drives the transcriptional upregulation of many ER-resident proteins and folding assistants. Ire1 activates XBP-1, which in turn induces transcription of factors that facilitate ER-associated degradation (ERAD). The two-step activation of XBP-1 (transcriptionally induced by ATF6 and post-transcriptionally regulated by Ire1) guarantees the proper timing of the UPR [unfolded protein response] attempts to fold proteins precede the decision to degrade them. If the response fails to clear the ER, apoptosis is induced through several pathways.” The authors explain that “The UPR is multi-faceted and regulates proteins involved in quality control, ERAD and many aspects of the secretory pathway.”
    14. Balancing act: The quality control mechanisms walk a tightrope, with serious consequences for falling off: “Quality control must be a balance between retaining and degrading potentially harmful products and not preventing export of biologically active proteins. CFTR mutants in cystic fibrosis illustrate an overzealous quality control, where biologically active mutants cannot leave the ER. In this case, relaxing the quality control could cure the patient. But disease can also originate from defective degradation. If the rate of synthesis of a protein exceeds the combined rates of folding and degradation, a fraction of it will accumulate intracellularly.” Misfolded proteins have to make it across the ER membrane in time, and get degraded by the proteasome in time, or else aggregations (aggresomes) can build up inside or outside the ER. These are implicated in a number of “ER storage diseases.”

    The authors put this all into perspective: “Over the past few years, much has been learned about how proteins are handled by the ER folding and quality-control machineries, and some of this knowledge has begun to be translated to industry and to the clinic. Yet, many questions remain....” They hope that further elucidation of these complex, coordinated systems will allow drug designers to target faulty elements that cause degenerative diseases, or induce apoptosis in tumors to make them self-destruct. Clearly, though, in spite of the complexity already described, much remains to be learned about cell quality control.
    1Robert Sitia and Ineke Braakman, “Quality control in the endoplasmic reticulum protein factory,” Nature 426, 891 - 894 (18 December 2003); doi:10.1038/nature02262
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    How Do Plants Know When to Bloom? 01/07/2004
    Scientists like to use big words to impress the rest of us, so they have a term for how a plant decides when to bloom: vernalization. But making up a word for a phenomenon is not the same as explaining it.
    Everybody observes that plants seem to just “know” that spring is here, when they put forth their glorious blossoming colors, but think about it: how can a plant, without eyes or a brain or a calendar, judge when it is safe to send out flowers? Through all the vagaries of weather they have an uncanny sense of timing. It’s especially puzzling how winter annuals do this, and biennials, which only bloom in the second year. How can a plant have a memory, and sense the seasons? What goes on in the genes, at the molecular level? How can the memory be preserved through multiple cell divisions?
    This was the subject of two scientific papers in the the Jan. 8 issue of Nature,1,2 and an analysis by Christopher Surridge.3 The process is very complex and still mysterious in many respects. It involves quite a few genes and proteins, particularly histones which are part of the chromatin that wraps DNA, and additional signaling molecules like acetyl and methyl groups. Biochemists have found that, in many cellular processes, there are starters and stoppers: genes and proteins that initiate or suppress an action, and other genes and proteins that stop or re-enable them. For instance, a molecule might clamp onto a gene, making it impossible for the translation machinery to read it, and another molecule will remove the suppressor, allowing the gene to be read and transcribed into a protein. The complex dance of activators and repressors and signalling molecules can be triggered by the external environment and by other activities inside the cell.
    If you can keep this all straight, vernalization goes something like this: a gene named FLC prevents flowering, and is normally expressed during the off-season. A cold snap induces the VIN3 protein to remove acetyl groups from the histones on the chromatin near this gene, signalling two other molecules (vernalization proteins VRN1 and VRN2) that this gene is silenced. Their job is to keep it that way, so that suppression of flowering is itself suppressed. The FLD gene, which promotes flowering, is then expressed. Somehow, FLD tells the molecules at the apical meristem (see 11/20/2003 headline), to send out the buds. Surridge explains, “Silencing is an effective means of controlling long-term gene expression, as it persists even after cells divide. In animals, switching silencing on or off is a well-known way to control development. It seems that plants share this system, using it to preserve the memory of winter’s passing.”
    How does cold cause these reactions? What is known so far is just part of a more involved process. One of the papers2 admits, “How cold results in low FLC RNA and whether any post-transcriptional regulation occurs that feeds back to cause reduced transcription is unknown at present.” The other paper1 says, “The additional components that interact with VIN3, and VRN1 and VRN2, to repress FLC during and after vernalization are not known.” Undoubtedly there are other environmental cues that affect vernalization, such as length of daylight and nutrient availability.
    A popular-level account from Reuters on these results can be found on
    1Sibum Sung and Richard M. Amasino, “Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3,” Nature 427, 159 - 164 (08 January 2004); doi:10.1038/nature02195.
    2Ruth Bastow et al., “Vernalization requires epigenetic silencing of FLC by histone methylation,” Nature 427, 164 - 167 (08 January 2004); doi:10.1038/nature02269.
    3Christopher Surridge, “Plant development: The flowers that bloom in the spring,” Nature 427, 112 (08 January 2004); doi:10.1038/427112a.

    How Enzymes Work – But Don’t Ask Where They Came From: Just Believe 01/12/2004
    Enzymes are protein machines in the cell that speed up reactions that normally would proceed very slowly or not at all. Four biochemists publishing in the Jan. 9 issue of Science1 describe the exquisite power of these biological catalysts: “Enzyme catalysis, which can produce rate accelerations as large as a factor of 10exp19, involves molecular recognition at the highest level of development.” That figure represents a speed increase of 10 quintillion (see also 05/06/2003 headline). After a brief review of efforts to understand enzymes, they remark that “An overview of our present understanding of enzyme catalysis is particularly timely because of the increasing number of articles that propose a variety of origins for enzyme catalysis,” of which they list the names of some proposals. Their paper offers a framework that incorporates these proposals.
    They used rate theory and computer simulations to characterize some of the methods enzymes use to perform their specific reactions. Their table lists sixteen different mechanisms used by sample enzymes from plants and animals. Here is an example for tyrosine-tRNA synthetase:
    Enzyme-transition state and enzyme-intermediate complementarity help to stabilize the transition state of tyrosine activation and to shift the chemical equilibrium by seven orders of magnitude in the direction of the intermediate. Loop motions induced by the chemical process are essential in creating these interactions and permitting access to the active site. (Emphasis added; for more on the tRNA-synthetase family of enzymes, see 07/21/2003 and 06/09/2003 headlines.)
    After providing detailed mathematical analyses of these mechanisms, they conclude, “Evolutionary selection makes possible the development of enzymes that use a wide range of molecular mechanisms to facilitate reactions. Although, in principle, such rate enhancements could arise from lowering the quasithermodynamic free energy of activation or increasing the generalized transmission coefficient, the present analysis shows that the former plays the dominant role” (emphasis added). They feel that modern transition state theory is adequate to describe these processes.
    1Garcia-Viloca, Gao, Karplus, and Truhlar, “How Enzymes Work: Analysis by Modern Rate Theory and Computer Simulations,” Science 09 Jan 2004, 10.1126/science.1088172.

    Centromere Shows More Gems in “Junk DNA” 01/12/2004
    A biochemist at University of Wisconsin-Madison and a colleague sequenced a hard-to-sequence part of the rice genome, the centromere, and found four genes in it. Previously, it was thought to be a vast wasteland of repetitive, non-coding DNA. The scientist, Jiming Jiang, thinks his work provides a “window to evolution” of the centromere, according to writer Terry Devitt: “The evolutionary progression [sic] of the centromeres, Jiang suggests, may be analogous to how temperate forests evolve [sic] from more diverse ecosystems to climax forests where a single species of tree dominates. In the rice centromere, it may be that evolution has not yet purged active genes to be replaced by the long and repetitive blocks of DNA that mark the centromeres of most organisms”.

    Live at the Improv: DNA Polymerase 01/20/2004
    When a DNA reader hits an unfamiliar line, it improvises, reports EurekAlert:
    Prof. Zvi Livneh and Ph.D. student Ayelet Maor-Shoshani of the Biological Chemistry Department cut a DNA strand — from the bacterium E. coli — and inserted material similar to that which composes crude oil in between both its ends. As expected, the regular DNA polymerase stopped working when it reached the foreign material. Yet to the scientists’ amazement, a specialized DNA polymerase jumped in to rescue the stalled replication process, and continued the copying process, inserting nonexistent genetic components into the “printout’ when it encountered the foreign material. This can be compared to a person who forgets some words in a song and makes up new ones to be able to continue to sing.
    The scientists believe this capability provides resilience against damaged DNA, except in the most extreme cases:
    True, when DNA polymerase improvises a tune, errors (i.e. mutations) may occur in the new cells’ DNA. Yet Livneh explains that the body cannot feasibly let all cells with damaged DNA die, for there are too many of them. “Only if the DNA contains a very high level of damage will the cell’s machinery ‘give up’ and let the cell die.”

    “Utmost Precision” Found in DNA Repair Enzyme 02/13/2004
    The cell has many helper enzymes that can repair DNA damage. One such enzyme, named MutY, has been described in the Feb. 12 issue of Nature.1 Reviewer Tomas Lindahl sets the stage: “Damaged DNA must be removed with the utmost precision, as mistakes are costly. The structure of a repair enzyme bound to its substrate provides a welcome clue to how this is achieved.”
    This particular enzyme is able to recognize its particular error target, an adenine incorrectly paired to an oxidized guanine, because of “extensive and precise contacts” it makes with that specific erroneous pair. These contacts prevent it from mistakenly removing a correct pair. In a paper in the same issue, Fromme et al.2 describe “an ingenious way by which this specificity is achieved” through these multiple, precise contacts.
    Lindahl describes how this enzyme works. Details of the jargon are not essential for appreciating the precision of this molecular machine’s lifesaving activity:

    “MutY belongs to a group of enzymes known as DNA glycosylases, which recognize altered bases in DNA and help to remove them. Like other DNA glycosylases, it generates a sharp bend in the DNA at the site of the mismatch. The new structural data provide a suitable explanation for why — as is desired — MutY doesn’t recognize and remove an adenine opposite its normal base partner, thymine (T): the extensive and precise contacts between MutY and an A•xoG pair are entirely absent in a normal AT pair. Similarly, the enzyme’s active site does not accommodate a cytosine opposite an oxoG; for coding reasons, it is important that the oxidized base rather than the normal base is repaired in this partnership.”

    Lindahl notes that mutations in this enzyme put humans at risk of colorectal cancer. Other oxygen-altered bases, if not repaired, are implicated in tissue degeneration and ageing.
    1Tomas Lindahl, “Molecular Biology: Ensuring error-free DNA repair,” Nature 427, 598 (12 February 2004); doi:10.1038/427598a.
    2Fromme et al., Nature Feb 12, 2004, p. 652.

    Your Internal Motors Can Run Nanotech 02/13/2004
    In each cell in your body, and in that of every living thing, there exists a tiny motor named ATP synthase that Science News1 calls “the ultimate molecular machine.” It converts electrical to chemical energy, writes Alexandra Goho, “with amazing efficiency.” Now, Japanese have harnessed some of these motors (only 12 millionths of a millimeter high) to power artificial machines. They attached hundreds of the motors to a glass surface and attached little magnetic beads to the rotor part. With an electromagnet, they induced them to spin, and were able to make them rotate both clockwise and counterclockwise.
    1Alexandra Goho, “Nature’s tiniest rotor runs like clockwork,” Science News, Week of Feb. 7, 2004; Vol. 165, No. 6, p. 94; see also article by Jessica Gorman, “Nanotech Switch: Strategy controls minuscule motor,” Science News, Week of Nov. 9, 2002; Vol. 162, No. 19.

    DNA Is a Code Operated by Another Code 02/17/2004
    The discovery in the 1950s that DNA stored a coded language was amazing, but recently a new level of complexity has come to the awareness of biochemists. Apparently, another code determines which DNA genes will be opened for expression and which should be suppressed.
    The Feb. 14 issue of Science News1 describes the history of the discovery of the so-called “histone code.” These are patterns of “tails” attached to the histones around which DNA is tightly wrapped. Within the last eight years, scientists have been discovering that the histones do not merely spool the DNA, they regulate which genes get expressed.
    The pattern of acetylation and methylation on the histone tails appears to form a code that is heritable through cell divisions. Compared to the well-known DNA genetic code, “A histone code may be much more complex,” writes John Travis. Shelley Berger (Wistar Institute) exclaimed, “There are all kinds of sites [on histone tails] that can be modified. The possibilities for a code are quite enormous. It’s not going to be a simple code.” After summarizing the literature, Travis concluded, “With such designer histones, it seems that researchers are on their way to having in their hands all the words of the histone code. But, it may still be a stiff challenge to figure out what those words mean.”
    For a previous story on the histone code, see 11/04/2002 headline, “Cell Memory Borders on the Miraculous.”
    1John Travis, “Code Breakers: Scientists tease out the secrets of proteins that DNA wraps around,” Science News, Vol. 165, No. 7, Feb. 14, 2004, p. 106.

    Cellular Cowboys: How the Cell Rounds Up Chromosomes Before Dividing 03/04/2004
    Two cancer researchers from UC San Diego describe mitosis (cell division) in the Mar. 4 issue of Nature.1 Pulling together the latest findings about this elaborate and important process, they begin by describing the puzzle that the cell needs to solve:

    “At the beginning of mitosis, the process of cell division, chromosomes are organized randomly — like jigsaw puzzle pieces spread out on the floor. Their constituent two ‘sister chromatids’, each of which contains one of the two identical DNA molecules produced by replication, must be oriented such that they will be pulled in opposite directions into the two newly forming cells. Like a jigsaw, the solution for correctly orienting all chromosomes comes partly through trial and error. Mechanisms must exist to eliminate wrong configurations while selecting the right ones.”

    In the article, they describe how cables (microtubules) connect to handles (kinetochores) on the chromosomes and start pulling them in opposite directions. Another enzyme dissolves the molecular “glue” in the centrosomes that hold the sister chromatids together, so that the opposite poles of the spindle can pull them apart into the daughter cells.
    A newly-described “highly-conserved enzyme” (i.e., identical in yeast and vertebrates), named Aurora B kinase, somehow finds chromosomes that lack an attachment to the other pole of the spindle, and fixes them. Apparently this enzyme is able to identify chromosomes that are incorrectly lassoed to the same pole (syntelic attachment) and therefore are not under tension. Only when there is tension on each chromosome, pulling the sister chromatids toward opposite poles, will the process continue. “Finding out how Aurora B identifies and corrects them is an obvious next step,” the authors say.
    Ian M. Cheeseman and Arshad Desai, “Cell division: Feeling tense enough?”, Nature 428, 32 - 33 (04 March 2004); doi:10.1038/428032b.

    Cell Networks 03/22/2004
    A team of Chinese scientists analyzed protein interactions in yeast cells, and titled their paper in PNAS1 “The yeast cell-cycle network is robustly designed.” They “demonstrated that the cell-cycle network is extremely stable and robust for its function,” and “able to survive perturbations.” The beginning of the paper expresses the wonder the stimulated their research:

    “Despite the complex environment in and outside of the cell, various cellular functions are carried out reliably by the underlying biomolecular networks. How is the stability of a cell state achieved? How can a biological pathway take the cell from one state to another reliably?

    After analyzing the stable states, “big attractors” and checkpoints in the yeast cell cycle, the scientists remind us that this network is part of an even bigger marvel:

    “Note that the network we studied ... is only a skeleton of a larger cell-cycle network with many ‘‘redundant’’ components and interactions.... Thus, we expect the complete network to be even more stable against perturbations.
    ... Furthermore, our results suggest that not only do biological states correspond to big fixed points but the biological pathways are also robust.
    Functional robustness has been found in other biological networks, e.g., in the chemotaxis of E. coli (in the response to external stimuli) and in the gene network setting up the segment polarity in insects development (with respect to parameter changes) . It has also been found at the single molecular level, in the mutational and thermodynamic stability of proteins. In some sense, biological systems have to be robust to function in complex (and very noisy) environments.”

    And now to the climax. In the closing statement, after claiming several times that these networks are “robustly designed” (their term), they suggest that all this complexity, all this robustness, all this control and regulation is the product of time, chance and contingency. In fact, the very robustness might even help evolution make it better:

    “More robust could [sic] also mean more evolvable, and thus more likely to survive [sic]; a robust ‘‘module’’ is easier to be modified, adapted, added-on, and combined with others for new functions and new environments. Indeed, robustness may provide us with a handle to understand the profound driving force of evolution.”

    1Fangting Li, Tao Long, Ying Lu, Qi Ouyang, and Chao Tang, “The yeast cell-cycle network is robustly designed,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0305937101, published online before print March 22, 2004.

    Quick Picks03/31/2004
    Too many stories came in too fast at the end of March. Here are some we would have liked to explore in more detail. They’re all interesting and some have amazing facts and quotes.

    DNA vs. Evolution: A paper in the Royal Society Biology Proceedings1 warned that pleiotropy, the antagonistic effect of genes that need to mutate together, inhibits natural selection more than is usually realized. Sarah P. Otto writes,

    “Pleiotropy is one of the most commonly observed attributes of genes. Yet the extent and influence of pleiotropy have been underexplored in population genetics models. ... Under the assumption that pleiotropic effects are extensive and deleterious, the fraction of alleles that are beneficial overall is severely limited by pleiotropy and rises nearly linearly with the strength of directional selection on the focal trait. Over a broad class of distribution of pleiotropic effects, the mean selective effect of those alleles that are beneficial overall is halved, on average, by pleiotropy.”

    Thus the simplistic notion that a beneficial mutation will be acted on by natural selection is “severely limited” by the effect of pleiotropy.

    Another Thing You Can’t Live Without: David Carling (Imperial College) provides a quick review of AMPK in the 23 March issue of Current Biology5. If you don’t know what AMPK is (AMP-activated protein kinase), just be glad you (and everything else alive) has it:

    “AMPK has been dubbed the cellular fuel gauge, because it is activated by a drop in the energy status of the cell. If ATP is used up faster than it can be re-synthesized, ATP levels fall and this leads to a rise in AMP. The increase in the AMP:ATP ratio triggers the activation of AMPK and leads to the phosphorylation of a large number of downstream targets. The overall effect of AMPK activation is to switch off energy-using pathways and switch on energy-generating pathways, thus helping to restore the energy balance within the cell. The conservation of AMPK throughout evolution emphasises its importance: homologs have been identified in all eukaryotic species examined to date, including plants.”

    Other recent articles have focused on this cellular “fuel gauge” as a means of controlling appetite and obesity (see, for instance, Nature April 1, 2004). When asked “Can we live without it,” Carlin answers immediately, “Almost certainly not.” Mice without it die in embryo, and it cannot be mutated much: “Although a complete loss of AMPK activity is lethal, subtle changes in AMPK activity can lead to serious clinical consequences.”

    Genome Size: In Current Biology6 Brian Charlesworth and Nick Barton examine the question of why genome sizes differ so much between organisms, and offer a suggestion:

    “Genome sizes vary enormously. This variation in DNA content correlates with effective population size, suggesting that deleterious additions to the genome can accumulate in small populations. On this view, the increased complexity of biological functions associated with large genomes partly reflects evolutionary degeneration.”

    Intron Origins: Another paper in the same issue of Current Biology7 attempts to put forward a hypothesis about intron origin and evolution (see 09/23/2003 headline). “Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns.”
    1Sarah P. Otto, “Two steps forward, one step back: the pleiotropic effects of favoured alleles,” Proceedings: Biological Sciences, The Royal Society, Issue: Volume 271, Number 1540, April 07, 2004 Pages: 705 - 714 DOI: 10.1098/rspb.2003.2635 (published online before print).
    5David Carling, “:Magazine: AMPK,” Current Biology, Vol 14, R220, 23 March 2004.
    6Brian Charlesworth and Nick Barton, “Genome Size: Does Bigger Mean Worse?” Current Biology, Vol 14, R233-R235, 23 March 2004.
    7Sadusky et al., “Exon Junction Sequences as Cryptic Splice Sites: Implications for Intron Origin,” Current Biology Vol 14, 505-509, 23 March 2004.
    Last edited by bob b; October 25th, 2006 at 08:15 AM.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Virus: Like DNA in a Hard Plastic Shell 05/07/2004
    A European team of biophysicists studied the mechanical properties of a virus and found the shell, made of protein, to act like hard plastic. Writing in PNAS,1 they described the coat of a bacteriophage they studied:

    “The protective proteinaceous shells (capsids) of viruses are striking examples of biological materials engineering. These highly regular, self-assembled, nanometer-sized containers are minimalistic in design, but they combine complex passive and active functions. Besides chemical protection, they are involved in the selective packing and the injection of the viral genetic material.”

    The capsids look like oblong, geometric shapes with pointy ends. The DNA is packed inside under pressure, and the coat can withstand indentations of 30%. “The measured Young’s modulus,” they found, “is comparable with that of hard plastic.” They seemed to admire the little cases: the bacteriophage capsid is :

    “remarkably dynamic yet resilient and tough enough to easily withstand the known packing pressure of DNA (~60 atmospheres). These capsids, thus, not only provide a chemical shield but also significant mechanical protection for their genetic contents. Viral shells are a remarkable example of nature’s solution to a challenging materials engineering problem: they self-assemble to form strong shells of precisely defined geometry by using a minimum amount of different proteins.”

    The team is looking at these miniaturized packages for inspiration in the burgeoning field of nanotechnology.
    1Ivanovska et al., “Bacteriophage capsids: Tough nanoshells with complex elastic properties,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0308198101, published online before print May 7, 2004.

    Botulinum Toxin Deactivated by One Slight Change 05/10/2004
    A researcher at Brookhaven National Laboratory mutated a botulinum enzyme by just one amino acid, and abolished its toxicity. The mutation, a change from a glutamate to a glutamine at one position, increased the distance from a zinc atom to a water molecule by 0.6 angstrom, less than one tenth of a billionth of a meter. This was enough to prevent the botulinum enzyme from cleaving its target protein, a neurotransmitter. The modified enzyme could still bind to it, but not cleave it.

    Former “Junk DNA” Now Considered Essential 05/10/2004
    The term “junk DNA” seems to be fading with each new discovery. Helen Pearson, reporting for [i\Nature Science Update[/i], leads with the line “‘Junk’ DNA reveals vital role: Inscrutable genetic sequences seem indispensable.” They don’t know what it does yet, but the assumption is it must be important for evolution to hang onto it for so long. Pearson writes,

    “If you thought we had explored all the important parts of our genome, think again. Scientists are puzzling over a collection of mystery DNA segments that seem to be essential to the survival of virtually all vertebrates. But their function is completely unknown.
    The segments, dubbed ‘ultraconserved elements’, lie in the large parts of the genome that do not code for any protein. Their presence adds to growing evidence that the importance of these areas, often dismissed as junk DNA, could be much more fundamental than anyone suspected.”

    Researchers found 480 sequences that are identical between humans, mice and rats, and “largely match up with chicken, dog and fish sequences too,” but do not exist in invertebrates such as sea squirts and fruit flies.
    Scientists can only guess what these sequences do. One idea is that they “control the activity of indispensable genes.” Another is that they may slice and splice RNA into different forms. Or perhaps they may control embryo growth. Pearson describes the initial reactions to the discovery that junk DNA is not junk after all:

    To solve the conundrum, experts predict a flurry of studies into the enigmatic DNA chunks. “People will be intrigued by this [finding],” says Kelly Frazer who studies genomics at Perlegen Sciences in Mountain View, California. “It is the kind of stuff that blows people away.”
    She quotes one researcher who said, “It absolutely knocked me off my chair.” It was hard to believe these sections could be 100% identical. Some thought they must have contaminated their samples. “The presence of exact copies in different animals suggests that even tiny changes in the sequence of these segments destroy whatever they do,” Pearson surmises, “and have been weeded out during evolution” [sic] whereas other parts have been free to accumulate mutations.
    Clearly there is a lot of work ahead, Pearson says. Finding the function of the ultraconserved elements is just the tip of the iceberg. There are other vast tracts of similar so-called “junk DNA” whose functions await discovery.

    On a related subject, Current Biology has news on introns (see 09/03/2003 headline). A dispatch by Arlin Stoltzfus begins, “The evolutionary origin of spliceosomal introns remains elusive. The startling success of a new way of predicting intron sites suggests that the splicing machinery determines where introns are added to genes.” New techniques show the splicing sites are not random, because observers can predict where they will be found with uncanny accuracy. The “putative benefits” of introns that “justify their existence” are still unknown. Apparently, the cell has “mechanisms of targeted intron gain.”
    See also the May 12 BBC News report on this finding.

    Male Imparts More to Embryo than Just DNA 05/12/2004
    A team of biologists have confirmed that male sperm RNAs are delivered to the oocyte along with the DNA. Specifically, paternal messenger RNAs are delivered to the egg. These might influence development and put the male’s imprint on the developing zygote. Writing in Nature,1 the researchers speculate what the finding means:

    “Why should spermatozoa messenger RNAs be transferred to the oocyte? Messenger RNAs encoding proteins that bind nucleic acids, such as protamine-2, are likely to be deleterious and are probably degraded following entry, and a similar fate may await other RNAs that gain access. But some may have a role in the developing zygote: for example, clusterin (also known as sulphated glycoprotein-2, or SGP-2) is delivered to the oocyte and has been implicated in cell-cell and cell-substratum interactions, enhancement of fertility rate, lipid transportation, membrane recycling, stabilization of stress proteins, and promotion or inhibition of apoptosis. These may therefore be required in the early zygote but unnecessary in the oocyte. Alternatively (or in addition), these and other unidentified molecules, such as small interfering RNAs (siRNAs), may participate in processes such as pronuclear formation, the orchestration of events leading to oocyte activation, the transition from maternal to embryonic gene control, and the establishment of imprints in early embryos.”

    But haven’t cloning and parthenogenesis experiments shown the male contribution to the zygote is dispensable?

    “However, the success of such experiments and of somatic-cell nuclear transfer is limited, as is the production of human embryonic stem cells after somatic-cell nuclear transfer. This may be because sperm RNAs contribute to early development. Transcripts that are specific to male germ cells play a role in the differentiation of embryonic stem cells and their function may not be easily replaced.”

    They conclude that these accessory RNAs delivered in sperm may be necessary for fertility, and may influence the developing embryo with a signature only the male can provide.
    1Ostermeier et al., “Reproductive biology: Delivering spermatozoan RNA to the oocyte,” Nature 429, 154 (13 May 2004); doi:10.1038/429154a.

    Cell Requires Two Keys to Let Cargo Pass 05/13/2004
    For high-security environments, guards sometimes require two independent authentication methods. Before humans came up with this trick, the cells in their bodies were already using it. Itoh and Camilli explain in the May 13 issue of Nature:1

    “Our cells contain a series of distinct compartments that do different jobs and have different properties. The membranes that clad each of these compartments – like the plasma membrane that encases the cell – are defined by precise molecular compositions, which are preserved despite the continuous influx and efflux of components in transit to and from other cellular locations. Precision is the hallmark of this flow of traffic, too, which must be directed appropriately between compartments. All of this is achieved, in part, by the reversible recruitment of regulatory proteins from other parts of the cell to specific membranes or membrane regions. A growing amount of evidence hints that membrane lipids cooperate with membrane proteins to control this recruitment.”

    They refer to work by Godi et al. in Nature Cell Biology that shows a dual-key authentication mechanism in the cell. With “at least two independent, but synergistic, mechanisms,” cargo is only allowed to bind to a membrane if it binds correctly to two cytosolic proteins. This can be envisioned as a kind of code:

    “The interaction of cytosolic proteins with both lipids and proteins on a target membrane is an efficient dual-key strategy to control their recruitment to membranes. Only when both the lipid-binding and protein-binding sites are engaged is the interaction with the membrane strong enough. The two elements of the code can be controlled independently, affording the possibility of fine-tuning the spatial and temporal regulation of recruitment.”

    Itoh and Camilli provide no suggestions on how such a system might have evolved.
    1Toshiki Itoh and Pietro de Camilli, “Membrane trafficking: Dual-key strategy,” Nature 429, 141 - 143 (13 May 2004); doi:10.1038/429141a.

    Fish Antifreeze Provided by “Pseudogene” 05/13/2004
    Freezing water forms crystals that can rip and tear at cells. Yet there are fish in arctic waters that can survive even below the freezing point of sea water. They accomplish this by means of special “antifreeze proteins” that interfere with the damaging effects of water crystals.
    Scientists knew about AFP (anti-freeze protein) Type I in winter flounder, and knew its properties. They became puzzled how the fish survived temperatures lower than the protection AFP type I could provide. They suspected another antifreeze protein was at work, and found the gene that codes for it. They explain why this gene, named 5a, had escaped detection for 30 years:

    “The two proteins differ slightly in their amino-terminal sequence and amino-acid composition. At the time of its discovery, the 5a gene was dismissed as an antifreeze-protein pseudogene, largely because the protein it encodes would have been grossly different from type I AFP and had never been detected in the flounder”

    The protein is normally present in low concentrations and degrades at room temperature. At low temperatures, however, it roars into action. It becomes extraordinarily hyperactive, providing more protection against freezing than the previously-known AFP by an order of magnitude.
    1Marshall, Fletcher, and Davies, “Hyperactive antifreeze protein in a fish,” Nature 429, 153 (13 May 2004); doi:10.1038/429153a.

    Mitochondrial Clock Untrustworthy 05/16/2004
    A major assumption of the “molecular clock” dating method has been called into question. If so, Science Now describes the impact on current theories:

    “ ‘Mitochondrial Eve’, the hypothetical mother of all modern humans who lived about 150,000 years ago, might be lying about her age. A key assumption in determining how long ago she lived—that molecules of mitochondrial DNA do not swap segments with one another—is false, researchers now say. Their findings call into question a multitude of findings in evolution, early human migration, and even the relations between languages.”

    The mitochondria in our cells, organelles that provide the ATP power supply, contain small amounts of DNA. You may have heard that we inherit this mitochondrial DNA only from our mothers. Now, scientists have found evidence that male mitochondrial DNA can be inherited, and might be mixed in with the rest of the mitochondrial DNA. Since “the implications are that this is going on all the time in our cells,” that would render it untrustworthy as a genealogical tracer and dating method.
    An announcement about evidence for recombination in human mitochondrial DNA was published in the May 14 issue of Science.
    1Kraytsberg et al., “Recombination of Human Mitochondrial DNA,” Science, Vol 304, Issue 5673, 981, 14 May 2004, [DOI: 10.1126/science.1096342].

    Selfish Genes Turn Cooperative 05/19/2004
    Nature1 has reported evidence that transposons help to regulate gene expression. Transposons are genetic material that insert themselves into the DNA of a host, and were thought to represent “selfish genes” that only had their own propagation in mind, “without regard for the consequences.” Some new studies on the L1 retrotransposon, which makes up about 17% of the human genome (mainly within non-coding introns), have shown, however, that they may do us some good.
    The studies “suggest that the insertion of L1 elements into introns can also diminish cellular gene expression in a graded fashion,” the News and Views piece says. “In the words of Han, Szak and Boeke, such L1 insertions provide a ‘molecular rheostat’ with which to govern gene activity — and their bioinformatics analysis establishes that the mechanism is widely used.”
    (For more on the molecular rheostat concept, see 01/10/2003 headline).
    1Frederic Bushman, “Gene regulation: Selfish elements make a mark,” Nature 429, 253 - 255 (20 May 2004); doi:10.1038/429253b.

    Gene Regulation: When Nonsense Makes Perfect Sense 06/03/2004
    Nature June 31 reports on another use for “junk DNA.” A portion of previously-considered “nonsense” genetic code, which does not produce a protein as does a gene, nevertheless has an important role: it regulates the expression of the neighboring gene. This opens a whole new realm of function for portions of our genetic material that were thought to be useless leftovers of evolution: it’s a new kind of gene that regulates other genes (see the Reuters summary on MSNBC News).
    Molecular biologists have been intrigued by the fact that the DNA translation machinery seems busier than required to produce proteins: “Why is there such a heavy traffic of RNA polymerases, the enzymes that copy DNA into RNA, and the production of large quantities of apparently non-coding and non-functional RNAs?” the reporters say. The new work by Martens et al. reported in the same issue shows that “RNA polymerases are evidently doing more than we thought.” The resulting “nonsense” RNAs produced by reading non-coding segments act as regulators, controlling the amount of protein that is expressed by the true genes by a process of “transcriptional interference.” What was considered nonsense, therefore, actually makes perfect sense on a higher level:

    “Taken together, these studies highlight the importance of intergenic transcription in regulating gene activity, even in the relatively densely packed genome of yeast. It seems that RNA polymerases are not only required for the production of particular RNA species, but by travelling along DNA they can also control the occupancy of regulatory sites by transcription factors. Widespread transcription of intergenic sequences has also been described in the human genome. Surprisingly, many of these non-coding transcripts seem to be regulated in a manner that is intimately connected to the transcription of protein-coding genes. So the high proportion of non-coding regions in the genomes of higher organisms is probably not due to the accumulation of nonsense DNA, but rather represents the evolution of ever more complicated gene-regulatory systems.”

    EurekAlert puts this finding into perspective:

    “If so, the findings would carry an important message for the post-human genome era-namely, that researchers’ attempts to turn the masses of data churned out by the Human Genome Project into an understanding of what actually happens in the human body may be even more complex than they anticipated. One of the main challenges for that effort is to figure out how and when genes are turned on and off during normal development and disease. Rather than look only at how genes are regulated by proteins, they would have to turn their attention to a new, and possibly more-difficult-to-detect form of control. And given that junk DNA makes up 95 percent of chromosomes, the mechanism could be fairly common.”

    The article gives the bottom line to one of the researchers, Fred Winston of Harvard Medical School: “Every time we thought we understood everything going on here, we have been wrong. There are additional layers of complexity.”
    1Sabine Schmitt and Renato Paro, “Gene regulation: A reason for reading nonsense,” Nature 429, 510 - 511 (03 June 2004); doi:10.1038/429510a.

    DNA Folds With Molecular Velcro 06/07/2004
    Many have heard how the inventor of Velcro got the idea from plant seeds that stick to clothing, but now Carlos Bustamente and team of Howard Hughes Medical Institute have found a velcro-like principle operating at a scale millions of times smaller. Small proteins called condensins are involved in the elaborate folding that DNA undergoes as it is wrapped into chromosomes. The team developed an ingenious method of gently pulling on DNA strands compacted with condensin. Bustamente relates, “when we began to pull it apart carefully, we saw it extend in a sawtooth pattern of force, like the click-click-click of Velcro unzipping.” When they relaxed the force, it collapsed back, then repeated the same pattern when pulled apart again. “That perfect reproducibility strongly suggested to Bustamante and his colleagues that they were seeing a condensed structure with a well defined organization,” the press release explains. Surprisingly, this reversible reaction did not require the expenditure of ATP.

    This is just one of the clever design features in the cell that allows over six feet of fragile DNA to be folded and compacted into a nucleus a few millionths of an inch wide. … Even more amazing is that this tight packing still allows the translation machinery to find the right gene, gain access, and do its work. For a glimpse of this additional complexity, see the 06/13/2002 and 03/08/2002 headlines.

    How Molecular Trucks Build Your Sensors 06/14/2004
    In the film Unlocking the Mystery of Life, biochemist Michael Behe, describing the intricacies of cells as we know them today, claimed that there are “little molecular trucks that carry supplies from one end of the cell to the other.” If that seems an overstatement, you should look at the illustration in Cell June 11 in a Mini-review called “Cilia and flagella revealed” by Snell, Pan and Wang.1 They not only describe trucks, they’ve found a train of boxcars and a whole crew of engineers, conductors and brakemen.
    Cilia are appendages in the cell membrane that wiggle. Everybody’s got them; they are ubiquitous in organisms, from bacteria to humans. They line our respiratory tract, cleaning debris from our lungs. They help our senses of smell and eyesight. They are important for kidney function. They may look simple, but only recently are scientists beginning to appreciate the complexity inside. The authors begin:

    “Our view of cilia has changed dramatically in the decade since Joel Rosenbaum and his colleagues discovered particles rapidly moving (2-4 micrometers/s) up and down within the flagella of the biflagellated green alga, Chlamydomonas (Kozminski et al., 1993). Once cell biologists identified the cellular machinery responsible for this intraflagellar transport (IFT), it became clear that IFT is essential for the assembly and maintenance of cilia and flagella in all eukaryotes (Rosenbaum and Witman, 2002). As we will outline in this brief review, the increased focus on these organelles has revealed that nearly all mammalian cells form a cilium, that the ciliary apparatus (a cilium plus its basal body) is somehow connected with cell proliferation, and that cilia play key (and as yet poorly understood) roles in development and homeostasis.”

    Michael Behe in his book Darwin’s Black Box had a whole chapter on how cilia move. Recently, however, it has been appreciated that nonmotile cilia can also act as sensory probes. The authors explain:

    “Several properties of cilia recommend them for use as sensory transducers. They project a cell type-specific distance from the cell body, making them exquisitely designed probes of the external milieu; both their overlying membrane and their cytoplasmic contents are relatively well isolated from the cell body, thereby offering all of the advantages of compartmentalization; the machinery for their assembly makes possible rapid, regulated transport of proteins between the organelles and the cell body; and, the assembly machinery seems exploitable for use directly in signaling pathways.”

    Now that we know cilia are vital, it’s what goes on inside the narrow shafts during construction that is truly remarkable. The authors mentioned IFT, or intraflagellar transport, a class of proteins that operate the transportation system. During construction of a cilium or flagellum, parts need to be transported to the growing tip, or axoneme. The IFT particles move up and down the inside walls of the shaft. They describe how this works. Watch for the word trucks:

    “This flow of materials is driven by the IFT machinery. Flagellar proteins synthesized in the cell body are carried to the tip of the flagellum (the site of assembly of the axoneme) by IFT particles, which are composed of at least 17 highly conserved proteins that form A and B complexes. The plus end-directed microtubule motor protein kinesin II is essential for movement of particles and their cargo toward the tip (anterograde transport) of the flagellum, and a cytoplasmic dynein carries IFT particles back to the cell body (retrograde transport). Thus, IFT particles function as constantly moving molecular trucks on a closed loop. The tracks they travel on are the microtubule doublets of the ciliary/flagellar axoneme, microtubule motors power them, and the individual structural components (e.g., microtubule subunits, dynein arms, and radial spoke proteins) of the cilium/flagellum are their cargo.”

    The construction system they describe next is reminiscent of a gondola at a ski resort, a series of ore carts in a mine shaft, or a conveyor at a rock quarry. If you can picture architects building a tall structure like the Seattle Space Needle or the Eiffel Tower, imagine the engineers first devising a way to get the raw materials to the growing top. Suppose they design a double trackway that can be extended in length as the structure grows. Attached to this track are self-propelled dump trucks that can climb up the tracks, and another set of dump trucks that can climb down. Each truck can carry a load of cargo. New trucks are constantly added at the bottom, and old ones upon reaching the base are removed. A pool of trucks and drivers is always available to traverse this vertical highway. With this automated system running, workers at the top can take the cargo and build with it, and send waste products down the other side. This two-way transportation system works not only to build the tower, but to dismantle it.

    “Figure 2 presents a model for regulation of assembly, disassembly and for regulation of flagellar length. In this model, the rate of particle entry and the number of particles per unit length are independent of length, and cargo loading is regulated. Thus, in a rapidly growing flagellum (in the extreme case), every particle entering carries cargo, and every particle returning to the cell body is empty. Once the proper length is attained, length control mechanisms engage. At this steady-state length, the number of IFT particles entering and leaving per unit time is unchanged, but the proportion of cargo-loaded IFT particles that enters the flagella comes to equal the proportion of cargo-loaded IFT particles that leaves the flagellum. In a disassembling flagellum, the situation is reversed from that of a growing flagellum, and (in the extreme case) every particle that enters the flagellum is empty and every particle that leaves the tip is full. Thus, by regulating cargo binding to particles at both the base and the tip, and by controlling of assembly and disassembly of axonemal components at the tip (presumably driven by mass action and regulatory proteins), cells specify assembly/growth, steady-state length, or disassembly/resorption.”

    The diagram in their figure shows what look like little ore-carts climbing up to the tip and back. The authors describe next how these tall structures function not only as oars and outboard motors, but as chemical antennae. Experiments have “called to the attention of cell biologists the under-appreciated but hardly insignificant role of cilia in sensory transduction.” Here are some of your body parts that depend on these miniature probes that extend out from the cell into the surrounding environment, sensing what’s out there:

    “Humans experience the environment through cilia in major sensory organs. The outer segments of retinal rod cells are modified, nonmotile cilia, replete with photoreceptors for interacting with light; and the odorant receptors in the olfactory epithelium are peppered over the surface of the cilia of olfactory neurons. Moreover, almost every mammalian cell contains a solitary cilium, called a primary cilium, whose most likely function is in signaling (Pazour and Witman, 2003). For example, many of the neurons in brain contain primary cilia, some of which express receptors for somatostatin and serotonin (Pazour and Witman, 2003). Perhaps the most striking example of the importance of primary cilia in homeostasis [i.e., dynamic equilibrium] comes from work on the epithelial cells of the collecting tubules in the kidney. The primary cilium on each renal tubule cell functions as a flow sensor both in vivo and in MDCK cells in vitro. Bending the cilium causes a large, transient increase in intracellular calcium concentration and a consequent alteration in potassium conductance (references in Boletta and Germino [2003]).”

    Each of these cilia, and many more, are constructed by this molecular transportation system. How many parts are involved in building a cilium? If this system were magnified a hundred million times, children might find this the ultimate Lego toy:

    “New proteomic and genomic studies may finally provide a platform for discovery of most of the as yet unidentified genes that encode ciliary/flagellar proteins. A proteomic analysis of the axoneme of human cilia identified over 200 potentially axonemal proteins (Ostrowski et al., 2002). Several of the proteins were previously identified as being in the axoneme, but many have no homologs or are of unknown function.”

    … From genomic studies, they estimate it would require at least 362 genes to build a motionless cilium, and “more than 400-500 genes that are predicted to be needed for forming and regulating the ciliary apparatus” One team measured the proteome (set of proteins) required to build the basal body (the bottom foundation of the structure) and flagellum to consist of 688 genes. “There is no doubt,” they say, “that the FABB [flagellar and basal body] proteome represents an incredibly rich resource.”
    Failure of cilia and flagella to develop properly are implicated in many diseases (see “Don’t mutate this gene, or else” in the 10/01/2003 headline). Even some human obesity disorders might be traced to ciliary breakdown, as well as hypertension, diabetes and other “seemingly unrelated clinical problems”.
    The authors do not speculate on how such a complex system with so many parts might have evolved, other than to assume that it did: for instance, “Paralogs of other mitotic proteins have also evolved [sic] to play roles [sic] in cilia.” They also claim that plants unevolved them: they seem to have lost the 400-500 genes needed for building cilia or flagella, if they ever had them. The authors examine studies in comparative genomics to determine how many of the cilia/flagella genes are ancestral, going back to the original machinery in the simplest alga or bacterium. One study compared the IFT genes in several organisms with those in fruit flies:

    “Using a large number of genomes provided stringent criteria and identified 187 candidate ancestral ciliary genes. Sixteen are conserved in all ciliated organisms examined and absent in all nonciliated organisms; 18 are present only in organisms with motile cilia; 103 are common to organisms that utilize only conventional ciliogenesis; and 50 are shared only by organisms that form motile cilia in the ciliary compartment.”

    Other studies are cited; 67% of the basal body genes in green algae and 90% of their flagellar and IFT genes were present in the full FABB proteome. It appears, therefore, that this transportation system evolved early on, if it did, and has not changed much since.
    1William J. Snell, Junmin Pan, and Qian Wang, “Minireview: Cilia and Flagella Revealed: From Flagellar Assembly in Chlamydomonas to Human Obesity Disorders,” Cell, Vol 117, 693-697, 11 June 2004.

    Biochemists Mutate Protein, Make a Catalyst 06/25/2004
    “Enzymes are among the most proficient catalysts known,” wrote three Duke University scientists, “and they catalyze a wide variety of reactions in aqueous solutions under ambient conditions with exquisite selectivity and stereospecificity.” The team set out to rationally design their own enzyme. Their work is reported in the June 25 issue of Science.1 Building on a non-enzymatic ribose-binding protein, they introduced 18 to 22 mutations at specific points, imitating the active site of triose phosphate isomerase (TIM). They succeeded in getting a million-fold increase in catalytic activity, and showed their NovoTim invention to be biologically active in E. coli bacteria. To them, not only does this demonstrate scientists’ ability to understand and imitate “naturally evolved” enzymes, but the “introduction of TIM activity into RBP is therefore equivalent to convergent evolution by computational design.” Their enzyme was less thermally stable than the wild type, however, and the reaction rate was 220 times lower.
    1Dwyer, Looger and Hellinga, “Computational Design of a Biologically Active Enzyme,” Science, Vol 304, Issue 5679, 1967-1971, 25 June 2004, [DOI: 10.1126/science.1098432].
    Last edited by bob b; October 26th, 2006 at 07:50 AM.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Cell Technology Celebrated 07/01/2004
    Humans are just beginning to imitate the manufacturing techniques cells use all the time, right under our noses. A book just came out about the subject, entitled Bionanotechnology: Lessons from Nature by David S. Goodsell. It’s hard to tell if Christof M. Niemeyer was more impressed with the book or with the living machines themselves, in his review in the July 1 issue of Nature.1 He writes,

    “Nanotechnology is perfectly realized in biological systems. Cells are essentially biological assemblers that build thousands of custom-designed molecules and construct new assemblers. In Bionanotechnology, structural biologist David Goodsell describes what biology can teach us about engineering and manufacturing at the nanometre scale.”

    “Small wonder,” reads a caption whimsically; “antibodies ... are just one example of the way nature uses nanotechnology.” Niemeyer mentions a few more examples of “the composition and structural principles of biomolecules harnessed in the cell” :

    • the machinery of DNA transcription and translation
    • biomolecular motors
    • the information-driven synthesis of biological molecules
    • the energetics and regulation of biological processes
    • the traffic across membranes and signal transduction along them
    • the interplay of myosin and actin filaments within the muscle sarcomere

    Although the book focuses on how humans can tinker with this biological nanotechnology, reviewer Niemeyer enjoyed every page of this “fascinating journey.”
    1Christof M. Niemeyer, “Living Machinery,” Nature 430, 20 (01 July 2004); doi:10.1038/430020a.

    Archaea Have Their Own Proofreading Mechanism 07/07/2004
    A team of Yale biochemists investigated a proofreading mechanism in one-celled organisms from the domain Archaea and found it different, but just as effective, as its counterpart in domains Bacteria and Eukarya (the latter including all plants and humans). Their work was published online in PNAS July 6.1
    The particular instance involved the ability to discriminate between two similar amino acids, threonine and serine, on the molecule that connects the amino acid to the transfer RNA (aminoacyl-tRNA synthetase, or aaRS). Members of Archaea have an enzyme that bears no sequence similarity, but is “functionally conserved” (i.e., does the same thing), to that of the other domains. The archaeal gene is “unrelated to, and absent from,” bacterial and eukaryotic genomes. The authors term this an instance of “functional convergence of unrelated domains” that “assures specificity” of the correct amino acid to the tRNA molecule. This “appears to be the first aaRS found to use two evolutionarily unrelated editing domains,” they state. “The functional convergence between the two ThrRS editing domains is highlighted by the observation that both depend on an absolutely conserved set of histidine residues for their function.”
    1Korencic et al., “A freestanding proofreading domain is required for protein synthesis quality control in Archaea,” Proceedings of the National Academy of Sciences USA , 10.1073/pnas.0403926101.

    Cell Cargo Speeds On Bidirectional Highways 07/12/2004
    As reported here numerous times (e.g., 06/14/2004, 12/04/2003, 04/14/2003, 03/28/2003, 02/25/2003, 12/17/2002, 09/26/2002, 03/26/2002, 02/01/2002, 12/06/2001, 08/17/2001, 06/19/2001, 02/21/2001), cells have an elaborate interstate highway system with molecular trucks hauling cargo back and forth. Scientists have known that the cellular highways have polarities labeled plus and minus, and that molecular motors typically go one way. Some motors, like kinesin, drive only in the plus direction, while others, like dynein, go in the minus direction. Now, it is becoming apparent that most pieces of cargo have at least one of each kind of motor, with a stickshift that allows it to drive in forward or reverse. The state of our knowledge about bidirectional transport is explored by Michael Welte in the July 13 issue of Current Biology.1
    Welte examines the evidence that many, maybe all, moving cargoes have bidirectional ability. In the microscope, certain organelles like mitochondria and melanosomes are seen to move back and forth rapidly, eventually making it to their target. Why is this, and how is it done? Does the organelle grab motors out of the cytoplasm? Are both motors working in a tug-o’war? Welte cites evidence against these possibilities, and suggests (although hard evidence needs to be found), that the cargo carries both motors, and a “complex coordination machinery ... ensures that when one motor is actively engaged with the microtubule, the other motor is turned off.” Moreover, this coordination machinery, whatever it is, may be under the influence of regulatory enzymes. “If the coordination machinery can attach to cargo independent of the motors,” he surmises, “distinct variants of the coordination machinery could be targeted to different cargoes, thus allowing cargo-specific coordination and regulation.”
    It seems odd, though, that cargoes would undergo a back-and-forth random walk instead of making a beeline to the target. Welte figures there must be biological justification for this behavior, so he examines some possibilities:

    Economy: “If cargoes always carry motors for both directions, net transport can easily be adjusted or even reversed by simply tweaking the relative activity of the two motors. This is likely to be much quicker than assembling a new set of motors on a cargo, and also allows transport to be abruptly altered depending on cellular needs. It even makes it possible to tune the overall speed of transport by altering the relative contribution of trips in the non-dominant direction.”
    Setting Up Polarized Distributions: “Sometimes it is necessary to set up a distribution rather than to confine the organelles to a single point .... Even if cargoes accumulate at a certain point (e.g. near plus-ends when motion is biased in the plus-end direction), trips in the non-dominant direction will tend to spread the cargoes out along the tracks, away from the point of accumulation. Modeling shows that by altering the relative contributions of plus- and minus-end trips, a wide range of steep to flat steady-state distributions can be achieved.”
    Avoiding Obstacles and Exploring Space: “As cytoplasmic dynein often steps sidewise to adjacent proto-filaments, a bidirectional cargo could find itself on the opposite side of the microtubule even after a short minus-end excursion. If it now switches back to kinesin I, it can pass the obstacle. Bidirectionally moving cargoes should, therefore, be less likely to contribute to disastrous traffic jams .... The random walk of bidirectional cargoes allows a single cargo to explore a large region of cellular space, especially if tracks are disordered.”
    Error Correction: “During unidirectional transport, the critical event that determines directionality of motion is the attachment to either a plus- or minus-end motor. A wrong attachment will cause misdelivery of the cargo. During bidirectional transport, the net direction of transport is determined by the balance of plus- and minus-end trips and can, therefore, be continually evaluated and even altered if physiological conditions change. Thus, bidirectional transport may facilitate error correction.

    It must be remembered that these motors are operating in the dark without eyes, like automated railroad cars. They don’t have sentient drivers on radios, but rather respond to chemical signals in the environment. Apparently these behaviors achieve the best solution to many complex problems. “Bidirectional transport by opposite-polarity microtubule motors is just one example of multiple motors working together to achieve carefully choreographed transport,” Welte says, as he concludes with a list of open problems needing further elucidation.
    1Michael A. Welte, “Bidirectional Transport along Microtubules,” Current Biology, Volume 14, Issue 13, 13 July 2004, Pages R525-R537,

    Plant “Evolutionary Leftover” Now Deemed Vital 07/22/2004
    Photorespiration, “a biological process in plants, thought to be useless and even wasteful” and “just an evolutionary leftover” from an age when carbon dioxide was more prevalent, has been found to be “necessary for healthy plant growth and if impaired could inhibit plant growth,” according to a UC Davis study published in PNAS. (see also summary on EurekAlert). It functions as a way to inhibit nitrate assimilation. Some agricultural scientists assumed it was an unnecessary process to be genetically engineered out of plants because it was wasteful, “But the new UC Davis study suggests that there is more to photorespiration than meets the eye and any attempts to minimize its activity in crop plants would be ill advised.”

    Spaghetti in a Basketball: How the Cell Packs DNA for Controlled Access 07/28/2004
    The beginning sentence of an article in Current Biology1 can’t help but grab your attention:

    “Imagine trying to stuff about 10,000 miles of spaghetti inside a basketball. Then, if that was not difficult enough, attempt to find a unique one inch segment of pasta from the middle of this mess, or try to duplicate, untangle and separate individual strings to opposite ends. This simple analogy illustrates some of the daunting tasks associated with the transcription, repair and replication of the nearly 2 meters of DNA that is packaged into the confines of a tiny eukaryotic nucleus. The solution to each of these problems lies in the assembly of the eukaryotic genome into chromatin, a structural polymer that not only solves the basic packaging problem, but also provides a dynamic platform that controls all DNA-mediated processes within the nucleus.”

    The article by Craig L. Peterson and Marc-André Laniel is otherwise boringly titled “Histones and histone modifications,” but after this appetizing start, goes into detail about how the tangled mess of alphabetized pasta is exquisitely controlled, folded, unfolded and copied continuously inside the cell, with the help of numerous protein and RNA parts.
    Of special importance are the histone proteins that comprise chromatin. Scientists have been discovering for several years now that these histones have “tails” of amino acids that can be altered through numerous ways. These alterations, called “post-translational modifications,” seem to influence the DNA wrapped around them in many important ways. They signal genes to activate for transcription, places needing DNA repair, places to start or repress DNA elongation or replication, where to silence telomeres, places to deposit more chromatin, and more. A table in the article lists 95 histone modifications and their functions that are known so far. Some are involved in mitosis (cell division), spermatogenesis, X-chromosome inactivation (silencing one of the two X-chromosomes in the female), apoptosis (programmed cell death), DNA “memory” and other important cell processes. Some have said these modifications constitute a “histone code” (see “Cell memory borders on the miraculous,” 11/04/2002 headline). These authors term it differently, but no less amazing: “rather than a histone code there are instead clear patterns of histone marks that can be differentially interpreted by cellular factors, depending on the gene being studied and the cellular context.” Activities like DNA repair or replication are often accompanied by histone modifications, for instance, as if one enzyme leaves its mark on a histone to signal a follow-up function. Complexes of small RNAs and enzymes depend on these markers to know where to go and what to do; the histone tails serve as attachment points for specific enzymes. And if that is were not amazing enough, the interplay of neighboring histone markers, or cross-talk, can have “a profound effect on enzyme activity.” The authors explain, “Thus, in many ways histone tails can be viewed as complex protein-protein interaction surfaces that are regulated by numerous post-translational modifications. Furthermore, it is clear that the overall constellation of proteins bound to each tail plays a primary role in dictating the biological functions of that chromatin domain.” Finally, since some of these histone states can survive cell division, they augment what’s inherited beyond DNA alone. The authors provide no suggestions on how this system might have evolved.
    On a related subject, three geneticists from Scotland describe, in the same issue of Current Biology,2 how DNA packs itself so tightly and efficiently. There are specialized proteins called condensins that perform this job. They are members of a set of hairpin-shaped enzymes called “structural maintenance of chromosomes” enzymes (SMCs, see 08/07/2002 headline). The authors remind us that “These extraordinary molecules are conserved [i.e., unevolved] from bacteria to humans.” Scientists are beginning to be able to watch condensin do its amazing work in real time (see “DNA folds with molecular velcro,” 06/07/2004 headline). Condensin produces “supercoils” of DNA, one of many steps in packing the delicate DNA strands into a hierarchy of coils that results in a densely-packed chromosome. “It is not entirely clear how the DNA is held in this supercoiled state,” they say, “but several studies suggest that the V-shaped arms of the condensin complex may loop and clamp the DNA in place.” This clamping is “rapid and reversible.” Scientists watching the process in both bacteria and humans are “showing that both vertebrate and bacterial condensins drive DNA compaction in an ATP-dependent fashion with a surprising level of co-operativity that was not fully appreciated.” The condensin molecules work as a team; if not enough condensin is around, nothing happens.
    These authors point out also that condensin is just one of many enzymes involved in chromosome formation. Think about how remarkable it is that during each cell division, the chromosomes are structured so reliably that they can be labeled and numbered under the microscope. “Our own proteomic analysis,” they claim, “has identified over 350 chromosome-associated proteins, so there is clearly more work to be done.”
    1Peterson and Laniel, “Histones and Histone Modifications,” Current Biology, Volume 14, Issue 14, 27 July 2004, Pages R546-R551, doi:10.1016/j.cub.2004.07.007.
    2Porter, Khoudoli and Swedlow, “Chromosome Condensation: DNA Compaction in Real Time,” Current Biology, Volume 14, Issue 14, 27 July 2004, Pages R554-R556, doi:10.1016/j.cub.2004.07.009.

    Gymnastic Enzyme Acts Like Logic Gate 07/31/2004
    An enzyme named vinculin undergoes “drastic” conformational changes, reports William A. Weis in the July 29 issue of Nature.1 Vinculin, with over a thousand amino acid links, is important at membrane junctions for transporting materials in and out of the cell. It helps cellular “glue” exit the membrane so that neighboring cells can adhere to one another, such as in epithelial tissues.
    Weis reports on recent studies that show vinculin undergoes radical conformational changes during its action. It will only build the adhesive junction when the necessary components are in place. Nothing happens unless the participants are ready; “the binding energy of several partners is needed to overcome the thermodynamic and perhaps kinetic barriers to activation,” he says. “Viewed in this way, vinculin functions as a logical AND gate, in which binding of two partners is required to generate an output, in this case a stable multi-protein complex”. What’s more, this automatic regulation is essential for its function; it prevents inappropriate assembly if the amount of product is unstable.
    1William A. Weis, “Cell biology: How to build a cell junction,” Nature 430, 513 - 515 (29 July 2004); doi:10.1038/430513a.

    Cell Nucleus Complexity Baffles Evolutionists 08/06/2004
    In her inimitable way, Science reporter Elizabeth Pennisi has once again portrayed a scientific controversy undergoing active ferment. This time it’s about the evolutionary origin of cell nuclei, which she terms “specialized, DNA-filled command centers.”1 At the conclusion, she gives prominence to a “provocative, but circumstantial and controversial” suggestion that viruses taught cells how to wrap their DNA in double membranes with controlled access. Since the idea presupposes that viruses preceded all three domains of life – prokarya, eukarya and archaea – “If this is true, then we are all basically descended from viruses,” as a believer puts it. The idea is unpalatable to some. “I do not believe [it],” a German molecular biologist retorts. “The idea of the viruses ‘inventing’ [eukaryotic cells] from scratch is hard for me to conceive.”
    Pennisi treats the new viral theory as tentative at best. What’s more revealing in her article are the problems with previously-popular ideas, and why. According to her, the key insight at a meeting in France last month on the subject was: “They had underestimated the complexity of the eukaryotic cell’s 1.5-billion-year-old [sic] precursor. The data presented indicated that this ancestral cell had more genes, more structures, and more diverse biochemical processes than previously imagined”. For a glimpse why, look at Pennisi’s brief description of the nucleus:

    “Each nucleus in a eukaryotic cell consists of a double lipid-based membrane punctuated by thousands of sophisticated protein complexes called nuclear pores, which control molecular traffic in and out of the organelle. Inside, polymerases and other specialized enzymes transfer DNA’s protein-coding message to RNA. Other proteins modify the strands of RNA to ensure that they bring an accurate message to the ribosomes outside the nucleus. The nucleus also contains a nucleolus, a tightly packed jumble of RNA and proteins that are modified and shipped out of the nucleus to build ribosomes.”
    (For more on the nuclear pore complex, see 06/17/2002 and 01/18/2002 headlines.)

    Eukaryotes are distinguished from bacteria by their double-membrane nuclei. “The nuclear distinction between prokaryotes and eukaryotes shaped early speculation about the development of complex life,” Pennisi says about ideas floating around up to the 1970s. Some thought eukaryotes were evolved prokaryotes, and others thought prokaryotes were degenerate eukaryotes. But then Carl Woese created new woes by identifying bacteria-like cells that were distinct from both prokaryotes and eukaryotes: so different, in fact, to warrant classification in their own domain – archaea. Others soon were surprised to find that eukaryotes appeared to have genes from both bacteria and archaea.
    So another story was born, the endosymbiont or merger hypothesis. This proposed that eukaryotes arose from “the ancient symbiotic partnership between bacteria and archaea.” That theory came under fire from the discovery of faint but distinct nuclei in an unusual group of bacteria, named planctomycetes, that live in soil and fresh water. Some of these planctomycetes have organelles and double-membraned sacs of DNA and RNA. According to a critic of the merger model, these observations “turn the dogma that ‘prokaryotes have no internal membranes’ upside down” Now, it seems no one is sure which way is up.
    There’s more to cause vertigo for evolutionists: the complexity of the nuclear pore complexes (NPCs). “Explaining these structures has always posed a sticking point for nuclear evolution [sic].” For one thing, “without pores, the nucleus can’t function.” But for another thing, Pennisi continues, the same planctomycetes, and possibly some other archaea and prokaryotes, apparently possess structures resembling these complex traffic-control gates. “Bacteria with nuclear pores and internal membranes, features typically considered eukaryote-specific, suggest that the nucleus was born much earlier than traditionally thought.”
    For some, that leaves as the leading contender the controversial theory that viruses first invented the nucleus. This, however, only pushes the complexity of nuclei and their pores farther back in time, and foists a huge design problem on earth’s most primitive biological entities. That is why the molecular biologist quoted earlier can’t believe that simple viruses created such complex structures from scratch. Pennisi shares a few speculations, based on circumstantial evidence, how it might have happened. But when she ends by pushing the answer to the future, it underscores the fact that no current theory accounts for the origin of the nucleus:

    “Did a virus provide the first nucleus? Or was it something an early bacterial cell evolved, either on its own or in partnership with an archaeum? To resolve the origin of the nucleus, evolutionary biologists are exploring new techniques that enable them to determine relationships of microorganisms that go much further back in time.... “

    The biologists in France argued and discussed many ideas. “But when it came to accounting for how the nucleus was born,” Pennisi admits, “no single hypothesis bubbled to the top.” She quotes French molecular biologist Patrick Forterre who said, “It’s like a puzzle. People try to put all the pieces together, but we don’t know who is right or if there is still some crucial piece of information missing.”
    1Elizabeth Pennisi, “Evolutionary Biology: The Birth of the Nucleus,” Science, Vol 305, Issue 5685, 766-768, 6 August 2004, [DOI: 10.1126/science.305.5685.766].

    ATP Synthase: Another Unexpected Case of Fine Tuning 08/10/2004
    ATP synthase, the miniature rotary motor that powers our cells, has been a subject of great interest since the elucidation of its rotary function won three scientists a Nobel prize in 1997. As an example of a precision-crafted, true electric rotary motor in living systems (another being the larger bacterial flagellum), it also provides a classic case study in intelligent design vs. evolution. It has been the subject of frequent updates in these pages (start at 02/13/2004 and work backwards). Now, another discovery about this ATP-synthesizing engine has revealed a deeper level of fine tuning. Japanese scientists publishing in PNAS1 found a precision coupling between two components that was unexpected, yet apparently essential.
    For review, recall that ATP synthase has two functional domains, named F0 and F1. The F1 part that actually synthesizes ATP from ADP + P is now fairly well understood. It is composed of three pairs of lobes that spring-load ATP with every 120o turn of the camshaft, each pair of lobes either loading, catalyzing or ejecting an ATP molecule. The F0 domain, however, has been harder to study. Scientists knew it looks like a carousel of identical proteins, labeled c subunits. Linked to it is a camshaft, named the gamma subunit, that drives the synthesis of ATP in F1. Scientists knew the F0 carousel runs on protons delivered by a gumball-like mechanism named the a subunit (see 12/22/2003 headline). But up till now, they were not sure how many c subunits comprised the carousel – or even if the number mattered. Some studies had hinted that the F0 motor contained anywhere from 8 to 13 c subunits, depending on the species. Now, the team of Mitome et al. found the answer: it is 10, and it must be 10 and only 10. Other numbers don’t work. That’s strange. It means that F0 needs 10 protons per revolution, but F1 produces 3 ATP per revolution. The ratio 10:3 is not an integer. How can that be?
    The scientists arrived at the number 10 by customizing F0 rings with fixed numbers of c subunits, 2 through 14. Then they linked them to the F1 domains and watched how much ATP was synthesized. Results were obtained for only c=2, 5, and 10, which is interesting, considering that 2 and 5 are factors of 10. The c=2 and c=5 cases produced a little ATP, and c=10 produced the maximum. All the other numbers produced none. The team deduced, therefore, that 10 (or one of its factors) is essential to match the proton-loading mechanism of the a subunit.
    The scientists also measured the proton flow through their custom carousels when disengaged from F1 and found, again, that 10 was the only number that worked. Without 10 c subunits, no protons flowed. Divide a circle of 360o by 10, and you get a 36o angle per c subunit during a complete revolution of the F0 motor. The F1 domain, by contrast, produces ATP for each 120o turn, or 3 ATP per complete revolution. The scientists seemed surprised that the proton-ATP ratio, “one of the most important parameters in bioenergetics,” is not an integer. It’s as if three protons are sufficient to generate an ATP sometimes and four other times, because one cannot have a third of a proton. Wouldn’t it be more logical if the number of c subunits was a multiple of three, say 6, 9, or 12? With c=9, for instance, the camshaft angle would regularly line up with the F1 lobes every 3 protons, yielding one ATP every time, nice and neat. The fact that it does not means that the coupling between F0 and F1 is not strict, as with toothed gears, but “permissive” – as if the two domains rotate according to their own structural needs, and are coupled together by a adaptor mechanism that has some degree of freedom to either twist or slip.
    The scientists ruled out slippage. They knew that the camshaft can only produce an ATP in the F1 domain when it is lined up perfectly at the 120o steps. Instead, they found that there is enough elastic flexibility in the camshaft to permit twist up to 40o during its rotation. This flexibility allows the two domains to work separately, each according to its optimum configuration, with the twisting camshaft able to rock back and forth a little to give the F1 lobes time to complete their work. In scientific lingo, “The flexibility of gamma allows both the F0-gamma and F1-gamma interfaces at the free-energy minima to stay in conformations adequate for the proton transport in F0 and the catalysis in F1 despite the step-size mismatch, providing sufficient time for those events to take place.”
    One more thing. There isn’t much tolerance for error in this system. The team found that a single point mutation at a spot named E56 in the c subunit was enough to quench all proton flow and all ATP synthesis: “This result provides evidence that each of all 10 E56 in the c-ring is indispensable.” Also, the quantity of 10 subunits in the c-ring is critical, because 8, 9, 11, 12 and other numbers did not fit the gumball proton-delivery system of the a subunit: “Thus, the proton transport through F0 requires very strict arrangement of contact surface between F0-a and F0-c in the F0 assembly and even a rotary displacement as tiny as 3.3o (360o / 10 – 360o / 11) seems to be enough to disable a proton transfer between them.”
    The team made their measurements on ATP synthase motors from a species of thermophilic (heat-loving) bacteria. They feel they have found a coupling strategy in living systems that could demonstrate a general principle: “Here, we report the permissive nature of the coupling between proton transport and ATP synthesis of F0-F1, but such nature of the coupling can be general among other biological motor systems to connect critical well tuned microscopic events in the large domain motions.”
    1Mitome et al., “Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403545101, published online 8/09/04.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Is the Evolution of Bacterial Resistance a Just-So Story? 09/12/2004
    Evolutionists frequently point to the emergence of bacterial resistance to antibiotics as an example of Darwinian evolution occurring right under our noses. Bruce R. Levin of Emory University, writing in the Sept. 10 issue of Science,1 is not so sure about that. He points out that cells might just have a built-in mechanism to shut down growth and reproduction in times of stress (the SOS response), to minimize the damage from toxins in the environment. He points to two studies in the same issue that indicate how noninherited resistance to antibiotics can be generated without reference to Darwinian natural selection.
    What’s more interesting in his report is his rebuke against fellow Darwinists who leap to unsubstantiated tales of evolution to explain how these mechanisms come about. His final paragraph states:

    “It is easy to concoct just-so stories to explain the evolution of a mechanism that, like the SOS response, produces quiescent cells that are refractory to lethal agents. Yet it seems unlikely that ampicillin was the original selective force [sic] responsible for the evolution [sic] of the induction mechanism observed by Miller and colleagues. A bigger challenge to those in the evolution business is to account for the generation of lower fitness cell types when they do not provide an advantage to the collective, like the persisters of Balaban et al. in the absence of antibiotics. Then again, just like people, bacteria do some seemingly perverse things that are not easy to account for by simple stories of adaptive evolution.

    1Bruce R. Levin, “Microbiology: Noninherited Resistance to Antibiotics,” Science, Vol 305, Issue 5690, 1578-1579, 10 September 2004, [DOI: 10.1126/science.1103077].

    Peering Into Paley’s Black Box: The Gears of the Biological Clock 09/15/2004
    William Paley’s famous “watchmaker argument” for the existence of a Designer, though intuitively logical to many, has been criticized by naturalists on the grounds that one cannot compare mechanical devices to biological ones. Biological “contrivances” might operate on totally different principles than mechanical ones made by humans we know.
    Michael Behe’s 1996 book Darwin’s Black Box was built on the theme that, until recently, the living cell was a “black box” to biologists: i.e., a system whose inner workings lay hidden from us. But now with the rapid advances in molecular biology, we are finding the cell to be a complex factory of molecular machines.
    These themes of Paley and Behe seemingly converge in a commentary by Susan S. Golden (Texas A&M) in PNAS about biological clocks.1 Golden works at the Center for Research on Biological Clocks in the Texas A&M Biology Department, and was struck by recent findings in two other papers in PNAS on the circadian rhythms of “primitive” blue-green algae (cyanobacteria). To her, they suggested we are opening the black box of biological clocks, and finding treasures that look remarkably familiar to the clocks we know:

    “A physiological black box is to a biologist what an ornately decorated package is to a small child: a mysterious treasure that promises delightful toys within. With fitting elan, a small community of scientists has ripped open the packaging of the cyanobacterial circadian clock, compiled the parts list, examined the gears, and begun to piece together the mechanism. Over the past 2 years, the 3D molecular structures have been solved for the core components of the cyanobacterial circadian clock: KaiA, KaiB, and KaiC. In a surprisingly literal analogy to mechanical timepieces, the protein that seems to be at the heart of the clock mechanism, KaiC, forms a hexameric ring that even looks like a cog: the escape wheel, perhaps. Previous work has shown that KaiC has an autophosphorylation activity, and that the presence of KaiA and KaiB modulates the extent to which KaiC is phosphorylated. In this issue of PNAS, Nishiwaki et al. biochemically identify two amino acid residues on KaiC to which phosphoryl groups covalently attach, and show the necessity in vivo of a phosphorylation-competent residue at these positions. By searching the crystal structure for evidence of phosphorylated sites, Xu et al. pinpoint a third residue that may “borrow” the phosphoryl group dynamically. Together, their work contributes richly to our understanding of what makes the gears mesh and turn to crank out a 24-h timing circuit....
    Because each of these components (at minimum) is a dimer [composite of two molecular chains], KaiC is known to be a hexamer [composite of six chains], and other proteins may be present as well, the cyanobacterial clock can be thought of as an organelle unto itself: a “periodosome” that assembles and disassembles during the course of a day, defining the circadian period.”

    The term “periodosome” means “time-keeping body” – i.e., clock. Her diagram shows KaiC as a six-sided carousel to which phosphate groups and other subunits attach and detach during the diurnal cycle. The feedback between the units provides the periodicity of the clock, similar to the back-and-forth pendulum in a grandfather clock or the escape wheel in a wristwatch. How is the clock tuned to the day-night cycle? Where do the parts come together, and how do the clock gears mesh with other cellular machines? We don’t know yet; the box has just been opened.
    The clocks examined in these papers are the “simple” clocks of blue-green algae, compared to the much more complex biological clocks in eukaryotes. Even about these relatively simple systems in cyanobacteria much remains to be understood, but our initial glimpses into the inner workings of a biological clock at the molecular level remind her of the delight of opening a chest of toys for the first time:

    “Identification of other potential components of the periodosome, intracellular localization of the clock parts, and elucidation of other potential modifications all may yield gears that are required to smoothly tick away the time and ensure that daughter cells do not run fast or slow.
    The cyanobacterial clock box, no longer black, is a chest filled with bioluminescence and attractive toys. Putting together the pieces to design a clock is a tedious task, but S. elongatus is a gracious host, and the guests at the party are hard at work.”

    1Susan S. Golden, “Meshing the gears of the cyanobacterial circadian clock,“ Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0405623101

    Secrets of the Spliceosome Revealed 09/17/2004
    A husband and wife team from Hebrew University has revealed the structure of the spliceosome, one of the most complex molecular machines in the cell (see 09/12/2002 headline), in more detail than ever before, says EurekAlert. The spliceosome is responsible for cutting out the introns in messenger RNA after it has transcribed DNA, and also for “alternative splicing” that rearranges the exons to produce a variety of proteins from the same DNA template: “Alternative splicing, which underlies the huge diversity of proteins in the body by allowing segments of the genetic code to be strung together in different ways, takes place in the spliceosome as well.”
    The Sperlings found a tunnel between the two major subunits of the machine where they believe the cutting and splicing operations take place, and also a cavity that might provide a safe haven for the messenger RNA strand, like a waiting room, before its surgery. Also, they found that four spliceosomes are bound together into a “supraspliceosome” which is able to do “simultaneous multiple interactions, rather than by a stepwise assembly” as inferred from other experiments in vitro. Their investigation in vivo (within a functioning, living cell) revealed even more complexity in the composite machine than had been seen in the individual machines:

    “Such a large number of interactions that the cell has to deal with can be regulated within the supraspliceosome. Having the native spliceosomes as the building blocks of this large macromolecular assembly, this large number of interactions can be compartmentalized into each intron that is being processed. At the same time, the whole supraspliceosome enables the communication between the native spliceosomes, which is needed for regulated splicing. The organization of the supraspliceosome, like other macromolecular assemblies that exist as preformed entities, avoids the necessity to recruit the multitude of splicing components each time the spliceosome turns over. In that sense, the overall coordination of the cellular interactions is reduced from the hard work of repeatedly placing each piece in the correct position of the puzzle to the relatively simpler work of coordinating the preformed puzzle.”

    In short, “The supraspliceosome represents a stand-alone complete macromolecular machine capable of performing splicing of every pre-mRNA independent of its length or number of introns.” They found that the individual spliceosomes are joined with a flexible joint like a hinge to provide flexible interactions and communication. Their work was published in Molecular Cell Sept. 10.1
    1Sperling et al., “Three-Dimensional Structure of the Native Spliceosome by Cryo-Electron Microscopy,” Molecular Cell, Volume 15, Issue 5, 10 September 2004, Pages 833-839; doi:10.1016/j.molcel.2004.07.022.

    Bacterial Flagellum Reveals New Structural Complexity 10/27/2004
    The bacterial flagellum, the unofficial mascot of the Intelligent Design movement, got more praise from the evolutionary journal Nature this week: Samatey et al.1 analyzed the hook region in detail and found that it is composed of 120 copies of a specialized protein that “reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.”
    Christopher Surridge, commenting on this paper in the same issue,2 adds that this joint must be able to bend up to 90 degrees in a millisecond or less while rotating at up to 300 times per second. He says that the researchers describe “how they determined the atomic structure of this super-flexible universal joint, and thereby how it achieves such a feat of engineering.”
    1Samatey et al., “Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism,” Nature 431, 1062 - 1068 (28 October 2004); doi:10.1038/nature02997.
    2Christopher Surridge, “Molecular motors: Smooth coupling in Salmonella,” Nature 431, 1047 (28 October 2004); doi:10.1038/4311047b.

    [b]“Crucial Evolutionary Link” Found for Eukaryotes 11/05/2004
    Often the opening words of a news story are what stick in the memory: “crucial evolutionary link.” The corroborating evidence, however, is buried in technical details of the press release from Rockefeller University, posted on NewsWise. In short, the researchers claim:

    “Scientists believe the emergence of organelles, compartments in the eukaryotic cell’s cytoplasm that perform such functions as energy production, waste removal and protein synthesis, and a nucleus evolved between 2 and 3 billion years ago.
    One hypothesis regarding the evolution of eukaryotic cells suggests that the endomembrane system developed because some ancient bacterial cells had the ability to sharply curve their membranes, allowing them to form internal membrane structures as well as to engulf other organisms. The findings reported by [Michael P.] Rout and colleagues [Rockefeller University] suggest that an ancestor of an NPC component, called the Nup84 complex, may have been a key molecular sculptor responsible for such a reshaping of the membrane.”

    To find out what the Nup84 complex is, you have to wade through the boring body of the article. For one thing, Nup84 is complicated:

    “...the scientists ... found that the Nup84 complex in yeast is composed of two types of protein structures, “alpha solenoids” and “beta propellers.” Two of the proteins are beta propellers, three are alpha solenoids and two are composed of beta propeller “heads” attached to alpha solenoid “tails.” The scientists showed that the architecture of the Nup84 complex also appears in the NPCs of human and plant cells and is therefore conserved throughout eukaryotes.”

    As our regular readers know, any functional protein is composed of a chain of amino acids, all left-handed, assembled by a complex factory of molecular machines (see online book). The function of a protein is dependent on the precise sequence of the amino acids and the way the chain is folded with the help of other machines named chaperones. When you have a complex of proteins working together (and most proteins work in complexes), the requirements for specified complexity are even higher. The authors are assuming that this protein complex Nup84 emerged through a Darwinian process.
    What’s the gist of the missing link claim? Basically, that Nup84 not only can curve a membrane, it is also involved in shuttling cargo around the cell. Since both prokaryotes and eukaryotes do that, but only eukaryotes curve their membranes to form organelles, they concluded that Nup84 is a missing link, a “crucial evolutionary link.”

    Bacterial Hypodermic Needle Examined 11/10/2004
    Those who have seen the film Unlocking the Mystery of Life might recall seeing the image of the “needle-nosed cellular pump” that some evolutionists claim was an intermediate for the bacterial flagellum. Those wishing to investigate this claim further might want to see the renditions that a Yale team produced of the pump, called a Type III Secretion System (TTSS), in the Nov. 5 issue of Science.1 Their introduction describes the machine:

    “TTSSs are composed of more than 20 proteins, including a highly conserved group of integral membrane proteins, a family of customized cytoplasmic chaperones, and several accessory proteins, placing TTSSs among the most complex protein secretion systems known.”

    Their images of the TTSS show parts resembling exquisitely crafted rings, gears, sockets, rods and tubes. The parts are flexible and undergo drastic conformational changes during assembly that amount to reprogramming of the parts. Here’s a small sample of what transpires during the assembly of this one molecular complex:

    “Contoured longitudinal sections revealed conformational changes that occurred during the transition from the base to the fully assembled needle complex (Fig. 3, A and B). The cuplike protrusion that emerged from the basal plate of IR1 moved down, while an inward, clamping movement of IR2 redefined the shape of the cavity that is located below the basal plate of the base (movie S2). These conformational changes may provide the structural basis for the functional reprogramming of the TTSS machinery, which upon completion of needle assembly, switches from secreting the needle protein PrgI, the inner-rod protein PrgJ (see below), and the regulatory protein InvJ ... to secreting the effector proteins that are delivered into the host cell. On the opposite side of the basal plate, the socketlike structure underwent an outward movement, which created an attachment point for the inner rod (movie S2). A similar outward movement was observed for OR1, which created space for the needle to dock at the outermost perimeter of the base (movie S2). These changes were complemented by an outward movement of OR2 and a drastic remodeling that flattened the septum, sealing the apical side of the base, against OR2 during needle assembly (Fig. 3, A and B; movie S2). This rearrangement of the septum is essential for creation of the secretion channel and transformed part of InvG from being a barrier into forming two scaffolds that enable assembly of the needle and the inner rod. Like the socket structure at the basal end of the chamber, these new scaffolds likely serve as adaptors, accommodating the symmetry mismatches between the base, the needle, and the inner rod.”

    Thus, the assembly of the TTSS involves not only parts coming together, but a coordinated series of shape changes of the parts relative to one another such that they fit together tightly, to enable the finished pumping action. We know the TTSS largely from “virulence of many Gramnegative bacteria pathogenic for animals and plants”.
    1Marlovits et al., “Structural Insights into the Assembly of the Type III Secretion Needle Complex,&148; Science, Vol 306, Issue 5698, 1040-1042, 5 November 2004, [DOI: 10.1126/science.1102610].

    Flagellar Oars Beat Like Galley Slaves In Synchronization 12/26/2004
    The Dec. 14 issue of Current Biology1 investigated another mystery in the operation of eukaryotic flagella:

    “Flagella are microtubule-based structures that propel cells through the surrounding fluid. The internal structure of a flagellum consists of nine parallel doublet microtubules arranged around a central pair of singlet microtubules (Figure 1). Force for propulsion is provided by thousands of dynein motors anchored in rows along one side of each doublet, which can walk along the microtubule of the adjacent doublet. In order to produce coordinated bending of the flagellum, these dynein motors — organized into multi-headed complexes called the inner and outer dynein arms — must produce their power strokes in synchrony, like the oarsmen on an ancient Mediterranean war-galley. But whereas oar-strokes were coordinated by a continuous drum-beat, it is much less clear how flagellar dynein motors are synchronized.”

    The authors of the paper consider growing evidence that the central microtubule pair provides the drumbeat, with the aid of “a protein complex called the dynein regulatory complex, located between the spokes and the dynein arms.” However, “The molecular mechanism by which the central pair regulates dynein is not known.”
    1Kimberly A. Wemmer and Wallace F. Marshall, “Flagellar Motility: All Pull Together,” Current Biology Volume 14, Issue 23, 14 December 2004, Pages R992-R993, doi:10.1016/j.cub.2004.11.019.

    Cells Find Signal in the Noise 12/20/2004
    Parents at an amusement park know the challenge of picking out their child’s voice, or even hearing their own hollering, in the noise of the crowd. Yelling won’t help much if the rest of the crowd is yelling also. Acoustic engineers know that raising the volume while playing back a noisy tape amplifies the noise as well as the signal. Cells have a novel way of meeting this challenge, as two Japanese mathematical biologists discuss in PNAS.1 Cells are continuously sending and receiving chemical messages, a process called signal transduction. Treating the cell signal transduction network like a physical system of receivers and amplifiers, the researchers noted that a cell, like an amusement park, is an intrinsically noisy place, yet some of the reactions are very sensitive. “How cells respond properly to noisy signals by using noisy molecular networks is an important problem in elucidating the underlying ‘design principle’ of cellular systems,” they say in the introduction. How do the sensitive reactions get their messages through all that noise?

    “Because intracellular processes are inherently noisy, stochastic reactions process noisy signals in cellular signal transduction. One essential feature of biological signal transduction systems is the amplification of small changes in input signals. However, small random changes in the input signals could also be amplified, and the transduction reaction can also generate noise. Here, we show theoretically how the abrupt response of ultrasensitive signal-transduction reactions results in the generation of large inherent noise and the high amplification of input noise. The inherently generated noise propagates with amplification through intracellular molecular network. We discuss how the contribution of such transmitted noise can be shown experimentally. Our results imply that the switch-like behavior of signal transduction could be limited by noise; however, high amplification reaction could be advantageous to generate large noise, which would be essential to maintain behavioral variability.”

    They categorized the noise as intrinsic, coming from the reaction itself, to extrinsic, coming from other reactions. This is somewhat like hearing your own voice vs. the yelling of those around you. The intrinsic noise has higher frequency than the extrinsic noise. As one source of noise becomes dominant, it reaches a crossover point where the other source is less dominant. This provides a kind of signal, or switch, which the cell can use to advantage:

    “From our result, it can be further suggested that if the extrinsic noise dominates, the upstream reactions affect the fluctuation of the most downstream reaction, which determines the cellular behavior. As a result, the behavioral fluctuations are made up of the contributions of the fluctuations of several upstream reactions. On the other hand, if the intrinsic noise dominates, only the intrinsic noise of the most downstream reaction determines the behavioral fluctuations. As a result, the behavior could be simpler than the case in which extrinsic noise is dominant....
    ....Consequently, the low-frequency modulations in the downstream reactions can be affected by the behaviors of upstream reactions, whereas the high-frequency modulations are expected to be independent of upstream reactions.”

    As a result, a bacterium can respond to chemicals in the environment, the hemoglobin in your blood can respond to changing conditions in the capillaries, genes can respond correctly to requests for expression, and complex cascades of cellular reactions can respond to the signal from any reaction in the series, in the midst of all the noise. “Therefore,” they conclude, “the result implies that the extrinsic noise is essential to maintain the behavioral variability in wild-type bacteria.” Their experiments related to three relatively simple reactions, and their analysis considered primarily linear response. Many cellular reactions involve nonlinear behavior. “In these cases,” they admit, “the relation between the response and the fluctuations can be more complicated than the relations we studied.”
    1Tatsuo Shibata and Koichi Fujimoto, “Noisy signal amplification in ultrasensitive signal transduction,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403350102, published online before print December 29, 2004.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Cellular UPS Gets Right Packages to Chloroplasts 01/01/2005
    If all your packages were sent correctly over the holidays, consider the job a plant cell has getting 3000 proteins into a chloroplast. Mistakes are not just inconvenient. They can be deadly, or at least bring photosynthesis to a halt. To guarantee proper delivery of components, plant cells have a remarkable shipping system, described in Current Biology by two UK biologists, Paul Javis and Colin Robinson.1 Part of the challenge is getting polypeptides past the double membranes of the chloroplast. A remarkable crew of enzymes and molecular machines puts a shipping label (transit peptide) on each amino acid chain, reads it, routes it to the correct destination, and then removes it:

    “Over 90% of the ~3000 different proteins present in mature chloroplasts are encoded on nuclear DNA and translated in the cytosol [cell fluid outside the nucleus]. These proteins are synthesized in precursor form – each bearing an amino-terminal targeting signal called a transit peptide – and are imported into the organelle by an active, post-translational targeting process (Figure 1). This process is mediated by molecular machines in the outer and inner envelope membranes, referred to as ‘translocon at the outer envelope membrane of chloroplasts’ (Toc) and ‘translocon at the inner envelope membrane of chloroplasts’ (Tic), respectively. Upon arrival in the stroma [chloroplast interior], the transit peptide is removed and the protein either takes on its final conformation or is sorted to one of several internal compartments in a separate targeting process.”

    The authors believe, like most evolutionists, that plastids (including chloroplasts) arose when a primordial cell engulfed another and took over its light-harvesting machinery, a process called endosymbiosis (see 10/01/2004, 09/09/2004, 08/06/2004 and 10/07/2003 headlines). They believe the former cell that became the chloroplast retained only a stripped down version of its genetic code, and most of the DNA instructions for building these 3000 chloroplast proteins got transferred to the nucleus. Yet this means that a tremendous amount of machinery had to be developed to get the proteins to their destinations:

    “Chloroplasts are complex organelles comprising six distinct suborganellar compartments: they have three different membranes (the two envelope membranes and the internal thylakoid membrane), and three discrete aqueous compartments (the intermembrane space of the envelope, the stroma and the thylakoid lumen). One of the consequences of this structural intricacy is that the internal routing of chloroplast proteins is a surprisingly complex process. While envelope proteins may employ variations of the Toc/Tic import pathway to arrive at their final destination, proteins destined for the thylakoid membrane or lumen employ one of four distinct targeting pathways (Figure 1). Thylakoid membrane proteins are targeted by the signal recognition particle (SRP)-dependent and spontaneous insertion pathways, whereas lumenal proteins are targeted by the Sec and Tat pathways....”

    Each of these “pathways” is an assembly-line process involving multiple proteins dedicated to these tasks. Several points brought out in the article make it challenging to perceive of a smooth transition from endosymbiosis to today’s complex shipping and handling pathways (numbering ours):

    1. The transit peptide needs to fit the receptor on the membrane, and another protein has to be ready to cleave it (remove it).
    2. The transit peptides have to be precise to avoid having the protein arrive at the wrong organelle, like the endoplasmic reticulum, mitochondrion or peroxisome – organelles which also accept polypeptides with shipping labels.
    3. Transit peptides are varied. “One might therefore expect chloroplast transit peptides to share well-defined primary or secondary structural motifs,” they say. “On the contrary, transit peptides are remarkable in their heterogeneity. They vary in length from 20 to >100 residues, and have no extended blocks of sequence conservation.”
    4. The transit proteins “do not seem to form secondary structure in aqueous solution” but once they arrive at their target membrane, they seem to take on a characteristic structure.
    5. The polypeptides (precursor proteins) are threaded through the needle of specialized gates in the membrane. There, additional molecular machines (chaperones) make sure they do not fold prematurely.
    6. To get a polypeptide through a membrane involves three steps: contact, docking, and translocation, when the transit peptide is cleaved. This requires energy: a high concentration of ATP must be present for the operation.
    7. The Toc and Tic squads, like a delivery organization with a variety of employees skilled in particular tasks but working on common goals, is made up of multiple proteins, each with its own task to perform, all working in coordination.
    8. Once inside the outer membrane, the polypeptide has to get past the inner membrane. Another set of specialized proteins are available for that task.
    9. A third import apparatus has to complete the task of getting the polypeptide to its final destination. Many go to the thylakoid membrane, rich with light-harvesting structures and ATP synthase (see 08/10/2004 headline).
    10. Those polypeptides bound for the thylakoid membrane have a secondary shipping label (transit peptide). In addition, they may have a “stop-transfer” signal to indicate their destination.
    11. Removal of the secondary transit peptide can occur by “one of two very different pathways,” called Sec and Tat. Sec transports proteins in an unfolded state, but Tat can transport them in a folded state. Each pathway involves multiple proteins working together.
    12. In the Tat pathway, “There is even evidence that some proteins are exported in an oligomeric form” [i.e., several proteins bound together in a complex], “which points to a remarkable translocation mechanism,” they remark. Is this like squeezing a completed sweater through the eye of a needle? “...we currently know very little about this mechanism,” they say. “Somehow, this system must transport a wide variety of globular proteins – some over 100 kDa [kilodaltons] – while preserving the proton motive force and avoiding loss of ions and metabolites.” Their surprise at this indicates it is quite a feat.
    13. The translocation process can expend 30,000 protons, “a substantial cost by any standard.” According to current theory, a pH difference between inner and outer membrane provides the proton flow, but that pH balance must be carefully monitored and regulated.
    14. Another pathway named SRP inserts proteins into the lumen. The authors claim this pathway was “clearly inherited from the cyanobacterial progenitor of the chloroplast,” but admit that there are differences in the insertion pathways and events at the thylakoid membrane in chloroplasts. “ is fair to state that, while the major players in this pathway have been identified, their modes of action remain unclear and we do not understand how such highly hydrophobic proteins are bound by soluble factors, shuttled to the membrane and then handed over to membrane apparatus and inserted.”
    15. Evolutionists who expected the SRP pathway from E. coli bacteria to act the same in chloroplasts, where homologous proteins were detected, learned otherwise: “Surprisingly, this is not the case. In vitro assays for the insertion of a range of membrane proteins have shown that the vast majority of such proteins do not rely on any of the known protein transport machinery, including SRP, FtsY, Alb3 or the Sec/Tat apparatus, for insertion.” Nor do they rely on nucleoside triphosphates or proton flow.
    16. Speaking of the apparent spontaneous insertion of the thylakoid proteins, they comment, “This unusual pathway for membrane protein insertion appears to be unique to chloroplasts.” Though the typical insertion components are not involved, they believe it would be “overly simplistic” to assume that this pathway requires no “complex insertion apparatus.”
    17. Other pathways than those described above are used for other proteins to get inside the chloroplast. Some are encoded by the chloroplast DNA, translated in the interior, then transported to their destinations.
    18. Chloroplasts have to transport not only the essential light-harvesting proteins, but also “housekeeping” proteins for structural maintenance. These must be imported at their own separate rates depending on the stage of development or the environmental conditions, and have their own specific transit peptides.
    This represents the state of our knowledge on protein transport in chloroplasts. It is only a partial picture of a varied and complicated picture with many players, as their final paragraph makes clear:

    “The Tat pathway manages the remarkable feat of transporting large, folded proteins without collapsing the delta-pH, and we currently know very little about this mechanism. Most membrane proteins use a possibly ‘spontaneous’ insertion mechanism that just does not make sense at the moment – why do these proteins need so little assistance from translocation apparatus, when membrane proteins in other organelles and organisms need so much? And how do these thylakoid proteins avoid inserting into the wrong membrane? We have gone some way toward understanding the rationale for the existence of all these pathways, but the thylakoid may still have surprises in store.”

    By contrast, another paper in the same issue of Current Biology2 makes confident claims that the endosymbiosis theory has been demonstrated with diatoms (see 10/01/2004 and 07/21/2004 headlines about diatoms). They suggest that it was dangerous for genes to remain in the plastids, because of free radicals generated by the photosynthesis machinery, and because of higher mutation rates, and that’s why most of them wandered to the nucleus.
    1Paul Jarvis and Colin Robinson, “Mechanisms of Protein Import and Routing in Chloroplasts,” Current Biology, Volume 14, Issue 24, 29 December 2004, Pages R1064-R1077, doi:10.1016/j.cub.2004.11.049.
    2Nisbet, Killian and McFadden, “Diatom Genomics: Genetic Acquisitions and Mergers,” Current Biology Volume 14, Issue 24, 29 December 2004, Pages R1048-R1050, doi:10.1016/j.cub.2004.11.043.

    DNA Translators Cannot Tolerate Editor Layoffs 01/12/2005
    We’ve explained elsewhere about the family of molecular machines called aminoacyl-tRNA synthetases (see 05/26/2004 entry and its embedded links). Their job is to associate each word of DNA code (codon) with its corresponding piece of a protein (amino acid). In a very real sense, they translate the DNA code into the protein code. One amazing capability of these machines is that they proofread their work. They can differentiate between similar molecules, and edit out incorrect pieces inserted by mistake. Scientists from Scripps Institute writing in PNAS1 thought they would watch what happened when they gave one of these translators a mutation that diminished this editing ability. It wasn’t pretty:

    “The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain. A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids. Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase. Ambiguity retarded cell growth at most temperatures in rich and minimal media. These growth rate differences were seen regardless of the carbon source. Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function. Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors. Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.”

    In short, removing the editing created big problems. The poor bacteria were stunted and vulnerable to malfunctions. When the translator could not maintain high fidelity by editing out mistakes, crippled proteins were produced, and the organism became a sitting duck for the harsh realities of survival.

    Update 01/26/2005: This paper generated a commentary in PNAS by Randall Hughes and Andrew Ellington of the University of Texas.2 They agreed that “over the long run, there has been and will continue to be tremendous selective pressure to maintain the current genetic code.” But they surmise that, since not all the substituted amino acids produced fatalities, evolution might take advantage of them. “Taking advantage of protein misfolding might at first seem to be an improbable event,” they admit, “but this phenomenon is conceptually similar to other ways in which organisms take evolutionary advantage [sic] of even inclement environments.” Like citizens under siege scrounging for food, they envision a cell under stress with “a general need to explore a larger genetic space or a larger protein folding space or both.” Maybe the cell has already planned for such things through experience. “To the extent that organisms have encountered environmental stress intermittently over evolutionary time,” they write, “it may even be advantageous to establish some sort of regulatory feedback between stress and phenotypic exploration.” In the end, though, they agree that the cell works hard to prevent such errors and possesses exquisite means to eliminate typos. That means it will be difficult to find ways to change the genetic code in lab organisms: “simple substitutions will be an uphill battle.”
    1Bacher, Crécy-Lagard and Schimmel, “Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0409064102, published online before print January 12, 2005.
    2Randall A. Hughes and Andrew D. Ellington, “Mistakes in translation don’t translate into termination,” Proceedings of the National Academy of Sciences USA, February 1, 2005, vol. 102, no. 5, pp. 1273-1274.

    Simple Darwinian Theories Have to Be Abandoned 01/17/2005
    Mutate one gene and a cascade of changes can result. This effect is called pleiotropy (see 10/01/2003 entry). According to an article by Stephen Strauss reporting for the Canadian newspaper Globe and Mail, “The emerging richness of pleiotropy means that any simple Darwinian notion of what is going on during natural selection has to be abandoned.”
    Unless Darwinians can show that the positive changes outnumber the negative effects, pleiotropy seems to spell difficulty, if not doom, for neo-Darwinian theory, which relies on beneficial mutations. But if beneficial mutations are rare to begin with, how can evolutionary theory face the new problem of pleiotropy? “The simplest answer,” Strauss writes, “is that nearly 150 years after Darwin first explained the theory of evolution, the richness of multiple effects from the same gene is such that existence itself seems problematic”.
    Strauss gives examples of a few more nuanced proposals for salvaging Darwinian evolution: “Faced with what amounts to a growing daily confusion of genetic effects, biologists are proposing new and more highly refined theories of evolution.” Some biologists hope that some mutations have only minor effects. Others are looking for examples of single mutations that might have a cascade of good effects. He ends on a hopeful note: “With modern genetics increasing the supply of data about the multiple functions of genes, evolutionary biologists are increasingly confident that they are going to be able to do what Darwin promised but couldn’t quite delivery [sic] -- truly explain the origin of species.”

    Ribosome Unties the Messenger-RNA Gordian Knot 01/19/2005
    Cells needing to translate their DNA into proteins have a problem. The messenger RNAs, the molecules that carry the genetic code from the nucleus to the translating machine called the ribosome, get tied up in knots. How does the ribosome untie them before they can begin translating? Takyar et al., writing in Cell,1 explored this problem and found that the ribosome has a novel solution.
    If you have seen the film Unlocking the Mystery of Life, you watched a messenger RNA molecule, nice and straight, exit the nuclear pore complex and neatly enter the ribosome, like a man reclining in a barber chair waiting to get a haircut. Unfortunately, things are not so simple. Because of chemical affinities between the bases of the RNA molecule, the bases attract other bases (base-pairing) or else fold over on themselves, forming amorphous lumps (secondary structure). Untangling this mess would be like straightening out a chain of several hundred magnets that has clumped together.
    The untangling problem is not unique to messenger RNA (mRNA). DNA in the nucleus also has to be unwound. Each of the processes of “replication, DNA repair, recombination, transcription, pre-mRNA splicing, and translation” have their own specialized enzymes, called helicases, that latch onto the nucleic acids and work their way down the helix, unwinding them for whatever subsequent operation is necessary. Until now, though, no helicase was found associated with the ribosome. It turns out the helicase activity is built-in.
    The ribosome has an entry tunnel and exit tunnel. As the mRNA strand enters, specialized proteins named S3, S4 and S5 are precisely placed to form a ring around the mRNA helix. They grab the phosphate groups on the side chains and separate the base pairs.2 There’s only room in the tunnel for a single strand. As the interior of the ribosome pulls the mRNA through, this entry-tunnel helicase, built into the walls of the tunnel, effectively “melts” the double strands, sending in a clean single strand for the translation machinery to work on. And how does the ribosome pull it in?

    “In their studies of ratcheting of the two ribosomal subunits between the pre- and posttranslocation states, Frank and Agrawal (2000) observed a reciprocal expansion and contraction in the diameter of the upstream and downstream tunnels, suggesting that these two features may alternately grab and release the mRNA during translocation of mRNA. This dynamic behavior in the downstream tunnel could also be related to its helicase function.”

    The action seems analogous to those old Dymo labelmakers people used to use for labeling household items. You remember: as your hand clicked the machine, the tape came in one tunnel and out another. In the case of the ribosome, the entry and exit tunnels alternately expand and contract, forcing the mRNA molecule to ratchet through the system. The ratchet prevents backward motion and also is delicate enough to prevent breakage of the single strand during the unwinding process.
    The placement of S3, S4 and S5 in the tunnel is critical. The researchers found that when they were mutated, the helicase activity stopped. Because it latches onto the phosphates, which are universal to RNA molecules, they can unwind any strand, regardless of the sequence of base pairs.
    The authors do not speculate on how this helicase system, which is unique to the ribosome, evolved. They only note that if it did, the unwinding puzzle needed to be solved by the very first living cell:

    “The inescapable presence of secondary structure within mRNA coding sequences must have been one of the first problems encountered in the transition from an RNA world to a protein world [sic] and may have resulted in coupling of ribosomal helicase activity with the fundamental mechanics of translocation.”

    How this was accomplished by a sequence of random changes, they do not explain.
    1Takyar et al., “mRNA Helicase Activity of the Ribosome,” Cell, Vol 120, 49-58, 14 January 2005.
    2It was not clear to the authors whether the helicase pulls the bases apart with the expenditure of energy. It may be that the helicase can take advantage of spontaneous separation. Base pairs tend to “breathe” as their weak hydrogen bonds stretch. The helicase may be able to latch onto the nucleotide during its spontaneous separation, as if saying “Aha! Gotcha!” and prevent the hydrogen bond from re-forming.

    Design Paper Published in PNAS 01/26/2005
    A team of Japanese and American biologists, from Caltech and University of California and elsewhere, describe the heat shock response in the cell. They not only compare this biological system to good engineering, but treat the engineering paradigm as a proper approach to the study of cellular systems: in fact, they say, “Viewed from this perspective, heat shock itself constitutes an integral functional module. Such a characterization of functional modules is extremely useful, because it provides an inventory list of cellular processes. An analogy would be a list of machines and their function in a factory.” For more design language, look at the abstract:

    “Molecular biology studies the cause-and-effect relationships among microscopic processes initiated by individual molecules within a cell and observes their macroscopic phenotypic effects on cells and organisms. These studies provide a wealth of information about the underlying networks and pathways responsible for the basic functionality and robustness of biological systems. At the same time, these studies create exciting opportunities for the development of quantitative and predictive models that connect the mechanism to its phenotype then examine various modular structures and the range of their dynamical behavior. The use of such models enables a deeper understanding of the design principles underlying biological organization and makes their reverse engineering and manipulation both possible and tractable. The heat shock response presents an interesting mechanism where such an endeavor is possible. Using a model of heat shock, we extract the design motifs in the system and justify their existence in terms of various performance objectives. We also offer a modular decomposition that parallels that of traditional engineering control architectures.”

    The paper is filled with design words: engineering, robustness, feedback loops, feed-forward loops, modularity, performance, functional criteria, and the like – all but the buzzphrase “intelligent design.” For example, “Biology and engineering share many similarities at the system level, including the use of complexity to achieve robustness and performance rather than for minimal functionality.”
    The only mention2 of biological evolution is a passing reference in the final discussion that, in the surrounding design language, seems almost irrelevant: “The formulation of such a problem aside, the physical implementation of any of its solutions seems to have been evolutionarily solved by using a number of recurring motifs...” How it was solved, and who solved it, is left unexplained. Instead, the authors seem enthusiastic that a design-theoretic approach, viewing cellular mechanisms the way a computer scientist would reverse-engineer software, can be a fruitful avenue for research:

    “However, to understand the operational principles of a certain machine, to repair it, or to optimize its performance, it is often necessary to consider a modular decomposition of the machine itself. Such a decomposition does not necessarily require stripping the machine down to the component level but rather identifying its submodules with their predefined functionalities. “A particularly successful such modular decomposition has been extensively used in the field of control and dynamical systems, where components of a system are classified in terms of their role with respect to the regulation objective. Similar decompositions exist in computer science, for example, because modularity is a basic principle of good programming.”

    The authors make no mention of a Programmer, or state their personal beliefs about origins. But that, again, supports a principle stated frequently in the intelligent design literature: the identity of the designer is not the issue. Design detection is a purely scientific question, and the design-theoretic approach is a fruitful avenue of research.
    1El-Samad, Kurata, Doyle, Gross and Khammash, “Surviving heat shock: Control strategies for robustness and performance,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0403510102, published online before print January 24, 2005.
    2The only other possible allusion states, “Indeed, in higher level languages, a complicated programming task is usually divided into a set of modules, subroutines, or objects, with simple well defined interfaces. This results in flexible and robust programs, whose modules can be designed almost separately and, as such, are more easily evolvable.” However, being in the context of computer program design, the statement implies guided evolution – i.e., upgrading – by intelligent design, not evolution by an undirected or Darwinian process.

    Your Motors Are Turbo-Charged 01/30/2005
    Think how fast 6000 rpm is. It would redline on most cars. Yet you have motors in your body that make that speed look like slow-mo.
    The Japanese have taken great interest in the cellular machine ATP synthase since its rotary operation was discovered in 1996 (see 12/22/2003 entry). Maybe it’s because they like rotary engines. ATP synthase is an essential protein complex that generates ATP (adenosine triphosphate), the energy currency of the cell. Found in the membranes of mitochondria and chloroplasts, it runs on an electrical current of protons, from sunlight (in plants) or digestion (in animals). It is a reversible engine: it can just as easily generate protons from the dissociation of ATP. It has five major protein parts, including a rotor, a stator, and a camshaft. The F0 domain runs like a waterwheel on protons and turns the camshaft. Three pairs of lobes in the F1 domain catalyze ATP from ADP and phosphate, in a three-phase cycle of input, catalysis, and output. Each revolution generates 3 ATP.
    Hiroshi Ueno and team, reporting in PNAS,1 have invented new techniques for studying and measuring the tiny motors. Now, with the aid of a high-speed camera running at 8,000 frames per second, they have clocked the rotational speed of the entire F0F1-ATP Synthase motor at 352 revolutions per second, a whopping 21,120 rpm.
    Although this molecular machine exists in all life forms, they used motors from a thermophilic bacterium. To monitor the action, the team fastened a microscopic bead to the carousel of c subunits. At 25° C, it ran at 230 rps. At 45° C, it ran at 650 rps. Extrapolating up to 60° C, the organism’s optimum growth temperature, they speculate that it could be running as fast as 1,600 rps – an unbelievable 96,000 rpm – and that with nearly no friction and almost ideal efficiency. While they caution that reservation is needed whether these “enormous numbers” are actually achieved, they do say with confidence that the rotation rates they measured are much higher than earlier claims. “It is intriguing to learn,” they say, “whether these rapid rotations are really occurring in living cells.”
    1Ueno et al., “ATP-driven stepwise rotation of F0F1-ATP synthase,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0407857102, published online before print January 24, 2005.

    Genes Evolving Downward 02/02/2005
    Those assuming the evolution of eukaryotic genomes has progressed upward in complexity may find the following abstract from PNAS1 startling:

    “We use the pattern of intron conservation in 684 groups of orthologs from seven fully sequenced eukaryotic genomes to provide maximum likelihood estimates of the number of introns present in the same orthologs in various eukaryotic ancestors. We find: (i) intron density in the plant-animal ancestor [sic] was high, perhaps two-thirds that of humans and three times that of Drosophila; and (ii) intron density in the ancestral bilateran [sic] was also high, equaling that of humans and four times that of Drosophila. We further find that modern introns are generally very old, with two-thirds of modern bilateran introns dating to the ancestral bilateran [sic] and two-fifths of modern plant, animal, and fungus introns dating to the plant-animal ancestor [sic]. Intron losses outnumber gains over a large range of eukaryotic lineages. These results show that early eukaryotic gene structures were very complex, and that simplification, not embellishment, has dominated subsequent evolution.”

    In their paper, Harvard biologists Scott Roy and Walter Gilbert used the maximum-likelihood phylogenetic method instead of maximum parsimony, and feel it provided a better ancestral tree. In fact, they used the same data as other scientists who used parsimony, and got very different results. They are emphatic about their conclusions:

    “These results push back the origin of very introndense genome structures over a billion years to the plant-animal split. Indeed, ancestors at the divergences between major eukaryotic kingdoms as well as the ancestral bilateran appear to have harbored nearly as many introns as the most intron-dense modern organisms. This is a sharp repudiation of the common assumption that intron-riddled gene structures arose only recently.
    In addition, our analysis shows that the majority of introns are themselves very old. Two-thirds of bilateran introns were present in the bilateran ancestor ; 40% of opisthokont introns were present in the opisthokont ancestor; and 40% of plant, animal, and fungal introns were present in the plant-animal ancestor. This is quite different from what is commonly assumed and surprising in light of relatively fast rates of intron turnover observed in nematodes and flies.”

    This bias toward intron loss instead of gain appears to be a general trend among eukaryotes, they conclude. What does this mean? The only way to rescue an evolution toward “improvement” with these results is to suggest that introns are bad, like parasites, and that over time, eukaryotes got better at ridding themselves of them. They reject that and other notions, assuming instead that “It seems much more likely that different selection or mutation regimes for introns along different lineages are driving the observed instances of gene streamlining.” Although intron function and evolution is still largely unknown, they leave only an admission of ignorance of what their results mean – only that geneticists had better re-examine their assumptions:

    “These results contradict the assumption that genome complexity has increased through evolution. Instead, species have repeatedly abandoned complex gene structures for simpler ones, questioning the purpose and value of intricate gene structures. These results suggest a reconsideration of the genomics of eukaryotic emergence.”
    1Scott W. Roy and Walter Gilbert, “Complex early genes,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0408355101, published online before print February 1, 2005.

    Molecular Machine Parts Stockpiled in Readiness for Assembly 02/06/2005
    A team from the European Molecular Biology Laboratory has done a “4D” time-and-materials study of molecular machines, analyzing the process of assembly, reports EurekAlert. They found that the cell stockpiles some parts and holds them in storage, but adds the crucial elements just in time.

    “The researchers discovered that in yeast, key components needed to create a machine are produced ahead of time, and kept in stock. When a new machine is needed, a few crucial last pieces are synthesized and then the apparatus is assembled. Holding off on the last components enables the cell to prevent building machines at the wrong times. That’s a different scenario from what happens in bacteria, which usually start production of all the parts, from scratch, whenever they want to get something done.
    “We saw a clear pattern as to how the complexes are assembled,” says Søren Brunak from DTU. It’s unusual to find such concrete patterns in biology, compared to physics for example, due to the evolutionary forces that change living systems. But using this new model, the underlying principle became very clear.”

    The authors next want to find out how long components stay around after use. Their results were published in Science1 Feb. 4; see also the brief on EurekAlert.
    1Lichtenberg et al., “Dynamic Complex Formation During the Yeast Cell Cycle,” Science, Vol 307, Issue 5710, 724-727, 4 February 2005, [DOI: 10.1126/science.1105103].

    Survival of the Fittest – or the Luckiest? 02/06/2005
    Evolutionists assume that bacteria spread because they evolve resistance to antibiotics and become more fit to survive. That’s apparently not true, says a story in EurekAlert about a study from Imperial College, London: the spread of bacteria appears to be due to chance alone.
    Here are two quotes from the article by team members explaining the finding:

    “Dr Christophe Fraser, from Imperial College London, a Royal Society University Research Fellow and one of the authors, says: “Microbiologists have assumed for some time that some disease strains spread more successfully than others. In fact we found that the variation in the communities we studied could be explained by chance. This was surprising, especially considering all the potential advantages one pathogen can have over another, such as antibiotic resistance and differences in host immunity.”
    Dr Bill Hanage, from Imperial College London, and also one of the authors, says: “When we look at a sample and see that some strains are much more common than others, it’s tempting to think that there must be something special about them. In fact, they could just be the lucky ones, and that’s what it looks like here. Most of the variation in the spread of these pathogens can be explained by chance alone.”

    The team studied three pathogenic bacteria and followed the social patterns of the humans they infected. There was no clear association between success at spreading and fitness for spreading .
    A related commentary by Dan Ferber in Science1 had another surprise about bacteria: they are not immortal. Reproducing strains in a culture apparently show their age. What does this mean? For one thing, the results “make it unlikely that natural selection produced an immortal organism.” For another, “It’s one of those exciting results that makes you take a fresh look at what you think you know.” One observer is not sure the populations that stopped growing were aging; maybe they were taking a break for repairs. But another said the new findings “put the onus of proof on anyone who claims that cells can be immortal.”
    1Dan Ferber, “Immortality Dies as Bacteria Show Their Age,” Science, Vol 307, Issue 5710, 656 , 4 February 2005, [DOI: 10.1126/science.307.5710.656a].

    Introns Engineered for Genetic Repair 02/18/2005
    Scientists at Purdue University are using bacterial machines to treat cancer and other diseases. These machines, called Group I introns, were thought to be useless:

    “Once thought of as genetic junk, introns are bits of DNA that can activate their own removal from RNA, which translates DNA’s directions for gene behavior. Introns then splice the RNA back together. Scientists are just learning whether many DNA sequences previously believed to have no function actually may play specialized roles in cell behavior.”

    Though the function of introns is still mysterious (see 02/02/2005 entry), they appear to be highly conserved in both archaea and eukarya, suggesting they are important. Bacteria have Group I introns that do self-splicing. Eukaryotes have Group II introns that are spliced by one of the most complex molecular machines in cells, the spliceosome (see 09/17/2004 entry).

    Clutch Enables Your Motors to Achieve 100% Efficiency 02/23/2005
    Those little ATP synthase motors (see 01/30/2005 entry) in your body and (in all living cells) made news again in Nature1 last week. Scientists in Tokyo performed an ingenious set of experiments to measure the efficiency of the F1 synthesizing domain. They attached a tiny magnet to the camshaft so that they could turn it with electromagnets at will, and they carefully measured the amount of ATP synthesized or hydrolyzed as the motor turned anticlockwise or clockwise under their control. In the hydrolysis cycle, they found that the motor did not waste ATP; each molecule was successfully hydrolyzed with perfect efficiency, to the limits of their detection.
    A particular focus of their investigation was the role of the eta subunit, which is attached to the gamma camshaft. During hydrolysis, the “downhill” function, it did not seem to matter whether eta was present or absent. But in the “uphill” process (synthesizing ATP), it made a dramatic difference. Without eta, each rotation produced, on average, only one product, but with it, they got three per revolution, with at least 77% efficiency. The actual efficiency was probably higher, but was hard to measure for such small entities. In best cases, it was 100%, they said: “Therefore our data point to an excellent mechanochemical coupling efficiency. In the best cases, we observed the postulated value of three ATPs synthesized per turn.”
    “These results are consistent with the ubiquity of this strategic enzyme that fuels most of the energy consuming biological processes,” they said. “The present work reveals the unexpected importance of the eta-subunit in the synthesis of ATP.” Though its precise function remains to be discovered, it was known to play a regulatory role; now, this team suspects it acts like a structural switch or clutch to lock the enzyme into synthesis mode. Without it, the tiny motor undergoes wasteful slippage.

    As a reminder to recent readers, you can find a wonderful animation of this molecular machine on the website of German biochemist Wolfgang Junge. It is labeled “F0F1-ATPSynthase (animation)” See also his Model Schematic.
    1Rondelez et al., “Highly coupled ATP synthesis by F1-ATPase single molecules,” Nature 433, 773 - 777 (17 February 2005); doi:10.1038/nature03277
    Last edited by bob b; October 27th, 2006 at 03:56 PM.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Tissues Build Firebreaks to Avoid Disease 03/09/2005
    An article in the March 3 issue of Nature1 explains how tissues communicate to fight off infection. As reported before, cells display samples of the proteins they contain on their outer membranes, a process called presentation. Killer T cells wander around, like cops, looking at the presentations. When they recognize alien proteins (antigens), they respond by killing the cell (see 06/27/2003 entry, “Cell to Phagocyte: I’m Dying – Eat Me”).
    Now, Dutch scientists Neijssen et al.2 have found that cells in tissues can also pass these flags to neighboring cells through passageways between them called gap junctions. The uninfected neighboring cells thus signal the cops that a firebreak needs to be constructed to avoid further damage. Australian biologists William Heath and Francis Carbone explain:

    “As well as providing another possible mechanism for initiating immunity by dendritic cells, the gap-junction-mediated cross-presentation described by Neijssen et al. offers an interesting method of efficiently limiting the spread of replicating virus. The authors show that not only will a cell expressing viral proteins be killed by T cells, but so will its closest neighbours – because they present viral peptides obtained through gap junctions. Extending the destruction to adjacent cells may provide a ‘fire-break’ around an infection, ensuring that if low levels of virus have spread to surrounding cells, but have yet to produce sufficient protein to allow recognition, such cells will still be eliminated.”

    The width of the firebreak is controlled, they explain: “The rapid degradation of peptides within the cell’s cytosol means that the spread of peptides through gap junctions will be rather limited, probably allowing the targeting of adjacent cells but not those more than one cell distant from the infection. Thus, the integrity of targeting should be maintained, with only limited bystander destruction.”
    1Heath and Carbone, “Coupling and cross-presentation, Nature 434, 27 - 28 (03 March 2005); doi:10.1038/434027a
    2Neijssen et al., “Cross-presentation by intercellular peptide transfer through gap junctions,” Nature 434, 83 - 88 (03 March 2005); doi:10.1038/nature03290.

    Bacterial Engineering On Par With Higher Life 03/11/2005
    Bacteria aren’t the simple life-forms microbiologists used to envision, writes Zemer Gitai in Cell.1

    “Recent advances have demonstrated that bacterial cells have an exquisitely organized and dynamic subcellular architecture. Like their eukaryotic counterparts, bacteria employ a full complement of cytoskeletal proteins, localize proteins and DNA to specific subcellular addresses at specific times, and use intercellular signaling to coordinate multicellular events. The striking conceptual and molecular similarities between prokaryotic and eukaryotic cell biology thus make bacteria powerful model systems for studying fundamental cellular questions.”

    This is different from the traditional picture of bacteria, he elaborates:

    “This traditional perspective [of bacteria as fundamentally different from eukaryotes (i.e., simpler] changed significantly in the past decade with dramatic advances in our understanding of bacterial cell biology. Work in multiple species has demonstrated that bacteria are actually highly ordered and dynamic cells. Much like their eukaryotic counterparts, bacterial cells are capable of polarizing, differentiating into different cell types, and signaling to each other to coordinate multicellular actions. The more recent surprises come from advances in fluorescence microscopy, demonstrating that bacterial cells exhibit a high level of intracellular organization. Bacteria dynamically localize proteins, DNA, and lipids to reproducible addresses within the cell and use this dynamic organization to tightly regulate complex cellular events in both space and time.”

    Gitai provides detail on the following examples: (1) Bacteria have homologs of the eukaryotic cytoskeleton, (2) bacterial cells are subcellularly organized (i.e., are not lacking organelles or a nucleus-like function), (3) several mechanisms underlie bacterial subcellular organization, and (4) bacteria are able to engage in multicellular activities.
    “Bacteria are wondrously diverse and resourceful, occupying virtually every environmental niche imaginable,” he writes in conclusion.
    1Zemer Gitai, “The New Bacterial Cell Biology: Moving Parts and Subcellular Architecture,” Cell, Volume 120, Issue 5, 11 March 2005, Pages 577-586, doi:10.1016/j.cell.2005.02.026.

    Flagellum Described in High-Performance Lingo 04/04/2005
    The bacterial flagellum, a virtual icon of the intelligent design movement, has been studied by many researchers, notably Howard Berg of Harvard, an expert on chemotaxis (the attraction of bacteria to chemical stimuli). Berg was interviewed in Current Biology1 and talked like a race car mechanic when discussing this molecular machine, though he is not involved in the ID movement and believes in evolution. Here are some excerpts:

    • “The modern era [of chemotaxis studies] began in the 1960s with Tetsuo Iino and Sho Asakura in Mishima and Nagoya, who began work on the structure of flagellar filaments (thought then to be primitive bending machines)...
    • the flagellar motor has several pistons and a novel torque-speed relationship....
    • We hope to understand how bacterial chemotaxis works, every nut and bolt. Who would have imagined: receptor complexes that count molecules and make temporal comparisons; activation of a diffusible signal that couples receptors to flagella; reversible rotary engines that drive propellers of variable pitch; force generators, rotors, drive shafts, bushings, and universal joints; a system with prodigious sensitivity, with amplification generated by receptor-receptor interactions? The biggest black box is the motor. We know a great deal about its electromotive and mechanical properties (torque, speed, changes in direction, and so forth) but we do not really know how it works. We need more structural information. This is hard, because essential components are membrane embedded. But even in an age of systems biology, one should not be embarrassed to focus on an isolated network controlling a particular molecular machine.”
    1Q&A: Howard Berg, Current Biology, Volume 15, Issue 6, 29 March 2005, Pages R189-R190, doi:10.1016/j.cub.2005.03.003.

    Molecular Motors Do Ballet 04/13/2005
    Scientists at University of Illinois studied dynein and kinesin – the tiny molecular trucks that ferry cargo inside the living cell – and found that they are not just individualists: they cooperate in a delicate yet effective performance.
    Some scientists had thought that the two machine types, which travel in opposite directions, were involved in a constant tug-o’war with each other. Instead, reports the university’s news bureau, “The motors cooperate in a delicate choreography of steps.”
    Using high-speed imaging techniques, they determined that “multiple motors can work in concert, producing more than 10 times the speed of individual motors measured outside the cell.” The machines move by “walking” on rails called microtubules in steps 8 billionths of a meter at a time. The team is measuring the force produced by the motion to “further understand these marvelous little machines.” There was no mention of evolution in the report.

    Bacterial Hydrogen Fuel Cell May Yield Cleaner World 04/24/2005
    Scientists at Penn State are working on a new, improved fuel cell. It’s secret? Bacteria that can be coaxed with a little electricity to produce “four times as much hydrogen directly out of biomass than can be generated typically by fermentation alone.” Will you someday be able to harness hydrogen from organic waste to drive your car? Their new electrically-assisted microbial fuel cell can theoretically be used to “obtain high yields of hydrogen from any biodegradable, dissolved, organic matter – human, agricultural or industrial wastewater, for example – and simultaneously clean the wastewater.”

    Genes Must Be Expressed in the Right Order 04/26/2005
    A team of scientists in Switzerland made neural cells switch on a transcription factor earlier during the embryo’s development. The result? Axons (long branches of nerve cells) refused to grow to the spinal cord and to the peripheral target. To the mice, this meant they couldn’t feel things on the skin due to stunted nerves. The paper is published in PLOS Biology. A synopsis of this paper in the same issue (published April 26) explains why the order of expression is important:

    “Building an embryo is like building a house: everything has to be done at the right time and the right place if the plans are to be translated faithfully. On the building site, if the roofer comes along before the bricklayer has finished, the result may be a bungalow instead of a two-story residence. In the embryo, if the neurons, for example, start to make connections prematurely, the resultant animal may lack feeling in its skin.
    On the building site, the project manager passes messages to the subcontractors, and they tell the laborers what to do and where. In the embryo, the expression of specific transcription factors (molecules that tell the cell which DNA sequences to convert into proteins) at different stages of development and in different places controls the orderly construction of the body.”
    1 Hippenmeyer, Arber et al., “A Developmental Switch in the Response of DRG Neurons to ETS Transcription Factor Signaling,”, [/i]Public Library of Science Biology[/i] Volume 3 | Issue 5 | May 2005, DOI: 10.1371/journal.pbio.0030159.

    World’s Smallest Rotary Motors Coming Into Focus 04/30/2005
    Science April 29 had three articles on the ATP synthase rotary motors that inhabit all living cells.1,2,3 Using creative techniques of extreme microscopy and crystallography, research teams are beginning to get more focused images of the carousel-like rotating engines of both F-type and V-type motors. (V-type enzymes pump ions into the cell to regulate acidity; see 2/24/2003 entry. F-type ATP synthase enzymes produce ATP, the energy currency of the cell; see 09/18/2003 entry.)
    The rotors look like elegant circular rings of helical units arranged at angles to the axis. From the side, they look like “concave barrel with a pronounced waist in the middle, and an inner septum that is probably filled with and electrically sealed by membrane lipids in vivo.” Scientists are still trying to figure out how the ions get into the active-site pockets in the subunits of the ring, and how they create torque to make the carousel go round. It may result from harnessing Brownian motion in a ratcheting manner that only allows rotation in one direction. All the researchers seem surprised that the gear ratio is not an integer, but rather 10:3 in some species, and 11:3 or 14:3 in others; it may be necessary that these motors have a non-integer ratio between the bottom carousel and the top catalytic engine for torque generation and catalytic activity (see 08/10/2004 entry). They are also beginning to understand the nature of the camshaft attached to the carousel that induces ATP production in the top.
    Whatever their mechanism, these little engines, only 12 nanometers tall, are effective. The review by Junge and Nelson says these motors can generate an acidity of pH 2 in lemons and 250 millivolts of electricity in insect guts. We humans also run on electricity. The constant action of quadrillions of these tiny generators running day and night in our bodies keeps all our energy systems humming at about 116 watts (see 02/05/2003 story).
    In another molecular-motor story, Current Biology4 reported about how actin and myosin work during cell division to pinch the two daughter cells apart. David R. Burgess in a review5 states, “Myosin II is the motor for cytokinesis, an event at the end of cell division during which the animal cell uses a contractile ring to pinch itself in half. New and surprising research shows that myosin, either through light chain phosphorylation or through its ATPase activity, also plays an important role in both the assembly and disassembly of the actin contractile ring.”
    1Wolfgang Junge and Nathan Nelson, “Structural Biology: Nature’s Rotary Electromotors,” Science Vol 308, Issue 5722, 642-644 , 29 April 2005, [DOI: 10.1126/science.1112617].
    2Murata et al., “Structure of the Rotor of the V-Type Na+-ATPase from Enterococcus hirae,” Science, Vol 308, Issue 5722, 654-659, 29 April 2005, [DOI: 10.1126/science.1110064].
    3Meier et al., “Structure of the Rotor Ring of F-Type Na+-ATPase from Ilyobacter tartaricus,” Science, Vol 308, Issue 5722, 659-662 , 29 April 2005, [DOI: 10.1126/science.1111199].
    4E. D. Salmon, “Microtubules: A Ring for the Depolymerization Motor,” Current Biology, Volume 15, Issue 8, 26 April 2005, Pages R299-R302, doi:10.1016/j.cub.2005.04.005.
    5David R. Burgess, “Cytokinesis: New roles for myosin,” Current Biology, Volume 15, Issue 8, 26 April 2005, Pages R310-R311, doi:10.1016/j.cub.2005.04.008.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Can Gene Duplication Promote Evolution? 05/15/2005
    Imagine you had no mouth but needed to eat. A hamburger comes flying at you. When it hits your body, your skin folds around it and pinches off, sealing it inside. Dozens of 3-armed parts form a geodesic dome around it and carry it to the stomach. Once delivered, all the parts are recycled for the incoming freedom fries.
    If this sounds bizarre, it’s kind of what really happens in your cells. Except for specialized channels that accept particular molecules, like water (12/20/2001 and salt (01/17/2002), a cell has no mouth; it is surrounded by a continuous membrane. When large nutrients need to get in, the membrane has acceptors on the outside that signal a cascade of events. The membrane dents inward and envelops the particle. On the inside, proteins called clathrins form a geodesic structure around the incoming vesicle as the membrane pinches off and seals the contents inside. Other proteins and enzymes stand at the ready to deliver the nutrient where needed. This process goes on continually and is called endocytosis. A press release from the University of Queensland says the cell eats its entire skin every 30 minutes.
    Progress continues to be made understanding clathrin-mediated endocytosis since our 10/07/2003 entry, but the evolutionary origin of this elegant system seems illusory. UC and Stanford biochemists writing in PNAS1 noted that two forms of clathrin are so different, being coded by different genes, they must have had separate evolutionary histories. They propose this happened during gene duplication events up to 600 million years ago.
    Andreas Wagner, however, publishing in Molecular Biology and Evolution,2 casts doubt on that method of evolutionary change:

    “I here estimate the energy cost of changes in gene expression for several thousand genes in the yeast Saccharomyces cerevisiae. A doubling of gene expression, as it occurs in a gene duplication event, is significantly selected against for all genes for which expression data is available. It carries a median selective disadvantage of s > 10?5, several times greater than the selection coefficient s = 1.47 x 10?7 below which genetic drift dominates a mutant’s fate. When considered separately, increases in messenger RNA expression or protein expression by more than a factor 2 also have significant energy costs for most genes. This means that the evolution of transcription and translation rates is not an evolutionarily neutral process. They are under active selection opposing them. My estimates are based on genome-scale information of gene expression in the yeast S. cerevisiae as well as information on the energy cost of biosynthesizing amino acids and nucleotides.”

    Whatever the origin of clathrin, its reputation as a versatile molecule is growing. In the April 28 issue of Nature,3 three Cambridge biologists wondered what it does when endocytosis is halted during cell division. They discovered that clathrin has another essential job:

    “Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia [complete 3-part clathrin proteins] but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin.”

    This is not just an incidental task for clathrin to do till cell division is over. “The importance of clathrin for normal mitosis,” they say, “may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.”
    1Wakeham et al., “Clathrin heavy and light chain isoforms originated by independent mechanisms of gene duplication during chordate evolution,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0502058102, published online before print May 9, 2005.
    2Andreas Wagner, “Energy Constraints on the Evolution of Gene Expression,” Molecular Biology and Evolution, 2005 22(6):1365-1374; doi:10.1093/molbev/msi126.
    3Royle et al., “Clathrin is required for the function of the mitotic spindle,” Nature 434, 1152-1157 (28 April 2005) | doi: 10.1038/nature03502.

    Rotary Clock Discovered in Bacteria 05/17/2005
    What could be more mechanical than a mechanical clock? A biochemist has discovered one in the simplest of organisms, one-celled cyanobacteria. Examining the three complex protein components of its circadian clock, he thinks he has hit on a model that explains its structure and function: it rotates to keep time. Though it keeps good time, this clock is only about 10 billionths of a meter tall.
    Scientists have known the parts of the cyanobacterial clock. They are named KaiA, KaiB, and KaiC. Jimin Wang of the Department of Molecular Biophysics and Biochemistry at Yale, publishing in Structure,1 has found an elegant solution to how the parts interact. He was inspired by the similarity of these parts to those in ATP synthase (see 04/30/2005 entry), a universal enzyme known as a rotary motor. Though structurally different, the Kai proteins appear to operate as another rotary motor – this time, a clock.
    We learned last time (see 09/15/2004 entry) that the parts interact in some way in sync with the diurnal cycle, but the mechanism was still a “black box.” Wang found that the KaiC part, a six-sided hexagonal cylinder, has a central cavity where the KaiA part can fit when it undergoes an “activation” that changes its shape, somewhat like unfolding scissors. Like a key, it fits into the central shaft and turns. The KaiB part, like a wing nut, fastens on KaiB at the bottom of the KaiC carousel. For every 120° turn of the spindle, phosphate groups attach to the outside of the carousel, till KaiC is fully saturated, or phosphorylated. This apparently happens to multiple Kai complexes during the night.
    How does this keep time? When unphosphorylated, KaiC affects the expression of genes. During the night, when complexed with the other two parts, it is repressed from acting, effectively shutting down the cell for the night. Apparently many of these complexes form and dissociate each cycle. As the complexes break up in the morning, expression resumes, and the cell wakes up. When KaiC separates from the other parts, it is destroyed, stopping its repression of genes and stimulating the creation of more KaiC. “In summary,” he says, “the Kai complexes are a rotary clock for phosphorylation, which sets the destruction pace of the night-dominant Kai complexes and timely releases KaiA.” The system sets up a day-night oscillation feedback loop that allows the bacterium to keep in sync with the time of day.
    Wang shares the surprise that a bacterium could have a clock that persists longer than the cell-division cycle. This means that the act of cell division does not break the clock:

    “The discovery of a bacterial clock unexpectedly breaks the paradigm of biological clocks, because rapid cell division and chromosome duplication in bacteria occur within one circadian period (Kondo et al., 1994 and Kondo et al., 1997). In fact, these cyanobacterial oscillators in individual cells have a strong temporal stability with a correlation time of several months.”

    Wang’s article has elegant diagrams of the parts and how they precisely fit together. In his model, the KaiC carousel resembles the hexagonal F1 motor of ATP synthase, and the KaiA “key” that fits into the central shaft resembles the camshaft. KaiB, in turn, acts like the inhibitor in ATP synthase. “The close relationship between the two systems may well extend beyond their structural similarity,” he suggests in conclusion, “because the rhythmic photosynthesis-dependent ATP generation is an important process under the Kai circadian regulation.”
    1Jimin Wang, “Recent Cyanobacterial Kai Protein Structures Suggest a Rotary Clock,” Structure, Volume 13, Issue 5, May 2005, Pages 735-741, doi:10.1016/j.str.2005.02.011.

    Design Language Gushes Out of Article Describing Cell Quality Control 05/18/2005
    Here are the design words found in a press release from Michigan State describing the editing mechanisms of the cell DNA-to-RNA transcription process: high fidelity, quality control, inner workings, genetic coding, exquisite nanotechnology in living systems, genetic control, blueprint for life, industrial assembly line, conveyor belt, preloading, criteria, backs up to correct the error, sensed and corrected, acceptable level of error required for the speed at which cells must reproduce, elegance of cell creation, fidelity mechanism, tried and true design, and enduring design.

    Here are the words in the press release describing the evolution of this system: [null].

    The aspect of transcription that so impressed the researchers was the ability of RNA polymerase (the main transcription machine) to preload bases before need: “Preloading of NTPs [nucleoside triphosphates, the “letters” of RNA code] hints at a previously unknown quality control station to maintain accuracy of RNA synthesis,” the article states (emphasis added in all quotes). “We’re able to show how an error will be sensed and corrected,” said Team member Zachary Burton. “The quality control system checks NTP loading several ways. If it doesn’t match the criteria, it gets booted out.” Details of the research were published in Molecular Cell.1 Another statement by Burton encapsulated the tone of their study: “RNA polymerase is one of nature’s great designs.”
    1Gong et al., “Dynamic Error Correction and Regulation of Downstream Bubble Opening by Human RNA Polymerase II,” Molecular Cell, Volume 18, Issue 4, 13 May 2005, Pages 461-470, doi:10.1016/j.molcel.2005.04.011

    Enzymes Chew Like Pac-Man 06/10/2005
    Evidence is growing that many enzymes have moving parts. They act like scissors, clamps and little pac-mans. When precisely-folded chains of amino acids emerge from the ribosome, they fold into unique shapes with the aid of chaperones. But those shapes are not static globs. They move, say Dmitry A. Kondrashov and George N. Phillips, Jr. (U. of Wisconsin). Writing in Structure,1 they describe some of the “molecular mastication mechanics” of these amazing machines:

    “Computational prediction of global protein motion... suggests that enzymatic active sites tend to be placed near the hinges of the “jaws” of enzyme structures.
    Proteins self-organize into exquisitely precise structures, but the actual conformation of a protein fluctuates, and almost never coincides exactly with the average structure observed via X-ray crystallography or other methods. Mounting evidence suggests that these induced motions play specific and essential roles in protein function....”

    Proteins are so tiny, the motions are very hard to observe. The authors describe the various techniques that try to shed light on “the central question: do these motions contribute to enzyme function?” It appears they do:

    “Stabilization of the transition state relative to the substrate is thought to be the key to enzymatic efficiency. Static effects clearly play a major part via the electrostatic contribution of the positioning of polar residues. The existence of a “dynamic effect,” however, is controversial, specifically the proposition that enzymes can channel thermal vibrational energy into modes co-directional with the reaction coordinate, thus making barrier crossing more likely. Nevertheless, evidence is accreting to indicate a link between well-defined global motions and catalysis.”

    After the technical jargon, they lighten up and explain this for the rest of us with some everyday comparisons:

    “Computation of the normal modes of motion allowed the determination of the “hinges” or pivot points that separate regions of the protein moving in opposite directions, much like the end of a nutcracker. In the vast majority of the enzymes studied, the catalytic residues were found to be located in a predicted hinge region.... This finding contributes a bioinformatic dimension to the field of functional protein dynamics and may allow improved functional annotation for the flood of newly solved protein structures. The results also suggest an enhanced role for the global protein structure, which often has been viewed as a scaffold supporting the active site. The study adds to the growing body of evidence that the fold determines global protein dynamics, suggesting a mechanism for allosteric signal transduction, functional impact of distant mutations, and other effects not explained by the chemistry of the active site. In this view, enzymatic structures resemble a Pac-Man icon, with active sites located in the wedge-shaped opening, and the structure responsible for the “chewing” motion of the “mouth.”

    What this means is that the whole protein – all the amino acids, even those distant from the active site, are involved. It is possible that they contribute to orienting the substrate into the active site and stabilizing it once it makes contact, like a vise grip. Moving parts might also contribute to the release of the substrate after catalysis is complete. The structure might strip off solvents before the substrate reaches the active site, resulting in more efficient catalysis. Even short fragments distant from the hinge might contribute an essential part of the overall function.
    Viewing enzymes as dynamic machines opens up new avenues for investigation, they envision. The specific sequences in all the parts of the enzyme would require closer scrutiny; they might have moving parts as well. At least, it is an idea to chew on, they conclude; “The relative importance of topology and sequence for protein dynamics and function needs to be investigated, in order to add more teeth to the masticating view of enzyme dynamics.”
    1Dmitry A. Kondrashov and George N. Phillips, Jr, “Molecular Mastication Mechanics,” Structure, Volume 13, Issue 6, June 2005, pages 836-837, doi:10.1016/j.str.2005.05.004.

    Cell Wonders Accelerate 06/14/2005
    Scientific papers on cell biology continue to uncover amazing things as techniques improve to peer into the workings of these units of life. Here are our Top Ten from the last few weeks:

    1. Immunity Tunes: A press release from Johns Hopkins talked about how, unlike other cells, immune cells undergo a “dizzying loop of activity” to generate huge varieties of antibodies through recombination. They liken the regulator of the recombination process to a band leader directing a jam session.
    2. Oxygen Sensor: “Cell’s Power Plants Also Sense Low Oxygen” announced a report from Howard Hughes Medical Institute. In summary, “Researchers have produced the strongest evidence yet that mitochondria – the organelles that generate energy to power the cell – also monitor oxygen concentration in the cell. If oxygen slips below a critical threshold, the mitochondrial ‘sensor’ triggers protective responses to promote survival.” Controlling oxygen levels is important. Both too little and too much can be deadly, not only to the cell, but to the whole organism.
    3. Reverse Gear: Nature1 June 9 talked about the myosin monorail trains that ride the microtubule rails. Out of the myosin superfamily of motor proteins, consisting of 18 classes, they were curious how Myosin VI is bidirectional, unlike most of its siblings. They studied its “lever arm,” “power stroke” and “converter” but did not come up with a final model of how it works. “Undoubtedly, this unique myosin family member has yet more surprises to reveal,” they concluded.
    4. Transporters: Aussie biologists talked about protein transport into mitochondrial membranes in [i]Current Biology[/b].2 Since there are two membranes, similar to those in chloroplasts (see 01/01/2005 story), there are two squads of transporters to get the cargo in and out. Named TOM and TIM for translocons of the outer and inner membranes, these are “a series of molecular machines” that know how to sort and authenticate objects needing to pass the gates. They envisioned an “entropic spring” mechanism that can help get the cargo passed through “no apparent input of energy.” This type of mechanism is “an emerging theme in biology” that harnesses the disordered motion of molecules to provide binding flexibility and low energy cost to accomplish “a range of functions.” “The TIM23 complex is a smart machine,” they say, describing its ability to grab a piece of cargo, insert it, respond to a stop-transfer signal and reject it, or pass the cargo to the next machine complex.
    5. Tissue Triage: Another paper in Current Biology3 discussed how epidermal cells repair damage. The phylogeny of this ability was a puzzle: “Amazingly, while the eyes and hearts of Drosophila and mammals are constructed in entirely different ways and are morphologically quite distinct, their development appears to be under the control of similar master-regulatory transcription factors,” they said. These operations on two vastly different types of organisms cannot be homologous, they suggest; they must be due to convergent evolution. However the repair mechanism arose, it involves signaling and a cascade of coordinated events involving molecular machines. The result? A stitch in time, and wounds that are self-healing. This is another “conserved repair response,” they say, meaning that it is found early in the history of life with little change since.
    6. Quality Control: A press release from Yale described a protein that “recognizes misfolded RNAs, creating a RNA quality control system for cells.”
    7. Kissing Chromosomes: A news story in Nature4 sheds light on a mystery of gene regulation. We all know chromosomes come in pairs, but how do the genes on each member get expressed together when they are separated by distance? Out of the “many strategies to orchestrate gene activation or repression” in the cell’s bag of tricks, “A three-dimensional examination of gene regulation suggests that portions from different chromosomes ‘communicate’ with each other, and bring related genes together in the nucleus to coordinate their expression.” It’s nice that the spouses are on speaking terms. “Such inter-chromosomal communication has been suspected for some time,” Dimitris Kioussis said, “but this is the first evidence that it actually takes place.” Our understanding of gene regulation has changed from a linear view “to an appreciation that genes are associated with groups of proteins, forming multimolecular complexes,” he said. We’re going to have to see the process not just in snapshots or just a movie: “Is it time to go 4D?” he jests with implicit seriousness. No one knows how the chromosomes are brought together. “How do genes find their appropriate location in the nucleus of a cell, and how are genes that must be expressed herded into active neighbourhoods?” he asks (see “Spaghetti in a Basketball,” 07/28/2004). Whatever the mechanism, “These remarkable findings will puzzle us for some time to come.”
    8. Inter-Agency Coordination: Cities have fire departments, police departments, ambulances, highway patrol, disaster response teams and other agencies that sometimes have overlapping duties. Cells do, too. There are multiple repair mechanisms able to respond to different kinds of DNA damage. Scientists writing in Molecular Cell5 discussed what is known about how they coordinate their actions during the emergency repair called TLS (trans-lesion DNA synthesis): “The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery.” It sounds like the first-aid squad knows how and when to patch up things enough to get the patient to the surgeon.
    9. Texas Tech: Scientists in Texas, publishing in Cell,6 found another multi-talented molecular machine. The rotor part of the V-type ATP synthase (see 02/24/2004 entry) does more than just help acidify vesicles. It also has “an independent function in membrane fusion,” they found. It is essential in the process of exocytosis – what neurons do to transmit their messages. They found that mutant embryos had severe defects in synaptic transmission of nerve signals. (This was found in fruit flies.) By the way, the other form of this rotary motor, the F-type ATP synthase, was called “The World’s Smallest Wind-Up Toy” by Richard Berry in Current Biology.7 Researchers have figured out how to make the motor turn, using magnets. He thinks scientists are on the verge of figuring out how the F0 rotor converts proton flow into torque.
    10. Ultimate Spa: Last but not least, scientists at the Salk Institute last month announced a surprising solution to the puzzle of how embryos start their left-right orientation. An “embryonic body wash” operated by cilia sweeps chemical signals across the embryo: “the foundations for the basic left-right body plan are laid by a microscopic ‘pump’ on the outer surface of the embryo’s underside that wafts chemical messengers over to the left side of the body. This sets up a chemical concentration gradient that tells stem cells how and where to develop.” The cilia rotate at a precise 40-degree angle to generate a current over the embryo. The original paper in Cell contains movies of the action.

    1Menetrey et al., “The structure of the myosin VI motor reveals the mechanism of directionality reversal,” Nature 435, 779-785 (9 June 2005) | doi: 10.1038/nature03592.
    2Perry and Lithgow, “Protein Targeting: Entropy, Energetics and Modular Machines,” Current Biology, Vol 15, R423-R425, 7 June 2005.
    3Stramer and Martin, “Cell Biology: Master Regulators of Sealing and Healing,” Current Biology, Vol 15, R425-R427, 7 June 2005.
    4Dimitris Kioussis, “Gene regulation: Kissing chromosomes,” Nature 435, 579-580 (2 June 2005) | doi: 10.1038/435579a.
    5Friedberg et al., “Trading Places: How Do DNA Polymerases Switch during Translesion DNA Synthesis?” Molecular Cell, Volume 18, Issue 5, 27 May 2005, Pages 499-505, doi:10.1016/j.molcel.2005.03.032.
    6Heisinger et al., “The v-ATPase V0 Subunit a1 Is Required for a Late Step in Synaptic Vesicle Exocytosis in Drosophila,” Cell, Volume 121, Issue 4, 20 May 2005, Pages 607-620, doi:10.1016/j.cell.2005.03.012.
    7Richard Berry, “ATP Synthesis: The World’s Smallest Wind-Up Toy,” Current Biology, Vol 15, R385-R387, 24 May 2005.

    Small Wonder: Tubulin Visualized Up Close 06/28/2005
    Science Daily printed a neat story about microtubules, complete with a 3D visualization of how the protein components are arranged. They are not just ropes or chains, but complex cylinders of precise parts. Scientists are starting to get an idea of why they continually grow and shrink within the cell. The process allows them to “explore their cellular environment to find their goals,” and is coordinated by numerous genes and protein parts. Microtubules form the cell’s superhighway (see 04/13/2005 and 12/04/2003 entries), and are also critical in cell division for winching chromosomes into the daughter cells (see 04/30/2005 entry).
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    “Junk” Cells Maintain the Brain 07/16/2005
    The most abundant immune cells in your brain are not the neurons, but microglia – spindly cells that were thought to be static and immobile, the smallest of the glia cells that were once considered mere scaffolding to support the more important gray matter (see 11/20/2001 and 01/29/2001 entries). When two scientists recently applied the new technique of two-photon microscopy to a live healthy mammalian brain, however, they were stunned at what they saw the microglia doing... “a static state is hardly what was observed,” reported Science magazine.1. They were the most motile cells in the brain.
    The little cells were observed to act like well-trained, active patrolmen doing a vital job. They extended probes into their environment to monitor the health of the brain, clean up debris and fight microbes. A caption explained:

    “Microglia continually extend ... and retract ... processes, surveying their immediate environment within the brain. The processes move rapidly toward a site of injury, such as a damaged blood vessel in the brain, in response to the localized release of a chemoattractant ... from the injured sited. Once at the target site, the processes form a barrier to protect healthy tissue.”

    Microglia comprise about 10% of cells in the central nervous system. This monitoring and disaster response apparently goes on continually. “These two elegant studies provide direct evidence for the highly dynamic nature of microglia, indicating that the brain is under constant immune surveillance by these cells.” Who knows what we would think without them.
    1Luc Fetler and Sebastian Amigorena, “Brain Under Surveillance: The Microglia Patrol,” Science, Vol 309, Issue 5733, 392-393, 15 July 2005, [DOI: 10.1126/science.1114852].

    Saddle Up Your Algae: Scientists Harness Flagellar Motors 08/19/2005
    1805: Beast of burden of choice: oxen.
    2005: Beast of burden of choice: algae.
    Science Now reported an unusual item: scientists have learned how to hitch their loads to a single-celled green alga named Chlamydomonas reinhardtii (see Yale description). Researchers are actually calling their little teams “micro-oxen.”

    “Scientists are increasingly interested in harnessing biological motors for use in micro- and nanotechnology, but recent research has mainly involved taking moving parts out of cells and adapting them for use elsewhere. It’s a complicated process that can require protein engineering. So, chemist Doug Weibel of Harvard University in Cambridge, Massachusetts, and colleagues wondered if they could simply use an intact organism as a beast of burden instead.”

    This alga contains whiplike flagella that propel them through liquid like motorized paddleboats (see U of Wisconsin description). “These algae are very reliable,” Weibel said. See also the BBC News report.
    In other flagellum news, Howard Berg of Harvard, writing in Current Biology,1 described how bacterial flagella (the rotary kind) receive feedback from the environment: “the flagellum senses wetness,” he reported. The wetness of the environment affects antagonistic regulatory proteins that control flagellum production. Research by Q. Wang et al. found that a suppressor is “pumped out of the cell by the flagellar transport apparatus once assembly of the basal part of the flagellum is complete,” Berg said. What for? “This prevents the cell from wasting energy on flagellin synthesis when this protein cannot be put to use.” The scientists sprinkled a little water on dry colonies for 90 seconds and, sure enough, got them to produce more and longer flagella that exhibited normal swarming behavior. Berg describes it:

    “Swarming is a specialized form of bacterial motility that develops when cells that swim in broth are grown in a rich medium on the surface of moist agar. The cells become multinucleate, elongate, synthesize large numbers of flagella, secrete surfactants and advance across the surface in coordinated packs.”
    1Berg, Howard, “Swarming Motility: It Better Be Wet,” Current Biology, Volume 15, Issue 15, 9 August 2005, Pages R599-R600.

    Molecular Motors Galore: How Did They Evolve? 08/26/2005
    Myosin is one of the cell’s little monorail motors that trucks cargo around the cell, pushes false feet into the surrounding environment, forces packages out the cell membrane, makes muscles move and wiggles hairlike cilia. Scientists reporting in Nature1 found twice as many varieties of myosin (37) than were previously known (17) and decided to plug them into the evolutionary tree of life and figure out how they diversified throughout eukaryotic lineages. Although they found many “synapomorphies” (apparent instances of “convergent evolution”), Richards and Cavalier-Smith think they reduced the diversity of myosins down to three ancestral types. They wrote, “We conclude that the eukaryotic cenancestor (last common ancestor) had a cilium, mitochondria, pseudopodia, and myosins with three contrasting domain combinations and putative functions” (emphasis added in all quotes). They did not elaborate, however, on how these mechanisms and functions arose in the hypothetical single-celled ancestor. Margaret Titus, commenting on this paper in the same issue of Nature,2 said, “Analysis of their sequences in a wide range of organisms reveals an unexpected variety of domains, and provides insights into the nature of the earliest eukaryotes.”
    In another molecular-machine story, three scientists found that the cellular powerhouse motors named ATP synthase come in pairs. Reporting in PNAS,3 they actually photographed pairs of the miniature machines – an incredible feat, considering they are only about 12 nanometers tall – and found them bridged together at 40° angles. They suspect that this arrangement helps in the formation of cristae (curved membranes within the mitochondria) and stabilizes the little rotary engines as they generate ATP: “This complex is assumed to improve the efficiency of ATP synthesis by substrate-product channeling.” The authors did not speculate on the evolution of the motors or of the larger structure that they call an “ATP synthasome complex.” Additional proteins and enzymes, whose functions are as yet unknown, appear to take part in the operation.
    1Thomas A. Richards and Thomas Cavalier-Smith, “Myosin domain evolution and the primary divergence of eukaryotes,” Nature 436, 1113-1118 (25 August 2005) | doi: 10.1038/nature03949
    2Margaret A. Titus, “Evolution: A treasure trove of motors,” Nature 436, 1097-1099 (25 August 2005) | doi: 10.1038/4361097a.

    Multi-Talented Telomerase: 08/31/2005
    Telomerase, the enzyme that keep DNA tips (telomeres) from unraveling, apparently does more than control the aging of a cell. Science Now reports that it also regulates stem cells and spurs cell growth. It can even grow hair on mice.

    Bacterial Parcel Service Discovered 09/14/2005
    Bacteria send letters and parcels to one another. Some of them are love letters, some of them are letter bombs. This amazing packaged system of communication, separate from the mere sending of diffusible chemicals, was described in Nature1 with the title, “Microbiology: Bacterial speech bubbles.” Stephen C. Winans described what is known about bacterial communication:

    “Many bacteria socialize using diffusible signals. But some of these messages are poorly soluble, so how do they move between bacteria? It seems they can be wrapped up in membrane packages instead.”

    He said that two research studies in the same issue of Nature, one on how bacteria talk to their friends, and another on how they attack their enemies, met in an “unexpected convergence.” One type of parcel, for instance, is “released in bubble-like ‘vesicles’ that also contain antibacterial agents and probably toxins aimed at host tissue cells as well.”
    Through this form of packaged communication, a community of microbes engages in “quorum sensing” to detect whether it is alone or surrounded by its own kind or other species. Some genes only turn on when there is a quorum reached. One of these Winans mentioned is bioluminescence – turning on the lights.
    The parcels can contain chemicals, proteins, toxins and other molecules in a lipid envelope. The packaging permits delivery of proteins and chemicals that otherwise might be insoluble. Some bacteria have three separate kinds of signal parcels. The packages form lipid bubbles around them as they emerge from the bacterial membrane. These can merge with a friendly neighbor or, depending on the need of the moment, deliver a toxin to an enemy – a package bomb on the scale of bacteria.
    To work, the system requires multiple parts: the contents, the packaging, the delivery method, and the response to received parcels. Winans did not speculate on how this system might have evolved, other than to say, “Various groups of bacteria use diffusible chemicals to signal to their own kind, and this method of communication seems to have evolved independently several times.”
    1Stephen C. Winans, “Microbiology: Bacterial speech bubbles,” Nature, 437, 330 (15 September 2005) | doi: 10.1038/437330a.

    Cell Has Automatic Jam-Clearing Proofreading Machinery 09/19/2005
    Findings at Rockefeller University have scientists excited. DNA copying machines work on a “sliding clamp” that can hold two repair machines at the same time. One is a low-fidelity repair tool, the other a high-fidelity repair tool. Usually, the high-fidelity one is active, but when it needs a bigger hammer that is perhaps more effective but less accurate, it automatically switches to the other. Here’s how the abstract of the paper in Molecular Cell by Indiani, O’Donnell et al.1 describes it in detail:

    “This report demonstrates that the beta sliding clamp of E. coli binds two different DNA polymerases at the same time. One is the high-fidelity Pol III chromosomal replicase and the other is Pol IV, a low-fidelity lesion bypass Y family polymerase. Further, polymerase switching on the primed template junction is regulated in a fashion that limits the action of the low-fidelity Pol IV. Under conditions that cause Pol III to stall on DNA, Pol IV takes control of the primed template. After the stall is relieved, Pol III rapidly regains control of the primed template junction from Pol IV and retains it while it is moving, becoming resistant to further Pol IV takeover events. These polymerase dynamics within the beta toolbelt complex restrict the action of the error-prone Pol IV to only the area on DNA where it is required.”

    The paper says this is like having a “toolbelt” with different tools depending on the need of the project. Bacteria have five DNA polymerase tools; humans have more. Pol III is like the perfectionist editor that cuts out the typos, but it can stall. Pol IV, like the plumber with a big wrench, isn’t as picayunish about the details but knows how to get the operation flowing again. “The findings by O’Donnell and his colleagues,” the press release explains, “show that, because both polymerases are bound simultaneously to the beta clamp, it can pull either of the polymerases out if its toolbelt as needed.” This apparently forms an automatic switchover mechanism where Pol III has priority. A stall either loosens the grip of Pol III, or triggers a change in the sliding clamp that lets Pol IV intervene for the brute-force repair.
    A paper in Cell2 earlier this month described how multiple parts work together to fix mismatched DNA. Since mismatched bases have serious health consequences, a suite of operations, still poorly understood, checks to detect and correct the error. The paper by Zhang et al. describes part of the process:

    “Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutS-alpha-MutL-alpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.”

    The cover of the issue humorously highlights the problem with a picture of a guy with unmatched socks. Mismatch in DNA is no joke, however; it can lead to cancer and genomic instability.
    1Indiani et al., “A Sliding-Clamp Toolbelt Binds High- and Low-Fidelity DNA Polymerases Simultaneously,” Molecular Cell, Volume 19, Issue 6, 16 September 2005, pages 805-815.
    2Zhang et al., “Reconstitution of 5'-Directed Human Mismatch Repair in a Purified System,” Cell, Volume 122, Issue 5, 9 September 2005, pages 693-705.

    Subway System Found in Immune Cells 09/20/2005
    The announcement of a “third form of intercellular communication” hit scientists like TNT: tunneling nanotubules, that is. Science Now reported that “Scientists have found what appears to be a whole new way for immune cells to communicate with one another: long, narrow tubes that enable them to connect and exchange molecules.” These subway tunnels between cells pass molecules quickly from cell to cell, including calcium ions that trigger actions in the cell, and possibly antigens. If so, this “may help explain how immune responses can be initiated so rapidly.”
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Muscle Motor Observed in Action 10/03/2005
    Myosin proteins have been heavily studied in recent years since they are critical to many cellular and tissue functions, including muscle. According to EurekAlert scientists from the Burnham Institute for Medical Research and the University of Vermont have captured the first 3-dimensional (3D) atomic-resolution images of the motor protein myosin V as it “walks” along trackways made of actin:

    “Myosins are a large family of motor proteins that interact with actin filaments for motor movement and muscle contraction. Myosin V is the workhorse of the myosin protein family. It exists to ferry a cargo of proteins needed in a specific place at a specific time. Fueled by hydrolysis -- the process of converting the molecule adenosine triphosphate (ATP) into energy -- myosin V travels in one direction using actin as a track to deliver its payload of cell vesicles and organelles. Myosin V is also involved in transporting proteins that signal and communicate with other cells.
    Myosin V has a two-chained “tail” that diverges to form two “heads” that bind to specific grooves on actin and walk hand over hand along the track, similar to the way a child moves along the monkey bars in a playground. Myosin V differs from the other myosin family proteins in that it is able to sustain this processive motion, enduring many hydrolysis cycles. The other myosins grab on tightly to actin and release after one hydrolysis cycle.”

    Using 3D electron cryo-microscopy, the Burnham team took snapshots of the action to put together a sequence that allowed them to visualize myosin in its natural state. They were able to see structural changes in the myosin and the actin, including movement of the “lever arm,” the scientists said.
    The tight specs of this molecular machinery were underscored in the press release. “The precise characterization of this myosin-actin interface is critical,” it stated, “evident by the way a single amino acid change in myosin leads to familial hypertrophic cardiomyopathy (FHC), an undetectable condition resulting in death by sudden cardiac arrest in otherwise healthy young adults.”

    Molecular Machine Updates 10/11/2005
    Scientists continue to make headway understanding the detailed workings of molecular motors. The two most famous rotary motors yielded additional secrets recently:

    ATP Synthase: “Making ATP” was the short title of a paper in PNAS this week.1 Xing, Liao and Oster came up with a model that linked the rotation of the gamma subunit (the camshaft) to the beta subunits in the F1 hexamer, where ATP synthesis occurs. They identified two “bumps” in the potential curve that prevent back-slippage of the rotor. The shaft is tightly coupled to the lobes, to produce a kind of “zipping” effect of hydrogen bonds as the beta subunits bend along a hinge during the catalytic function.
    The eta part of the stator is apparently also essential in preventing slippage, in order to couple the energy to the synthesis function. Mutations were shown to flatten the “energy bumps” on the potential curve, making slippage more likely.
    They also noted that in ATP hydrolysis mode (the reverse cycle) ADP tends to get stuck in the mechanism; “this is hardly surprising,” they said, “because F1 evolved to synthesize, and only under laboratory conditions does the eukaryotic F1 operate in hydrolysis mode.” The bacterial ATPase and vacuolar ion pump do operate in hydrolysis mode in vivo and presumably do not have this inhibition problem. Their lingo on this point mixes design and evolution: “The V1 motor of the vacuolar ATPase, being designed for ion pumping, may have avoided ADP inhibition by the evolution of additional subunits”.
    Bacterial Flagellum: A Japanese and UK team publishing in Nature2 found stepping behavior in the flagellar rotor by direct observation. The torque generation by the ion flux may be responsible for the rotation taking place in measurable steps. Their observations “indicate a small change in free energy per step, similar to that of a single ion transit.” They mentioned that this had been seen in ATP synthase, but never before in the bacterial flagellum. They measured about 26 discrete steps per revolution. There was no mention of evolution in the paper.
    Type III Secretion System (TTSS): The TTSS, a kind of molecular syringe embedded in the membrane of some bacteria that allows them to inject toxins in nearby hosts, was also described more fully in the same issue of Nature by two Yale scientists.3 They found that the protein ordnance is too large, so there are special chaperones on hand to unfold them before loading them into the barrel.

    1Xing, Liao and Oster, “Making ATP,” Proceedings of the National Academy of Sciences USA, published online before print October 10, 2005, 10.1073/pnas.0507207102.
    2Sowa et al., “Direct observation of steps in rotation of the bacterial flagellar motor,” Nature 437, 916-919 (6 October 2005) | doi: 10.1038/nature04003.
    3Akeda and Galan, “Chaperone release and unfolding of substrates in type III secretion,” Nature 437, 911-915 (6 October 2005) | doi: 10.1038/nature03992. See also the News and Views section by Blaylock and Schneewind, “Microbiology: Loading the type III cannon,” Nature 437, 821 (6 October 2005) | doi: 10.1038/437821a.

    Cellular Black Box Reveals Precision Guidance and Control 10/27/2005
    Amazing discoveries about the cell are being made each week. It’s a shame more people don’t hear about them. They are usually written up in obscure journals with incomprehensible jargon, but when explained in plain English, the findings are truly astounding. Not long ago, the cell was a “black box,” a mechanism of unknown inner workings that somehow survived and reproduced. Only recently have imaging techniques allowed us to peer inside the box at the nanometer scale (one nanometer is a billionth of a meter) and see what is going on. Prepare to be astonished.
    A fundamental shift in thinking about cellular processes has occurred since the structure of DNA was elucidated in the 1950s, and has been accelerating ever since. What used to be mere chemistry is now mechanics; what used to be imagined as fluids mixing in a watery balloon is now programmed robotic machinery. Cells don’t just perform chemical reactions like we did in high school, pouring mixtures together and seeing if they explode or not. It’s more like robotics, and is properly known these days as “biophysics.” Cells are not just tossing ingredients together, but guiding them into place with motors, pivots, guardrails and inspectors.5 The cell is engaged in precision manufacture with molecular machines and motorized transport. The coolness factor of these molecule-sized gadgets would blow away any competition in Popular Mechanics if they could be appropriately visualized and described. Let’s try with some recent examples.

    1. tRNA: Guided Trackways: A paper in PNAS1 took five pages describing one tiny segment of the DNA translation process: the moment when transfer RNA (tRNA) enters the inner sanctum of the protein-building machine, the ribosome (see also summary on Science Now). If you have seen the animations in the film Unlocking the Mystery of Life, you probably remember the climactic scene of tRNAs lining up in assembly-line fashion as their attached amino acids are fastened together. Stunning as that animation was, it was vastly oversimplified. The ribosome actually contains a precisely-molded entrance tunnel where each tRNA is inspected and guided into place before allowed into the active site. Each tiny movement along the track is authenticated by contacts with specific atoms at checkpoints along the way. A Los Alamos team achieved the highest-resolution images yet of this process and found that parts of the tRNA and the tunnel turnstiles actually flex as much as 20° as part of the guided entrance, called accommodation. Their diagrams show multiple precision contacts all along the four specific stages of accommodation they investigated. Whether able to follow their dense jargon-laden description or not, the reader is sure to get the sense that something incredibly precise is going on. And then to learn that it all takes place in two nanoseconds is almost too much to handle.
    2. DNA Copying: Tight Fit: Another paper in PNAS2 explored the fit of DNA bases in the copying machinery at the sub-angstrom level (an angstrom is 10-10 meter, about the width of a hydrogen atom). Stanford and MIT scientists investigated how thymine fits into DNA Polymerase I as the genetic code is transcribed. As in the tRNA case above, the fit is precise and guided. They were surprised to find a little bit of margin inside the active site, which they speculated might “allow for an evolutionarily advantageous mutation rate.” Nevertheless, their “results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity.” The tight fit ensures that illegal interlopers cannot make it into the active site. They also found that simple Watson-Crick base-pairing was not sufficient: the machines actually force the bases together in a coordinated way with error-checking. They remarked that this authentication and guidance system is speedy: “This choice, which occurs dozens of times per second, involves the selection of one nucleotide among four for insertion into the growing primer strand, opposite each DNA template base as it is addressed in turn.”
    3. Unzipping Acrobatics: A paper in Nature3 investigated helicases, the molecular machines that unwind and unzip DNA strands. “Helicase enzymes can move along DNA or RNA, unraveling the helices as they go,” said Eckhard Jankowsky in an analysis of this paper in the same issue.4 “But simply traveling along a nucleic acid in one direction seems not to be enough for some of these molecular motors.” They discussed how helicase repeatedly bends over, forms loops, and snaps back into position during the operation. These acrobatic machines don’t just plod along in one direction but undergo a complex choreography with moving parts as they consume ATP for energy. The “repetitive shuttling” the authors described has a purpose, possibly for “keeping the DNA clear of toxic recombination intermediates.”
    4. Cellular Oarsmen: Three German researchers imaged eukaryotic flagella with twice the resolution of previous attempts. The whiplike propellers, which beat with back-and-forth motion (unlike the rotary flagellar motors of bacteria), contain a 9+2 arrangement of microtubules that are tied together with motors and spokes. “Both the material associated with the central pair of microtubules and the radial spokes display a plane of symmetry that helps to explain the planar beat pattern of these flagella,” they wrote. Their paper in PNAS6 includes a stereo pair image that provides a 3D look down the flagellum shaft.

    The literature is filled with examples like these. They usually say little or nothing about how these machines evolved; in fact, more often, they are likely to mention that the machines are “highly conserved” (i.e., unevolved) between the simplest one-celled organisms and humans.
    Though the articles valiantly attempt to describe what happens at these submicroscopic levels, the subject matter would greatly benefit from top-notch animation. Microscopic imaging technology keeps improving, though; some day soon, it may be possible for scientists to watch the machinery of the cell at its own nanometer scale in real time.
    1Sanbonmatsu et al., “Simulating movement of tRNA into the ribosome during decoding,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0503456102, published online before print October 25, 2005.
    2Kim et al., “Probing the active site tightness of DNA polymerase in subangstrom increments,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0505113102, published online before print October 25, 2005.
    3Myong et al., “Repetitive shuttling of a motor protein on DNA,” Nature 437, 1321-1325 (27 October 2005) | doi: 10.1038/nature04049.
    4Eckhard Jankowsky, “Biophysics: Helicase snaps back,” Nature 437, 1245 (27 October 2005) | doi: 10.1038/4371245a.
    5This is not to say that biomolecular machinery looks like human machinery. Straight lines and geometric shapes are rare; tRNA entering a ribosome looks like spaghetti in a blender to an untrained eye. In addition, at the nanometer scale, molecules are subject to the random vibrations of Brownian motion. It has taken decades of careful research to tease out the order and intricacy of the cell’s moving parts. Nevertheless, the language of motors and machines in the literature is apt and ubiquitous, as is the language of physics (piconewtons of force, thermodynamics, translational motion in nm/s and rotational motion in Hz or rps). Human engineers are trying to emulate some of these machines in the new science of nanotechnology.
    6Nicastro et al., “3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0508274102, published online before print October 24, 2005.

    Red Blood Cells Are Master Contortionists 10/28/2005
    Biophysicists have analyzed why red blood cells are able to squeeze through tight spaces on their journeys through our tissues, reports the UCSD Jacobs School of Engineering. Their membranes contain a network of 33,000 hexagons arranged in a complex geodesic dome formation. Each hexagon vertex is joined with flexible lines to a central maypole-like proto-filament, giving it the ability to twist and contort without breaking. This contortionist ability serves another purpose beyond just enabling the cell to get through tight spaces: it also helps squeeze out the oxygen into the tissues. Despite being twisted, folded, flattened or stretched, the geodesic structure permits the cell to pop back into its familiar biconcave shape.
    The press release states, “Their paper in Annals of Biomedical Engineering uses aeronautical terms commonly used to describe the changing position of an airplane to explain how the six attached spectrin fibers make a proto-filament swivel and flip.” Science Now took note of this study on “bendable blood.”
    Last edited by bob b; October 28th, 2006 at 01:48 PM.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Bacterial Flagellum Visualized 11/02/2005
    Another visualization of the bacterial flagellum, the “poster child of the ID movement,” by Japanese researchers on NanoNet, the Nanotechnology Researchers Network Center of Japan. The 02/05/2004 NanoNet Bulletin features the bacterial flagellum with still images from a stunning movie they made, A Rotary Nanomachine, downloadable from the site. The movie contains crisp animations of the flagellar motor at work and features amazing facts about how the propeller is assembled, molecule by molecule, at the growing tip. The film (34 minutes, 36mb) also tells the story of how challenging it was for the team to image the nanometer-scale parts of the system.
    Another issue, 09/16/2004 NanoNet Bulletin tells how professor Masasuke Yoshido first visualized the rotation of another biomolecular rotary motor, ATP synthase. The entire website is concerned with nanotechnology, and many of the articles blur the distinction between biological and artificial machines.

    Living Wonders at a Glance 11/04/2005
    Here is an assortment of recently-reported biological marvels at the cellular level. Researchers into creation and evolution explanations may wish to delve into these more deeply.

    1. Clock Conductor: The brain is a “time machine,” reports EurekAlert on research at Duke University about the human biological clock. Each structure in the brain has a resonant frequency of oscillations, like the ticking of a clock. How do they get coordinated? Think of the tune-up at the beginning of a concert, says Catalin Buhusi of Duke: “It’s like a conductor who listens to the orchestra, which is composed of individual musicians. Then, with the beat of his baton, the conductor synchronizes the orchestra so that listeners hear a coordinated sound.”
    2. Molecular Scissors: MicroRNAs (miRNA) have been implicated in recent years with the regulation of genes (09/08/2005), including silencing genes that need to be slowed down or stopped. EurekAlert reported on work by Wistar Institute that detected a “molecular scissors”action involving three independent parts: “The two enzymes in the complex are like two scissors working together in a concerted fashion, connected and coordinated by the third member of the complex,” said Ramin Shiekhattar. The activity apparently occurs without any expenditure of ATP energy.
    3. Nerve Code: Scientists at Howard Hughes Medical Institute were surprised at an unexpected discovery: neuron development follows a code – “an organized relationship between Hox proteins, their chromosomal organization, and the differentiation and connectivity of motor neuron pools.” The discovery of a combinatorial code, which governs three levels of motor neuron organization, “shows how the nervous system can generate the huge diversity of neurons necessary for a complex task like locomotion.” Song and Pfaff of the Salk Institute reported on this surprising find in Cell, titling their article, “Hox Genes: The Instructors Working at Motor Pools.”
    4. Sprinting Motor: Like a sprinter crouching at the block before sprinting, kinesin stores up energy before its 7.8 nanometer leaps, reported Fisher and Kim in PNAS last month. And like a strong sprinter, it’s not a pushover: “sideways lurching is not supported.”
    5. Give Me Iron, or Give Me Death: Taylor et al., writing in PNAS, studied the structure of a yeast enzyme named Fet3p essential to oxidizing both iron and copper. The regulation of these metal ions is essential; Taylor et al. said, “Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death.”
    6. Gecko Rain Dance: Geckos have a billion spatula-shaped structures at the ends of the hairs on their feet that allow them to “adhere to nearly all surface topographies.” Huber et al. in PNAS explored the capillary action on a single spatula and found that “humidity contributes significantly to gecko adhesion on a nanoscopic level.” They were interested in learning about gecko feet “for the development of artificial biomimetic attachment systems.”
    7. Packaging into the Cell: Some cargoes get wrapped in membrane and are delivered right through the cell exterior; this is called clathrin-mediated endocytosis. Kaksonen, Toret and Drubin at UC Berkeley found that “four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis.” The clathrin itself (an interesting three-pronged protein that forms geodesic structures around the vesicle) “facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation.” The work was reported in Cell; see also EurekAlert summary.
    8. Not Just a Recycle Bin: The proteasome is getting more respect. This “large multiprotein complex” is critical to the degradation of proteins tagged for recycling. Baker and Grant reported in Cell that the proteasome was found involved in gene activation, adding to a “growing body of evidence indicating that the proteasome has nonproteolytic functions.”
    9. Sharper Image: Peter Moore in Science was glad about the “ribosomal coup” performed by Schuwirth et al. in the same issue, who imaged the bacterial ribosome at 3.5 angstrom resolution. This molecular machine, the protein assembly factory, has moving parts. Moore said, “The two subunits of the ribosome not only communicate during protein synthesis, they also engage in coordinated, relative motions.”
    10. Bacterial Centipedes: Did you know that bacteria can walk? They project little feet called pili that adhere to surfaces; as the bacteria retract them, they pull the bacteria along in a crawling motion. Researchers at UC Berkeley reporting in Science found a signaling molecule that they watched traveling from one end of the bacterium to the other when the organism needed to change directions. They figured that this enzyme, FrzS, constituted a chemosensory system that hops onto the intracellular highway and orchestrates the formation of the pili.
    11. Mr. Peabody Gains Respect: Little specks called P-bodies near the nucleus never had so much limelight. Jean Marx, writing in Science, told how scientists used to think they were just trash cans for used messenger RNAs (mRNA), a dead-end job. Now, it appears that these “tiny speckles at the heart of the cell’s machinery” are active, critical players in the regulation of protein synthesis. They act like routers, holding onto mRNA transcripts while deciding which get used or recycled. Are they important? When they go awry, cancer and autoimmune diseases can result.
    12. Bees Under the Floodlights: Humans can distinguish red, yellow and other colors under different lighting conditions, an ability called color constancy. Bees have this talent, too. To prove that weird lighting in a natural setting doesn’t throw them off, two London scientists put bumblebees in a specially-lit chamber. All the flowers had black backgrounds, and four colored lights could alter the ambience. They found that “bees can generate color-constant behavior by encoding empirically significant contrast relationships between statistically dependent, but visually distinct, stimulus elements of scenes” – spoken like a scientist, but the bees get the applause.

    These 12 brief glimpses at recent science literature hint at the stream of discoveries being made that uncover more and more complexity and coordinated design.

    Scientists Learning How to Harness Cellular Trucks 11/15/2005
    In an article that blurs the line between biology and technology, a press release from the Max Planck Institute (see EurekAlert for English translation) described the amazing performance of the nanoscopic trucks that ride the cell’s microtubule superhighways. Kinesin and myosin motors, fueled by ATP, usually “sprint” on the trackways for short distances, but working in concert like a relay team, can run marathons for centimeters or even a meter. This is especially important in neurons, some of which can have axons a meter long – in our spinal cord. The scientists are learning as much as they can about these molecular motors in order to harness the technology for directed chemical reactions and biomimetic applications. The Energizer Bunny would face stiff competition on this scale: the article comments, “in contrast to human sprinters, molecular motors don’t get tired.”

    Cell Ribosome Assembly Is Like Throwing Car Parts Together 12/01/2005
    Ribosomes are the protein-assembly machines in the living cell (11/24/2005, 07/26/2005, 01/19/2005). A bacterium can have thousands of them. They are composed of two large RNA complexes; the smaller one has 20 unique proteins that fit snugly in various parts of the apparatus, and the larger complex has even more. How do the parts all come together? That’s an area of intense study, reports Sarah A. Woodson in Nature:1

    “Many of the biochemical events that occur in a cell are performed by huge complexes of proteins and nucleic acids. A cunning approach promises to show how the components convene to make a functioning ‘machine’.
    The cell’s macromolecular machines contain dozens or even hundreds of components. But unlike man-made machines, which are built on assembly lines, these cellular machines assemble spontaneously from their protein and nucleic-acid components. It is as though cars could be manufactured by merely tumbling their parts onto the factory floor.”

    Clearly there is more to it than that, because the parts all fit together in the right places, at the right times. Woodson describes how researchers are trying to observe whether the assembly steps are strictly determined in a predefined sequence, or whether the parts can arrive via alternative paths, like band members in a scatter formation.
    Whatever happens, it needs to be reliable and energy-efficient. All the parts “interact through highly specific interfaces,...” she explains. “Actively growing cells demand many thousands of ribosomes, whose synthesis consumes a large fraction of the cell’s metabolic energy. So ribosome assembly must be efficient as well as precise.”
    Unlike car parts, protein and RNA parts have some flexibility. In a process called induced fit, they snap together snugly, like rubbery puzzle pieces:

    “In the soft world of biological materials, cooperativity and specificity are achieved by the induced fit of molecular interfaces; that is, as two or more components come into contact they mould around one another to create stronger, more specific junctions. The idea that ribosome assembly can follow more than one path is consistent with redundant cooperative linkages in the assembly map. These cooperative linkages ensure that individual complexes are assembled completely. They also create alternative kinetic paths that make the assembly process itself more robust.”

    Woodson spoke of machinery and machines five times, but only mentioned evolution twice, neither time explaining how the machinery and its precision assembly process came about. In her introduction, she merely said, “Knowing how cellular complexes organize themselves is crucial for understanding molecular evolution and for engineering materials that can mimic their properties.” The other mention of evolution was in her last sentence: “In the ribosome, these interactions have been fine-tuned through billions of years of evolution, providing a clear window into the world of cellular machines.”
    1Sarah A. Woodson, “Biophysics: Assembly line inspection,” Nature 438, 566-567 (1 December 2005) | doi:10.1038/438566a.

    Micro-RNAs are Cell’s Optimizers 12/12/2005
    “Unnoticed next to the main ingredients, microRNAs were considered to be ‘junk’ DNA, leftovers from millions of years of evolution.” That line comes from an article on EurekAlert telling about how dramatically that picture has changed. RNA molecules are now seen to be indispensable, with many roles in the cell. This article talked about how a certain microRNA has a “fail-safe” role in development, preventing birth defects. Researchers at the University of Florida found microRNA that acts “as protective mechanisms in healthy development not just by strategically turning off gene activity, but by making sure it stays turned off.” This is one way a hind limb is prevented from turning on genes that are only supposed to be expressed in the forelimb.
    Another article on EurekAlert claimed that RNAs have “shaped the evolution of the majority of mammalian genes,” but the connection to macroevolution is obscure. What scientists at the Whitehead Institute for Biomedical Research found is that most genes have microRNAs that regulate them. These RNAs don’t just switch them on and off; they finely-tune the expression, to help cells achieve the optimum levels of proteins for the tissues that need them. Many of these microRNAs are “evolutionarily conserved” (i.e., unevolved) from animals as different as humans and chickens. One researcher noted, “Our genomes have good reason to maintain the microRNA targeting sites necessary for turning down these genes at the appropriate place and time.”

    One-Celled Organism’s Spring Generates Enormous Forces 12/13/2005
    The pioneering Dutch microscopist Antony van Leeuwenhoek marveled at the miniature “animalcules” he witnessed darting through the water and spinning like a top. One such marvelous protozoan was Vorticella. The way it rapidly contracted and expanded on its little stalk must have reminded Leeuwenhoek of a spring. It turns out, it is a spring – a remarkable motorized spring made of molecules that generates “enormous forces,” according to a report on EurekAlert. In fact, this little spring sets the speed and power record for cellular nanomachines.
    Researchers presenting their findings at the annual meeting of the American Society for Cell Biology likened the spring to a stretched telephone cord that recoils rapidly – so rapidly, in fact, that size for size, it outperforms human muscles and car engines. The secret is a bundle of contractile fibers called the “spasmoneme” running through the center of the stalk. The researchers looked “under the hood” and found a calcium-fueled engine that uses spasmin, a protein in the centrin family. The exact mechanism of this engine is poorly understood, but scientists hope that by learning about it they can some day build nanomolecular machines of exquisite power and efficiency.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Minimal Cell More Complex Than Expected 01/03/2006
    Craig Venter’s lab has been working on an interesting project in theoretical biology: what is the minimum set of genes needed for life? They have taken one of the simplest organisms, Mycoplasma genitalium, and knocked out genes to see which ones are essential and which are nonessential for viability. (This is part of the “top down” approach to understanding the origin of life; the “bottom up” approach, by contrast, tries to build life from scratch). Their latest results, published in PNAS,1 showed a larger number of essential genes – 347 – than their earlier prediction in 1999. That’s 79% of the organism’s inventory.

    “This is a significantly greater number of essential genes than the 265-350 predicted in our previous study of M. genitalium, or in the gene knockout disruption study that identified 279 essential genes in Bacillus subtilis, which is a more conventional bacterium from the same Firmicutes taxon as M. genitalium. Similarly, our finding of 387 essential protein-coding genes greatly exceeds theoretical projections of how many genes comprise a minimal genome such as Mushegian and Koonin’s 256 genes shared by both H. influenzae and M. genitalium, and the 206-gene core of a minimal bacterial gene set proposed by Gil et al. One of the surprises about the essential gene set is its inclusion of 110 hypothetical proteins and proteins of unknown function. Some of these genes likely encode enzymes with activities reported in M. genitalium, such as transaldolase, but for which no gene has yet been annotated.”

    Since this organism, an obligate human parasite, is apparently stripped down to bare essentials, “it is likely that all its 482 protein-coding genes are in some way necessary for effective growth,” they said. The team hopes this information will lead to building synthetic free-living cells.
    1Glass, Venter et al., “Essential genes of a minimal bacterium,” Proceedings of the National Academy of Sciences USA, Published online before print January 3, 2006, 10.1073/pnas.0510013103.

    Health Depends on Robust Cell Machinery 01/05/2006
    When we think of health, we typically visualize the big things: firm muscles, energy, lack of a protruding stomach and the like. Cell biology, though, is showing us how our health depends on the proper functioning of countless myriads of molecular machines. Here are some recent samples from the science journals:

    1. Heroic Underdogs in the Brain: Neurons always got the glory in neurology studies, but now it appears that structural cells called astrocytes deserve more respect. A summary of work at U. of Rochester posted on EurekAlert says that these “housekeeping” cells actually perform critical functions in regulating blood flow. They “play a direct role in controlling blood flow in the brain, a crucial process that allows parts of the brain to burst into activity when needed.” When they malfunction, they might contribute directly to degenerative maladies like Alzheimer’s disease. See also LiveScience.
    2. The Vital Destroyer: When cancer spreads, hope shrinks. Friends and family of cancer victims know the agony of metastasis. At least in some kinds of cancers, metastasis may be traced to failure of a protein named caspase-8 that acts like a curfew cop. Normally, reported EurekAlert about work by St. Jude’s Research Hospital, caspase-8 patrols the surfaces of tissues looking for vagrant cells that have dislodged from their normal locations and are wandering into unsafe territory. When it finds them, it turns on their built-in self-destruct program, called apoptosis. When the cops are out sick, the vagrants get out and cause trouble. The paper was published in Nature.1
    3. Your Third Eye: A rare type of eye cell can see. Rods and cones, we know, do most of the real-time visualization, but scientists at Brown University found “intrinsically photosensitive retinal ganglion cells,” or ipRGCs, that respond to light and are hardwired to the brain. They are pretty sure these slower-acting light sensors are responsible for setting our biological clock and controlling the iris muscles, regulating how much light enters the eye. “These cells operate like a light meter on a camera,” said researcher David Berson. “They tell the brain to constrict the pupil based on the amount of light registered over time.” There are about 2000 of these cells in the eye, compared to millions of rods and cones.
    4. Don’t Bang the Eardrums: Our ears can tolerate many orders of magnitude in volume, but there are limits. Researchers at Ohio State found that “years of repeated exposure to loud noise increases the risk of developing a non-cancerous tumor that could cause hearing loss.” Please pass this warning along to your local fitness center.
    5. Watergate Scandal: Point mutations to our water gates, the water-regulating channels in cell membranes, can let the wrong substances in, reported Breitz et al. in PNAS.2 These elaborate channels made of protein, called aquaporins, depend on a precise amino-acid structure to authenticate water but keep other similar-size molecules out; they can even keep out tiny protons. The team inserted mistakes here and there and found that contraband like urea or glycerol could sneak in. One amazing factoid they mentioned is that a single red blood cell has as many as 200,000 aquaporins. For more on membrane channels, see 05/29/2002 and 12/20/2001. A reader found detailed powerpoint presentations and animations at the University of Illinois at Urbana-Champaign website, and more at the University of Maine.
    6. Gutfull Wonders The stomach is a lively place. Lots of organisms live there; hope you don’t mind. A team from Stanford and NYU decided to start surveying these one-celled companions, because “The microbiota of the human stomach ... remain largely unknown.” Their preliminary results, published in PNAS,3 began, “A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described.” Ten percent of them were previously unknown, and they come from at least five separate phyla. Surprisingly, the population in the stomach differs from that in the mouth and esophagus, and different people have different assortments. There are some known bad bugs like Helicobacter pylori that form ulcers, but most of them must be OK or even helpful, since we usually feel good after a big meal: “The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease,” they said.
    7. Clamp Champs: You have sliding clamps in your cells. Really. Current Biology4 talked about these wonderful machines that twist DNA during the copy process:

    “DNA sliding clamps were first characterized as DNA polymerase processivity factors: without their presence, cell division would be inconceivably slow; replication of long stretches of DNA would be hopelessly inefficient because DNA polymerases tend to fall off the DNA after elongating a strand by just a handful of bases. By tethering the polymerase to the DNA, such processivity factors enable the polymerase to add thousands of bases in a few seconds without detaching from the DNA.”

    They work kind of like magic Chinese linking rings. Somehow they melt around the DNA strand without harming it. This allows all the other machinery to get a grip during that heavy-duty copying cycle. Good thing we don’t have to wait so long for the copy operation or we might never grow up.
    8. DNA Gyrations During Packaging: Nature printed articles on two other DNA motors that deserve special notice: one is an acrobatic “gyrase” that generates negative supercoils in DNA (that’s important for packing and safety during cell division).5 In their words, “Negative DNA supercoiling is essential in vivo to compact the genome, to relieve torsional strain during replication, and to promote local melting for vital processes such as transcript initiation by RNA polymerase.” The little motor runs on the cell’s special fuel pellets, ATP. The scientists put beads on it and watched it spin around. They found it was quite sensitive to tension.
    9. More DNA Acrobatics: Another team publishing in Nature6 studied motors called DNA helicases, which are “involved in nearly all aspects of DNA and RNA metabolism.” Utilizing special techniques, they watched this incredibly tiny molecular motor and discovered that it “might move like an inchworm”. It also runs on ATP in a precise range of stresses. Without the helicase machinery, DNA unfolding would be very, very slow. This particular helicase, named NS3, is just one of many “helicases involved in many essential cellular functions.”

    1Stupack et al., “Potentiation of neuroblastoma metastasis by loss of caspase-8,” Nature 439, 95-99 (5 January 2006) | doi:10.1038/nature04323.
    2Breitz et al., “Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons,” Proceedings of the National Academy of Sciences USA, published online before print January 3, 2006, 10.1073/pnas.0507225103.
    3Bik et al., “Molecular analysis of the bacterial microbiota in the human stomach,” Proceedings of the National Academy of Sciences USA, published online before print January 4, 2006, 10.1073/pnas.0506655103.
    4Barsky and Venclovas, “DNA Sliding Clamps: Just the Right Twist to Load onto DNA,” Current Biology, Volume 15, Issue 24, 24 December 2005, pages R989-R992.
    5Gore et al., “Mechanochemical analysis of DNA gyrase using rotor bead tracking,” Nature 439, 100-104 (5 January 2006) | doi:10.1038/nature04319.
    6Dumont et al., “RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP,” Nature 439, 105-108 (5 January 2006) | doi:10.1038/nature04331.

    Soil Provides Library of Antibiotic Resistance 01/19/2006
    The “evolution of antibiotic resistance” is a staple in the creation-evolution debates, providing evolutionists with a living illustration of evolution taking place right before our eyes. What if all the information for antibiotic resistance, however, already exists in a library from which bacteria can find it? That seems to be the implication of a study by D'Carlo et al. in Science.1 A Canadian biochemical research team decided to survey the techniques of antibiotic resistance already present in soil bacteria. They were astonished. Every antimicrobial medicine, including some only recently developed, had a defensive weapon ready for it:

    “This study provides an analysis of the antibiotic resistance potential of soil microorganisms. The frequency of high-level resistance seen in the study to antibiotics that have for decades served as gold-standard treatments, as well as those only recently approved for human use, is remarkable. No class of antibiotic was spared with respect to bacterial target or natural or synthetic origin. Although this study does not provide evidence for the direct transfer of resistance elements from the soil resistome to pathogenic bacteria, it identifies a previously underappreciated density and concentration of environmental antibiotic resistance.”

    The authors could not determine whether “The presence of antibiotics in the environment has promoted the acquisition or independent evolution of highly specific resistance elements in the absence of innate antibiotic production,” and are not sure whether today’s resistant pathogens acquired their resistance from soil organisms. They could not rule it out, however: “The soil could thus serve as an underrecognized reservoir for resistance that has already emerged or has the potential to emerge in clinically important bacteria.” A frightening implication is that no matter what agents we throw at them, bacteria may be able to check out a defense from this “environmental resistome.”
    Alexander Tomasz commented on this study in the same issue of Science.2 He said that, “Actually, the majority of the most effective antibiotic-resistance mechanisms in human pathogens are acquired,” or gained not by evolution but by lateral gene transfer. The acquired resistance, he says, is superior to that gained by mutations:

    “The superiority of such acquired mechanisms is illustrated by the contrast between Staphylococcus aureus strains that have decreased susceptibility to vancomycin through mutations (so-called VISA strains) as compared to VRSA strains, S. aureus that acquired a complete vancomycin-resistance gene complex via the transposon Tn1546. The VISA strains have low-level resistance (the minimal inhibitory concentration of vancomycin is 6 to 12 g/ml), are often associated with reduced oxacillin resistance, and show abnormal cell wall synthesis; the multiple transcriptional changes documented by DNA microarray analysis reflect the complexity of this mechanism. In contrast, in VRSA strains, the Tn1546-based mechanism produces high-level vancomycin resistance (with a minimal inhibitory concentration of more than 500 g/ml) that does not interfere with oxacillin resistance, and cell wall synthesis proceeds with a depsipeptide cell wall precursor specific to these strains.”

    Though the transfer mechanism is not known, “Clearly, mobilization of a resistance mechanism must involve ‘packaging’ into a plasmid, phage, or some transposable element,” he believes. Tomasz called the sheer variety of resistance mechanisms catalogued by D'Carlo et al. “remarkable”. It appears that microorganisms might not only make antibiotic weapons in profusion, but also make a plethora of defenses against them.
    1D'Costa et al., “Sampling the Antibiotic Resistome,” Science, 20 January 2006: Vol. 311. no. 5759, pp. 374 - 377, DOI: 10.1126/science.1120800.
    2Alexander Tomasz, “Weapons of Microbial Drug Resistance Abound in Soil Flora,” Science, 20 January 2006: Vol. 311. no. 5759, pp. 342 - 343, DOI: 10.1126/science.1123982.

    Precision of Cell Quality Control Described 02/03/2006
    Two research papers in Molecular Cell give more glimpses into the precision of cellular controls to ensure mistakes are detected and weeded out before harm occurs. Vogel, Bukau and Mayer1 found that the molecular “chaperone” Hsp70 has a “proline switch,” found in all living organisms. This switch regulates when the polypeptide needing to be folded is attached for processing, then ejected:

    “Crucial to the function of Hsp70 chaperones is the nucleotide-regulated transition between two conformational states, the ATP bound state with high association and dissociation rates for substrates and the ADP bound state with two and three orders of magnitude lower association and dissociation rates. The spontaneous transition between the two states is extremely slow, indicating a high energy barrier for the switch that regulates the transition. Here we provide evidence that a universally conserved proline in the ATPase domain constitutes the switch that assumes alternate conformations in response to ATP binding and hydrolysis. The conformation of the proline, acting through an invariant arginine as relay, determines and stabilizes the opened and closed conformation of the substrate binding domain and thereby regulates the chaperone activity of Hsp70.”

    What is Hsp70 used for? “The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist folding of newly synthesized polypeptides, refolding of misfolded proteins, and translocation of proteins through biological membranes, and in addition have regulatory functions in signal transduction, cell cycle [i.e., cell division], and apoptosis [i.e., programmed cell death].”
    Another paper in the same issue by Gromadski, Daviter and Rodnina2 looked at a quality-control mechanism in the ribosome, where proteins are synthesized before going to the chaperone for folding. They found a way that the machine recognizes typos in transfer-RNA (tRNA) molecules, by authenticating each molecule in a series of precision molecular contacts at the docking site. Mismatches slow down the assembly line from 120-260 per second to 3-4 per second, and result in a thousandfold faster ejection of errors, regardless of their shape:

    “Ribosomes take an active part in aminoacyl-tRNA selection by distinguishing correct and incorrect codon-anticodon pairs. Correct codon-anticodon complexes are recognized by a network of ribosome contacts that are specific for each position of the codon-anticodon duplex and involve A-minor RNA interactions. Here, we show by kinetic analysis that single mismatches at any position of the codon-anticodon complex result in slower forward reactions and a uniformly 1000-fold faster dissociation of the tRNA from the ribosome. This suggests that high-fidelity tRNA selection is achieved by a conformational switch of the decoding site between accepting and rejecting modes, regardless of the thermodynamic stability of the respective codon-anticodon complexes or their docking partners at the decoding site. The forward reactions on mismatched codons were particularly sensitive to the disruption of the A-minor interactions with 16S rRNA and determined the variations in the misreading efficiency of near-cognate codons.”
    The scientists calculated that this one proofreading step reduces errors from somewhere between one in 1,000 to one in 100,000.3 There was no mention of evolution in either of these papers.
    1Vogel, Bukau and Mayer, “Allosteric Regulation of Hsp70 Chaperones by a Proline Switch,” Molecular Cell, Volume 21, Issue 3, 3 February 2006, Pages 359-367, doi:10.1016/j.molcel.2005.12.017.
    2Gromadski, Daviter and Rodnina, “A Uniform Response to Mismatches in Codon-Anticodon Complexes Ensures Ribosomal Fidelity,” Molecular Cell, Volume 21, Issue 3, 3 February 2006, Pages 369-377,
    3There are many other proofreading steps in the process. There are quality-control mechanisms when the DNA is decoded, when the messenger RNA is assembled, when it enters the ribosome, when the amino acids are attached to the proper transfer-RNA, when the tRNA enters the ribosome (as shown here), when the polypeptide exits the ribosome, when the polypeptide is folded in the chaperone, and even later, when post-translational modifications take place in the endoplasmic reticulum.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    In Praise of Fat 03/06/2006
    Well, great balls of fat. Cells have spherical globs of lipid (fat) molecules that never had gotten much attention nor respect. They have been called lipid droplets, oil bodies, fat globules and other names suggesting they were just the beer bellies of the cell. Not any more. Scientists have been taking a closer look at these globs and are finding them to be dynamic, functional sites of critical metabolic activity. No longer are they bags of superfluous undesirable molecules: they have been promoted to essential organelles, named adiposomes.
    Mary Beckman introduced two papers in Science with a summary of the new discoveries.1

    “Whatever their name, these intracellular blobs of triglycerides or cholesterol esters, encased in a thin phospholipid membrane, are catching the attention of more and more biologists. It turns out these lively balls of fat have as many potential roles within cells and tissues as they have names. Pockmarked with proteins with wide-ranging biochemical activities, they shuffle components around the cell, store energy in the form of neutral lipids, and possibly maintain the many membranes of the cell. The particles could also be involved in lipid diseases, diabetes, cardiovascular trouble, and liver problems.”

    Beckman discussed several recent findings demonstrating what happens when fat regulation by adiposomes is disrupted. Since there is still much to be learned about adiposomes, Beckman mainly teased the readers with the possibilities that lie ahead. She quoted one biologist who called the biology of lipid droplets “immense and untapped.”
    A Perspectives paper in the same issue by Stuart Smith introduced new findings about the machines that make fat.2 He summarized a paper by Maier, Jenni and Ban revealing, in unprecedented detail, the structure of mammalian Fatty Acid Synthase (FAS),3 and another by the same authors plus Leibundgut about the comparable FAS machine in fungi.4 The former looks somewhat like a flying saucer; the latter, like a wheel with spokes from the top, or a complex cage from the side. The diagrams of these machines point out “active sites” and “reaction chambers” where complex molecules are assembled in a specific sequence. The machines apparently have moving parts. The conclusion of the mammalian FAS paper hints how everything must be done in order and with the right specifications:

    “The overall architecture of mammalian FAS has been revealed by x-ray crystallography at intermediate resolution. The dimeric [two-part] synthase adopts an asymmetric X-shaped conformation with two reaction chambers on each side formed by a full set of enzymatic domains required for fatty acid elongation, which are separated by considerable distances. Substantial flexibility of the reaction chamber must accompany the handover of reaction intermediates during the FAS cycle, and further conformational transitions are required to explain the presence of alternative inter- and intrasubunit synthetic routes in FAS. The results presented here provide a new structural basis to further experiments required for a detailed understanding of the complex mechanism of mammalian FAS.”

    Even for the fungal machine, the authors spoke of the “remarkable architectural principles” it exemplifies. It’s a whole new world of fat. Let that go to your understanding, not to your waist.
    1Mary Beckman, “Great Balls of Fat,” Science, 3 March 2006: Vol. 311. no. 5765, pp. 1232 - 1234, DOI: 10.1126/science.311.5765.1232.
    2Stuart Smith, “Architectural Options for a Fatty Acid Synthase,” Science, 3 March 2006: Vol. 311. no. 5765, pp. 1251 - 1252, DOI: 10.1126/science.1125411.
    3Timm Maier, Simon Jenni, Nenad Ban, “Architecture of Mammalian Fatty Acid Synthase at 4.5 ŠResolution,” Science, 3 March 2006: Vol. 311. no. 5765, pp. 1258 - 1262, DOI: 10.1126/science.1123248.
    4Simon Jenni, Marc Leibundgut, Timm Maier, Nenad Ban, “Architecture of a Fungal Fatty Acid Synthase at 5 ŠResolution,” Science, 3 March 2006: Vol. 311. no. 5765, pp. 1263 - 1267, DOI: 10.1126/science.1123251.

    Introns Stump Evolutionary Theorists 03/09/2006
    This story is not about Enron and Exxon, but about introns and exons. The proportions of the scandals they are causing in evolutionary theory, however, may be comparable.
    Introns are spacers between genes. For several decades now, it has been a puzzle why they are there, and why a complex machine called a spliceosome takes them out and joins the active genetic parts – the exons – together. Only eukaryotes have spliceosomes, though; mitochondria have “group II introns” and some mRNAs may have them. Their presence and numbers in various groups presents a bewildering array of combinations. Figuring out a phylogenetic tree for introns has eluded evolutionary geneticists, as has understanding their origin and functions (02/18/2005). Why do genes come in pieces that have to be reassembled?
    William Martin and Eugene Koonin said in Nature1 that “The discovery of introns had a broad effect on thoughts about early evolution.” Some theories have been falsified, and others remain in the running. Consider the scope of the problems:

    “A current consensus on introns would be that prokaryotes do indeed have group II introns but that they never had spliceosomes; hence, streamlining in the original sense (that is, loss of spliceosomal introns) never occurred in prokaryotes, although it did occur in some eukaryotes such as yeast or microsporidia. An expansion of that consensus would be that spliceosomes and spliceosomal introns are universal among eukaryotes, that group II introns originating from the mitochondrion are indeed the most likely precursors of eukaryotic mRNA introns and spliceosomal snRNAs, and that many—conceivably most—eukaryotic introns are as old as eukaryotes themselves. More recent are the insights that there is virtually no evolutionary grade detectable in the origin of the spliceosome, which apparently was present in its (almost) fully fledged state in the common ancestor of eukaryotic lineages studied so far, and that the suspected source of introns—mitochondria, including their anaerobic forms, hydrogenosomes and mitosomes—was also present in the common ancestor of contemporary eukaryotes (the only ones whose origin or attributes require explanation).
    This suggests that intron origin and spread occurred within a narrow window of evolutionary time: subsequent to the origin of the mitochondrion, but before the diversification of the major eukaryotic lineages. This, in turn, indicates the existence of a turbulent phase of genome evolution in the wake of mitochondrial origin, during which group II introns invaded the host’s chromosomes, spread as transposable elements into hundreds—perhaps thousands—of positions that have been conserved to the present, and fragmented into both mRNA introns and snRNA constituents of the spliceosome.

    This means that a complex molecular machine, the spliceosome (09/17/2004, 09/12/2002), appeared fully formed almost abruptly, and that the intron invasion took place over a short time and has not changed for hundreds of millions of years. They submitted a new hypothesis:

    “Here we revisit the possible evolutionary significance of introns in light of mitochondrial ubiquity. We propose that the spread of group II introns and their mutational decay into spliceosomal introns created a strong selective pressure to exclude ribosomes from the vicinity of the chromosomes—thus breaking the prokaryotic paradigm of co-transcriptional translation and forcing nucleus-cytosol compartmentalization, which allowed translation to occur on properly matured mRNAs only.”

    But this means that the nucleus, nucleolus and other complex structures also had to appear in a very brief period of time. It means that the engulfed organism that somehow became mitochondria had to transfer its introns rapidly into a genome lacking a nucleus. It means the nucleus had to evolve quickly to segregate the new mitochondrial genes from the nuclear genes. A lot had to happen quickly. “This bipartite cell would not be an immediate success story: it would have nothing but problems instead,” they admitted, but they believed that natural selection would favor the few that worked out a symbiotic relationship with their new invaders.
    This is not the end of the problems. The group II introns would have had to embed themselves with reverse transcriptase and maturase without activating the host’s defenses, then evolve into spliceosome-dependent introns and remain unchanged forever after. Then those embedded group II introns would undergo mutational decay, interfering with gene expression. Will this work without some miracles?

    “A problem of a much more severe nature arises, however, with the mutational decay of group II introns, resulting in inactivation of the maturase and/or RNA structural elements in at least some of the disseminated copies. Modern examples from prokaryotes and organelles suggest that splicing with the help of maturase and RNA structural elements provided by intact group II introns in trans could have initially rescued gene expression at such loci, although maturase action in trans is much less effective than in cis. Thus, the decay of the maturase gene in disseminated introns poses a requirement for invention of a new splicing machinery. However, as discussed below, the transition to spliceosome-dependent splicing will also impose an unforgiving demand for inventions in addition to the spliceosome.”

    A spliceosome is not an easy thing to invent; it has five snRNAs and over 200 proteins, making it one of the most complex molecular machines in the cell. Not only that, they appeared in primitive eukaryotes and have been largely conserved since. Perhaps the miracles can be made more believable by dividing them into smaller steps:

    “It seems that the protospliceosome recruited the Sm-domain, possibly to replace the maturase, while retaining group II RNA domains (snRNAs) ancestrally germane to the splicing mechanism. While the later evolution of the spliceosome entailed diversification with the recruitment of additional proteins—leading to greater efficiency—the simpler, ancestral protospliceosome could, in principle, rescue expression of genes containing degenerate group II introns in a maturase-independent manner, but at the dear cost of speed.”

    Will a lateral pass from maturase to incipient spliceosome during a long field run lead to a touchdown? If a stumbling protospliceosome could survive, in spite of vastly decreased translation rate, it might have been able to run the distance with natural selection’s encouragement, they think. Players would be falling left and right in this “extremely unhealthy situation,” they say, and “the prospects of any descendants emerging from this situation are bleak.” How could the game go on, then? “The only recognizable mechanism operating in favour of this clumsy chimaera is weakened purifying selection operating on its exceptionally small initial population.” Purifying selection means weeding out losers, not adding new champions. “Finding a solution to the new problem of slow spliceosomes in the presence of fast and abundant ribosomes required an evolutionary novelty.”
    They winnow down the possibilities. Getting instant spliceosomes smacks too much of an improbable feat. Getting rid of spliceosomal introns from DNA apparently did not occur. Their solution? The invention of the nucleus, where slow spliceosomes could operate without competition from fast ribosomes.
    This adds new miracles, however. The nucleus has highly complex pores that permit only authenticated molecules into the inner sanctum. They think, however, that it must have happened, somehow: “Progeny that failed to physically separate mRNA processing from translation would not survive, nor would those that failed to invent pore complexes to allow chromosome-cytosol interaction.” So pick your miracles: since necessity is the mother of invention, “The invention of the nucleus was mandatory to allow the expression of intron-containing genes in a cell whose ribosomes were faster than its spliceosomes.”
    The near-miraculous arrival of the nucleus is underscored by other feats it performs: “In addition to splicing, eukaryotes possess elaborate mRNA surveillance mechanisms, in particular nonsense-mediated decay (NMD), to assure that only correctly processed mature mRNAs are translated, while aberrant mRNAs and those with premature termination codons are degraded.” How could this originate? Again, necessity must have driven the invention: “The initial intron invasion would have precipitated a requirement for mechanisms to identify exon junctions and to discriminate exons (with frame) from introns (without frame), as well as properly from improperly spliced transcripts. Thus, NMD might be a direct evolutionary consequence of newly arisen genes-in-pieces.” But then, if it is verified that some translation occurs in the nucleus, that would be “difficult to reconcile with our proposal.”
    They ended with comparing their hypothesis with others. “Our suggestion for the origin of the nucleus differs from previous views on the topic,” they boasted, “which either posit that the nuclear membrane was beneficial to (not mandatory for) its inventor by protecting chromosomes from shearing at division, or offer no plausible selective mechanism at all.” At least theirs is simpler and includes some requirements to select for the cells with the best inventors – or the ones with the luckiest miracles.
    1Martin and Koonin, “Hypothesis: Introns and the origin of nucleus-cytosol compartmentalization,” Nature 440, 41-45 (2 March 2006) | doi:10.1038/nature04531.

    Misfolded Proteins Cause Cascade of Harmful Effects 03/12/2006
    Understanding how proteins fold is at the leading edge of scientific research. Proteins begin as linear chains of amino acids (polypeptides), but end as complex shapes with loops, sheets, bumps, ridges and grooves that are essential to their functions. If you imagine a string of beads, some with electrical charges, magnets, oil droplets or other attraction-repulsion attributes on them, what would happen if you dropped it in water? It would seem there are a myriad ways it could collapse into a shapeless mass. How many of those possible shapes would make it a machine? That’s the kind of problem that protein-folding presents to the researcher.
    Normally, cells help the newly-assembled polypeptides fold properly with the aid of chaperones, the cellular “dressing rooms” where they can prepare for their debut (05/05/2003). Mistakes happen, however. A mutation might put a charge on the wrong amino acid, making it fold the wrong way. Here again, the cell usually deals with these badly-folded masses and destroys them as part of its “quality control” procedures. Once in awhile, however, misfolded protein machines get out of control, and some, like chain saws run amok, can cause harm. Here’s an excerpt from an article in Science by Gillian Bates (King’s College London School of Medicine). Describing recent work on this subject, he explains the consequences:

    “This work indicates that the chronic expression of a misfolded protein can upset the cellular protein folding homeostasis under physiological conditions. These results have implications for pathogenic mechanisms in protein conformational diseases. The human genome harbors a load of polymorphic variants and mutations that might be prevented from exerting deleterious effects by protein folding and clearance quality control mechanisms in the cell. However, should these mechanisms become overwhelmed, as in a protein conformation disease, mild folding variants might contribute to disease pathogenesis by perturbing an increasing number of cellular pathways.... Therefore, the complexity of pathogenic mechanisms identified for protein conformation diseases could in part result from the imbalance in protein folding homeostasis.”

    In other words, one mistake in one protein can have a cascading effect, causing a multitude of mistakes downstream. The normal dynamic equilibrium of the cell (homeostasis) turns into a disaster scene, as the quality-control cops become overwhelmed by victims, as in a natural disaster. Examples of degenerative diseases caused by misfolded proteins mentioned in the article: “Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis—these neurodegenerative disorders are among many inherited diseases that have been linked to genetic mutations that result in the chronic aggregation of a single specific protein.”
    1Gillian Bates, “Perspectives: One Misfolded Protein Allows Others to Sneak By,” Science, 10 March 2006: Vol. 311. no. 5766, pp. 1385 - 1386, DOI: 10.1126/science.1125246.

    Reviewer Stunned by Author’s Handwaving 03/31/2006
    David Nicholls appears to have suffered whiplash from a line in a book he was reviewing in Science,1 Power, Sex, Suicide: Mitochondria and the Meaning of Life by Nick Lane (Oxford, 2006). Though he liked the book in general, he said this about Lane’s explanation for how the first cell got its power generator:

    “The author is less convincing when he turns to the origin of life (at least he is not afraid to deal with big topics). Citing the work of Mike Russell2 and Alan Hall, Lane states that in order to generate a primitive cell from an iron sulphide vesicle “all that the cells need to do to generate ATP is to plug an [proton translocating] ATPase through the membrane.” Any bioenergeticist who has followed the elucidation of the extraordinary structure and mechanism of the mitochondrial ATP synthase over the past decade will pause at the word “all,” because the ATP synthase—with its spinning rotor massaging the surrounding subunits to generate ATP—is without doubt the most amazingly complex molecular structure in the cell.”3

    After that, Nicholls had mostly praise for the rest of the book.
    1David G. Nicholls, “Cell Biology: Energizing Eukaryotes,” Science, 31 March 2006: Vol. 311. no. 5769, p. 1869, DOI: 10.1126/science.1126251.
    2See 12/03/2004 on theories by Michael Russell.
    3The amazing structure and function of the universal ATP synthase motor has been discussed many times in these pages. See, for instance, 01/30/2005 and 12/22/2003, and animation mentioned on April 2002 page.

    How Much Can a Cell Do Without? 04/14/2006
    In an old high school game, the leader would call some unsuspecting boy to the front, put a sheet over him, and say, “Take off what you don’t need.” Perhaps a shoe would emerge from under the sheet. “Take off something else you don’t need,” the leader would continue, and the volume of giggling in the room would rise as socks, a shirt, and whatever would emerge from under the covers. If the young person was smart, he would realize the only thing he didn’t need was the sheet itself.
    Scientists play this game in a more sophisticated manner with cells, in a process called gene knockout. The idea is to disable a gene or protein and see what happens. They can also overexpress the gene, or mutate it, for additional data. If the cell gets by just fine, it must have been a nonessential part. Usually, however, something terrible happens, even when the gene or protein was previously unknown. Here are just a couple of examples from today’s PNAS:

    • Power Plant Sabotage: Scientists from Michigan State1 studied FZO, “dynamin-related membrane-remodeling protein that mediates fusion between mitochondrial outer membranes in animals and fungi.” In the model plant Arabidopsis, they knocked out the plant-specific member of the dynamin superfamily, FZL. This protein targets to the thylakoid membrane of the chloroplasts, the light-harvesting power plants of plants. Here’s what happened: fzl knockout mutants have abnormalities in chloroplast and thylakoid morphology, including disorganized grana stacks and alterations in the relative proportions of grana and stroma thylakoids. Overexpression of FZL-GFP also conferred defects in thylakoid organization. Mutation of a conserved residue in the predicted FZL GTPase domain abolished both the punctate localization pattern and ability of FZL-GFP to complement the fzl mutant phenotype. FZL defines a new protein class within the dynamin superfamily of membrane-remodeling GTPases that regulates organization of the thylakoid network in plants. Notably, FZL levels do not affect mitochondrial morphology or ultrastructure, suggesting that mitochondrial morphology in plants is regulated by an FZO-independent mechanism.”

    This means that this specific protein was essential for just the thylakoid membrane inner structure, and there must be another essential mechanism affecting the overlying structure. (Note: the capitalized acronym, FZL, refers to the protein, while the italicized lower-case acronym fzl refers to the gene that codes for it.) They found that mutating or deleting the gene causes disaster – but so does overexpressing it. This means that not only is FZL a key player, but the activity of its gene fzl must be regulated by something else.

    Centrosome Attack: Mitosis, or cell division, has been studied for many decades, but now another essential player has been identified. Scientists from Japan and Pennsylvania2 describe what happened when they played “take off what you don’t need” with a centrosome protein named Su48:

    “The centrosome functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation. In our efforts to identify the protein components of the centrosome and to understand the structure features involved in the assembly and functions of this organelle, we cloned and characterized a centrosome-associated protein called Su48. We found that a coiled coil-containing subdomain of Su48 was both sufficient and required for its centrosome localization. In addition, this structure also modulates Su48 dimerization. Moreover, ectopic expression of Su48 causes abnormal mitosis, and a mutant form of Su48 disrupts the localization of gamma-tubulin to the centrosome. Finally, by microinjection of an anti-Su48 antibody, we found that disruption of normal Su48 functions leads to mitotic failure, possibly due to centrosome defects or incomplete cytokinesis. Thus, Su48 represents a previously unrecognized centrosome protein that is essential for cell division. We speculate that Su48 abnormalities may cause aberrant chromosome segregation and may contribute to aneuploidy and malignant transformation.”

    These papers are just two out of a growing body of knockout experiments that find out, by examining the wreckage, that there’s not much a cell doesn’t need.
    1Gao et al., “FZL, an FZO-like protein in plants, is a determinant of thylakoid and chloroplast morphology,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0507287103, published online before print April 14, 2006.
    2Wang et al., “Characterization of Su48, a centrosome protein essential for cell division,”
    Proceedings of the National Academy of Sciences USA
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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    Will Genetics Be Neo-Darwinism’s Downfall? 05/04/2006
    … Most genetics papers … are finding degrees of order, regulation and coordinated action in the cell that challenge gradualistic explanations. Here are some examples from the past two months:

    Rapid Gradualism? New Scientist reported that many human genes must have “evolved recently” – even as recently as within the last 15,000 years. While some of the 700-odd genes they studied, they claim, appear to have been targets of natural selection after the human line diverged millions of years ago, “some of the newly identified genes fall into categories not previously known to be targets of selection in the human lineage, such as those involved in metabolism of carbohydrates and fatty acids.” (Ker Than at Live Science took this to mean humans are still evolving.)

    Transcript Complexity: PLoS Genetics had a special issue about the complexity of the “transcriptome,” the body of all transcribed DNA. The lead article’s teaser sounds pretty dramatic:

    “Besides revealing staggering complexity, analysis of this collection is providing an increasing number of novel mRNA classes, expressed pseudogenes, and bona fide noncoding variants of protein-coding genes. In addition, new types of regulatory logic have emerged, including sense-antisense mechanisms of RNA regulation. This high-resolution cDNA collection and its analysis represent an important world resource for discovery, and demonstrate the value of large-scale transcriptome approaches towards understanding genome function.”

    After the human genome was deciphered, scientists were puzzled by the seeming small number of genes – about 30,000. Now, it appears that the exons of genes can be assembled and reassembled in a modular way by alternative gene splicing (09/23/2005), yielding many protein variants from one gene. Not only that, the DNA “negative” on the opposite (antisense) strand can play a role in regulating the gene. These articles speak as if a whole new world of complexity is coming to light.

    Who Regulates the Regulators? Nature March 23 reported on important pathways that regulate the fate of RNA transcripts of genes. David Tollervey wrote in the introduction:

    “Cells alter their rates of mRNA transcription to change mRNA levels, and so rates of protein synthesis, in response to many stimuli. To adjust mRNA levels, cells must be able to rapidly get rid of normal mRNAs that were previously synthesized (turnover). In fact, different mRNAs differ radically in their rates of degradation, and this is subject to both metabolic and developmental regulation. In addition, cells must guard against the synthesis of abnormal mRNAs (surveillance), which can produce defective, potentially toxic, protein products.”

    The mechanisms described in the article, including “go/no-go” checkpoints unveil a higher level of complexity beyond the information contained in the genes themselves.

    Ring Job: The copies during cell division must be accurate. Many protein parts cooperate to ensure high levels of quality control. Nature reported March 23 on a discovery of a ring that slides along the microtubules in the all-important stage of separation of the paired chromosomes.

    High Fidelity Proofreading: Albertson and Preston talked about quality control of the DNA copying process in an article in Current Biology March 23:

    “Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.”

    This means that some polymerases (copy machines) have better fidelity than others, but they cooperate to ensure a precision product. A low-fidelity machine might be necessary to get past a bad break, for instance – like when a heftier wrench is needed (09/19/2005). How good is the system? Orders of magnitude better than a human copyist:

    “Normal cells replicate their DNA with remarkable fidelity, accumulating less than one mutation per genome per cell division. It is estimated that replicative DNA polymerases make errors approximately once every 104-105 nucleotides polymerized. Thus, each time a mammalian cell divides approximately 100,000 polymerase errors occur, and these must be corrected at near 100% efficiency to avoid deleterious mutations. This is accomplished through the combined actions of... exonucleolytic proofreading and post-replication mismatch repair.”

    New Uses for Junk: “Just because we don’t know what it does, doesn’t mean it’s really junk,” said Christina Cheng of non-coding DNA (U of Illinois) in an interview for Radio Netherlands. Her work has found that arctic cod produce antifreeze proteins (05/13/2004) from non-gene regions of DNA, “a gene that appears to have evolved out of this DNA that supposedly serves no purpose.” Yet “Preserving this rubbish seems an inefficient use of time and resources. Evolutionary pressures should favour creatures with less junk DNA” said author Marnie Chesterton. “So its conservation may be because it has functions that we don’t yet know.” Cheng said, “conventional thinking assumes that new genes must come from pre-existing ones because the probability of a random stretch of DNA somehow becoming a functional gene is very low if not nil.”

    No More Mr. Simple Guy: Embley and Martin in Nature March 30 had some words for those who tell simplistic tales about an ancient prokaryote being co-opted as a mitochondrion in the first primitive eukaryote (see 08/06/2004):

    “The idea that some eukaryotes primitively lacked mitochondria and were true intermediates in the prokaryote-to-eukaryote transition was an exciting prospect. It spawned major advances in understanding anaerobic and parasitic eukaryotes and those with previously overlooked mitochondria. But the evolutionary gap between prokaryotes and eukaryotes is now deeper, and the nature of the host that acquired the mitochondrion more obscure, than ever before.

    Modular Programming: An article in Nature March 30 by 37 European scientists found an exquisite example of modular programming – in yeast. They even spoke machine language:

    “The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.”

    Question is, what evolutionist would want to model 257 novel proteins and 491 complexes, all tightly regulated and “evolutionarily conserved” (i.e., unevolved)?

    Pas de Deux: We know that we have two copies of each gene, one from the father and one from the mother, but which copy leads and which follows? As in marriage, this process is surprisingly complicated. Spilianakis and Flavell explored this important question in a Perspectives article in Science April 14. They showed how the dance involves the help of many servants:

    “The genetic information of higher organisms is encoded in DNA that is not randomly dispersed within the cell nucleus, but is organized with nucleoproteins into different kinds of chromatin, the building blocks of the chromosomes. Each chromosome resides in a specific region of the nucleus when the cell is not undergoing cell division, and usually genes that are actively being expressed loop out from their condensed chromatin territory and localize to a region of transcriptional activity. These “transcription factory” areas are thus abundant with protein factors that initiate and regulate gene expression.”

    The dance gets really wild, but not chaotic, when a gene on one chromosome is regulated by factors on another chromosome.

    The Parallel Universe of RNA: The title of this article in PNAS hints at previously-unknown complexity: “Short blocks from the noncoding parts of the human genome have instances within nearly all known genes and relate to biological processes.” This article was discussed in more detail here 04/27; see also the 09/08/2005 entry.

    Guardian Spirits: In today’s Nature (May 4), Paul Megee titled an article, “Molecular biology: Chromosome guardians on duty.” He begins, “Curiously, in cell division the proper separation of chromosomes into daughter cells needs set periods when they are stuck together. So how do they come apart at the right time and place? Their ‘guardian spirits’ intercede.” Reminding the reader of the importance of high fidelity in cell division, he discusses work by Japanese scientists who “describe how proteins known as shugoshins – Japanese for ‘guardian spirits’ – and an associated regulatory enzyme temporally and spatially control the removal of cohesins from chromosomes.” Cohesins keep the chromosomes together while they line up on the spindle, but need to be broken at the right time (03/04/2004) in a coordinated way – thanks to their guardian spirits.

    These are just samples pouring out of the secular literature on genomics. Clearly, a great deal more choreographed complexity is being found in the nucleus than Watson and Crick could have imagined when the genetic code first began to be deciphered.
    Random changes are destructive to any carefully crafted piece of work, such as a computer program, a novel or the genome of a lifeform.
    Matt 23:24Ye blind guides, which strain at a gnat, and swallow a camel.

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