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bob b

Science Lover
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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.

“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.

bob b

Science Lover
Hall of Fame
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
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bob b

Science Lover
Hall of Fame
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.

bob b

Science Lover
Hall of Fame
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 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).

bob b

Science Lover
Hall of Fame
“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.”

bob b

Science Lover
Hall of Fame
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.”
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bob b

Science Lover
Hall of Fame
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.

bob b

Science Lover
Hall of Fame
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.

bob b

Science Lover
Hall of Fame
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

bob b

Science Lover
Hall of Fame
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.

bob b

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Protein Dressing Room Has Electronic Walls 06/07/2006
Properly folded proteins are essential to all of life. When a polypeptide, or chain of amino acids, emerges from the ribosome translation factory on its way to becoming a protein, it looks like a useless, shapeless piece of string. It cannot perform its function till folded into a precise, compact shape particular for its job. Some short polypeptides will spontaneously fold into their “native” state, ready for work, but many of the bigger ones need help. Fortunately, the cell provides a private dressing room called the GroEL-GroES chaperonin that not only gives them privacy, away from the bustle of colliding molecules in the cytoplasm, but actually helps them get dressed (see also 05/05/2003 entry). This chaperone or “helper” machine thus not only gets the actor ready for the stage faster, but prevents misfolding that could clutter the cell with useless or harmful aggregates of protein.
A team from the Max Planck Institute, writing in Cell,1 investigated how the internal structure of this barrel-shaped molecular machine overcomes energy barriers to proper folding and speeds up the process ten-fold. They found that the inside walls of the GroEL barrel and the inside walls of the GroES lid contain protrusions that generate electrostatic and hydrophobic forces on the interior space. When the unfolded protein enters, therefore, it is subjected to gentle pressures that coax it to fold. These forces are nonspecific enough to work on hundreds of different substrates that use this general-purpose machine.
Furthermore, they found that the forces change during the entry of the nascent protein. The interior is not barrel shaped when the actor approaches the door; the GroES lid, with the help of the energy molecule ATP, guides the protein in, and then the barrel pops into its shape, providing a safe haven for folding. Moreover, the electronic walls turn on to provide that gentle nudge to get the polypeptide over its energy barriers and into the right folding pathway. When the protein has properly completed its folding after about 10 seconds in the dressing room, the door opens and the protein pops out, ready for action.
How finely tuned is this machine? The authors did some experiments on mutating the chaperone to make the barrel looser and tighter. They found that volume changes as small as 2-5% slowed down the folding considerably. The barrel volume needs to be within certain narrow limits, yet general enough to handle a variety of small, medium and large proteins.

“The GroEL/GroES nano-cage allows a single protein molecule to fold in isolation. This reaction has been compared to spontaneous folding at infinite dilution. However, recent experimental and theoretical studies indicated that the physical environment of the chaperonin cage can alter the folding energy landscape, resulting in accelerated folding for some proteins. By performing an extensive mutational analysis of GroEL, we have identified three structural features of the chaperonin cage as major contributors to this capacity: (1) geometric confinement exerted on the folding protein inside the limited volume of the cage; (2) a mildly hydrophobic, interactive surface at the bottom of the cage; and (3) clusters of negatively charged amino acid residues exposed on the cavity wall. We suggest that these features in combination provide a physical environment that has been optimized in evolution [sic] to catalyze the structural annealing of proteins with kinetically complex folding pathways. Thus, the chaperonin system and its mutant versions may prove as useful tools in understanding how proteins navigate their energy landscape of folding.”

1Tang et al., “Structural Features of the GroEL-GroES Nano-Cage Required for Rapid Folding of Encapsulated Protein,” Cell

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Plant Hula-Hoop Railroads Build Cell Walls 06/09/2006
Solving a long-standing mystery about how plants build cell walls, Stanford scientists imaged molecular machines traveling along hoop-shaped rings around the inside of the cell. Publishing in Science, Paradez, Somerville and Ehrhardt proved that cellulose synthase (CESA), a machine that manufactures cellulose composed of six subunits arranged in rosettes, rides like a rail car on microtubules that encircle the inside of the plasma membrane. From there, the machine extrudes the complex molecule to the exterior, building the rigid cell wall.
Clive Lloyd, commenting on this finding in the same issue of Science,2 seemed happily astonished, not only at the scientific achievement, but at the plants themselves:

“In a remarkable series of biological transformations, green plants convert carbon dioxide into cellulose fibers stronger than steel. These thin threads of polymeric glucose are wrapped around growing cells, lending structural support to the plant as it extends further into the environment. The fibers are not simply secreted into the plant cell wall in a haphazard fashion but are deposited in ordered layers that still allow the cell to expand. For more than 40 years, it has been known that the alignment of these cellulose fibers (microfibrils) in the cell wall often coincides with cytoskeletal microtubules tethered to the cytoplasmic side of the plasma membrane... Despite this coincidence, there has never been direct proof that microtubules provide a guidance mechanism for the alignment of cellulose microfibrils. Now, on page 1491 of this issue, Paredez et al. (1) provide that proof.”

Lloyd described the cell-encircling hoops as a “microtubule railroad” providing tracks for the cellulose-synthesizing machines. Apparently these tubules can reorient themselves, perhaps in hula-hoop fashion, allowing the machines to stitch cross-hatch patterns of cellulose for added strength (see 01/16/2003) for analogous process).
1Paradez, Somerville and Ehrhardt, “Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules,” Science, 9 June 2006: Vol. 312. no. 5779, pp. 1491 - 1495, DOI: 10.1126/science.1126551.
2Clive Lloyd, “Microtubules Make Tracks for Cellulose,” Science, 9 June 2006: Vol. 312. no. 5779, pp. 1482 - 1483, DOI: 10.1126/science.1128903.

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Eukaryote Evolution Proceeded from Complex to Simple 06/09/2006
As if reprimanding simpletons, three scientists writing in Science1 preached that the old picture of evolution from simple to complex is simplistic. This is particularly true, they claim, for the story that eukaryotes were born from a blessed union. “Data from many sources,” they counter, “give no direct evidence that eukaryotes evolved by genome fusion between archaea and bacteria.” Further down, they remark, “Unfortunately, such a model has been tacitly favored by molecular biologists who appeared to view evolution as an irreversible march from simple prokaryotes to complex eukaryotes, from unicellular to multicellular.” The old picture harks back to obsolete views of straight-line evolution.

“Comparative genomics and proteomics have strengthened the view that modern eukaryote and prokaryote cells have long followed separate evolutionary trajectories. Because their cells appear simpler, prokaryotes have traditionally been considered ancestors of eukaryotes. Nevertheless, comparative genomics has confirmed a lesson from paleontology: Evolution does not proceed monotonically from the simpler to the more complex. Here, we review recent data from proteomics and genome sequences suggesting that eukaryotes are a unique primordial lineage....”

Out with the old, in with the new. What do they suggest to replace the old picture? Believe it or not, “sequence loss and cellular simplification.” Since these are “are common modes of evolution,” they argue that the first eukaryote was already a unique, complex creature. Like a predator or raptor, it acquired mitochondria by phagocytosis, and diversified from there.
Their view does not prohibit increases in complexity, yet they seem eager to distance evolutionary theory from visions of progress. “Genome evolution is a two-way street,” they say; “This bidirectional sense of reversibility is important as an alternative to the view of evolution as a rigidly monotonic progression from simple to more complex states, a view with roots in the 18th-century theory of orthogenesis.” They describe several life-forms that have reduced their genomes and slimmed down to the bare minimum: parasites, symbionts, organelle genomes, and anaerobes.
OK so far; evolution can move either toward complex or simple – but how does this explain eukaryotes (cells with nuclei and compartmentalized organelles)? Here, their explanation appears forced by the hard realities of the evidence. From the earliest possible ancestor, eukaryotes were already complex. They had introns (and complex spliceosomes, half of whose 78 proteins are unique to eukaryotes, to handle them), mitosomes, hydrogenosomes, mitochondria, nuclei, nucleoli, the Golgi apparatus, centrioles, and an endoplasmic reticulum, along with “hundreds of proteins with no orthologs evident in the genomes of prokaryotes.” (Simple Giardia, for example, has 347 eukaryote signature proteins.) Much of the article describes the unprecedented features of eukaryotes, which constitute a “unique cell type that cannot be deconstructed into features inherited directly from archaea and bacteria.”
This calls for alternatives to “hypotheses that attribute eukaryote origins to genome fusion between archaea and bacteria” (endosymbiosis), which they claim “are surprisingly uninformative about the emergence of the cellular and genomic signatures of eukaryotes.” Recognition of these realities must be “the critical starting point” for explaining where eukaryotes came from: i.e., a “larger and more complex cell” at the beginning when the three kingdoms – bacteria, archaea and eukaryotes – diverged.
Their picture can be summarized as follows: (1) the common ancestor was a raptor or predator on prokaryote mitochondria. (2) Cellular crowding and compartmentalization led to more efficient molecular interactions. (3) Extensive genome reduction followed. Darwin, of course, grins in the background; “This abbreviated account of genome reduction illustrates the Darwinian view of evolution as a reversible process in the sense that ‘eyes can be acquired and eyes can be lost’” (because of the two-way street of natural selection). Even Darwin would have agreed that “selection gives, and selection takes.” They concur with essential evolutionary doctrine without hesitation: “Genomes evolve continuously through the interplay of unceasing mutation, unremitting competition, and ever-changing environments.” Darwinism is safe, therefore; so now, let’s picture the new emerging story for the 21st century:

“For the reasons outlined above, we favor the idea that the host that acquired the mitochondrial endosymbiont was a unicellular eukaryote predator, a raptor. The emergence of unicellular raptors would have had a major ecological impact on the evolution of the gentler descendants of the common ancestor. These may have responded with several adaptive strategies: They might outproduce the raptors by rapid growth or hide from raptors by adapting to extreme environments. Thus, the hypothetical eukaryote raptors may have driven the evolution of their autotrophic, heterotrophic, and saprotrophic cousins in a reductive mode that put a premium on the relatively fast-growing, streamlined cell types we call prokaryotes.”

One problem. How this complex, predatory cell with most of its unique parts “emerged” is anyone’s guess. So get busy, everyone: “This scenario, which is not contradicted by new data derived from comparative genomics and proteomics, is a suitable starting point for future work.”
1Kurland, Collins and Penny, “Genomics and the Irreducible Nature of Eukaryote Cells,” Science, 19 May 2006: Vol. 312. no. 5776, pp. 1011 - 1014, DOI: 10.1126/science.1121674.

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Rubisco “Highly Tuned” for Fixing Atmospheric Carbon 06/22/2006
Rubisco sounds like a brand of cracker or something, but it’s actually an air cleaner your life depends on. It’s an enzyme that fixes atmospheric carbon for use by photosynthetic microbes and plants. In doing so, it sweeps the planet of excess carbon dioxide – the greenhouse gas implicated in discussions of global warming – making it a politically important molecule as well the most economically important enzyme on earth. Rubisco is the most common enzyme in the world, too; every person on earth benefits from his or her own 12 to 25 pounds of these molecular machines, which process 15% of the total pool of atmospheric carbon per year. For a long time, biochemists thought this enzyme was slow and inefficient. That view is changing. Rubisco now appears to be perfectly optimized for its job.
Rubisco’s cute name is a handy anagram for the clumsier appellation ribulose bisphosphate carboxylase. Tcherkez et al. first broke the paradigmatic logjam about this enzyme’s purported inefficiency with an article in PNAS,1 titled, “Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized.” Howard Griffiths commented this week in Nature2 about this paper and the new findings about its optimization. Though his article referred to evolution seven times, and only mentioned design twice, the latter word seemed the most valuable player.
There are four classes of Rubisco, some more efficient at fixing carbon than others. Its reputation as a slow enzyme (2-8 catalytic events per second) may be unfair. Carbon dioxide in gaseous form has to compete for access to the active site against the much more abundant and lighter oxygen. Griffiths shows what a difficult job this molecule has to perform; no wonder it leaks somewhat. But, as he explains, even the leaks are accommodated:

“It is curious that Rubisco should fix CO2 at all, as there is 25 times more O2 than CO2 in solution at 25°C, and a 500-fold difference between them in gaseous form. Yet only 25% of reactions are oxygenase events at this temperature, and carbon intermediates ‘lost’ to the carbon fixation reactions by oxygenase action are metabolized and partly recovered by the so-called photorespiratory pathway. Catalysis begins with activation of Rubisco by the enzyme Rubisco activase, when first CO2 and then a magnesium ion bind to the active site. The substrate, ribulose bisphosphate, then reacts with these to form an enediol intermediate, which engages with either another CO2 or an O2 molecule, either of which must diffuse down a solvent channel to reach the active site.”

This is a harder job than designing a funnel that will pass only tennis balls, when there are 500 times more ping-pong balls trying to get through. Not only is Rubisco good at getting the best mileage from a sloppy process, it may actually turn the inefficiency to advantage. Griffiths started by claiming, “evolution has made the best of a bad job,” but ended by saying that the enzyme’s reputation as “intransigent and inefficient” is a lie. Why? It now appears that “Rubisco is well adapted to substrate availability in contrasting habitats.” This means its inefficiency is really disguised adaptability.
Experimenters thought they could “improve” on Rubisco by mutating it. They found that their slight alterations to the reactivity of the enediol intermediate drastically favored the less-desirable oxygenase reaction. This only served to underscore the contortions the molecule must undergo to optimize the carboxylase reaction:

“Such observations provided the key to the idea that in the active site the enediol must be contorted to allow CO2 to attack more readily despite the availability of O2 molecules. The more the enediol mimics the carboxylate end-product, Tcherkez et al. conclude, the more difficult it is for the enzyme to free the intermediate from the active site when the reaction is completed. When the specificity factor and selectivity for CO2 are high, the impact on associated kinetic properties is greatest: kcat [i.e., the rate of enzyme catalytic events per second] becomes slower.
So, rather than being inefficient, Rubisco has become highly tuned to match substrate availability.”

Another finding about the inner workings of Rubisco bears on dating methods and climate models. Scientists have known that Rubisco favors the lighter, faster-moving carbon isotope 12C over 13C. By measuring the ratio of these stable isotopes in organic deposits, paleoclimatologists have inferred global carbon dioxide abundances and temperatures (knowing that Rubisco processes the isotopes differently). That assumption may be dubious:

“Several other correlates are also explained by this relationship. For instance, Rubisco discriminates more against 13C than against 12C, the two naturally occurring stable isotopes in CO2. But when the specificity factor is high, the 13C reaction intermediate binds more tightly, and so carbon isotope discrimination is higher (that is, less 13C is incorporated); in consequence, the carbon-isotope signals used to reconstruct past climates should perhaps now be re-examined. In contrast, higher ambient temperatures (30-40 °C) reduce the stability of the enediol, and Rubisco oxygenase activity and photorespiration rate increase.”

Those considerations aside, Griffiths is most interested in two things: how this enzyme evolved, and whether we can improve on it. If we can raise its carboxylation efficiency, we might be able to increase crop yields. So far, genetic engineers have not succeeded.3
As for the evolution of Rubisco, he mentions three oddball cases but fails to explain exactly how they became optimized for their particular circumstances – only that they are optimized. Yet their abilities seem rather remarkable. For instance, though the “least efficient” forms of Rubisco reside in microbes living in anaerobic sediments, where oxygen competition is not a problem, “One bacterium can express all three catalytically active forms (I, II and III), and switches between them depending on environmental conditions.” In another real-world case, “some higher plants and photosynthetic microorganisms have developed mechanisms to suppress oxygenase activity: CO2-concentrating mechanisms are induced either biophysically or biochemically.” In another example, “Rubisco has not been characterized in the so-called CAM plants, which use a form of photosynthesis (crassulacean acid metabolism) adapted for arid conditions.” These plants, including cacti and several unrelated species scattered throughout the plant kingdom, have other mechanisms for dealing with their extreme environments. In every mention of evolution, therefore, Griffiths assumed it rather than explaining it: viz., “The systematic evolution of enzyme kinetic properties seems to have occurred in Rubisco from different organisms, suggesting that Rubisco is well adapted to substrate availability in contrasting habitats.”
So, can we improve on it? If so, given all the praise for what evolution accomplished, Griffiths seems oblivious to the implications of his own concluding sentence:

“Other research avenues include manipulating the various components of Rubisco and cell-specific targeting of chimaeric Rubiscos. Potential pitfalls here are that the modified Rubisco would not only have to be incorporated and assembled by crop plants, but any improved performance would have to be retained by the plants. Finally, one suggestion is that we should engineer plants that can express two types of Rubisco – each with kinetic properties to take advantage of the degree of shading within a crop canopy. Such rational design would not only offer practical opportunities for the future, but also finally give the lie to the idea that Rubisco is intransigent and inefficient.”

What, students, is a synonym for “rational design”?
1Tcherkez et al., “Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized,” Proceedings of the National Academy of Sciences USA, published online before print April 26, 2006, 10.1073/pnas.0600605103 PNAS | May 9, 2006 | vol. 103 | no. 19 | 7246-7251.
2Howard Griffiths, “Plant biology: Designs on Rubisco,” Nature 441, 940-941 (22 June 2006) | doi:10.1038/441940a; Published online 21 June 2006.
3If and when they do, the benefit would be tuned for humans and their livestock, not necessarily for the ecology or atmosphere.

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Cell Untangles Its Own DNA 07/17/2006
DNA is packed like spaghetti in a basketball (07/28/2004), but must constantly be accessed by transcribers, duplicators and other molecular machines. Scientists at the Karolinska Institute, according to EurekAlert, have found a complex of protein machines that know how to untangle DNA. Machines that can keep DNA from separating too early (cohesins) and keep DNA coils compact (condensins) have been studied extensively, but these scientists looked more at another mechanism. When they artificially perturbed DNA strands, the machines went to work fixing the damage:

“The research group has studied the third, less well understood, protein complex, known as the Smc5/6 complex. This protein complex was found to bind to locations on the DNA strand that the researchers had artificially damaged, suggesting that it is directly involved in the repair process. Moreover, the Smc5/6 complex also seems to be required for the disentanglement of undamaged chromosomes before cell division. If these tangles, which are a natural consequence of the DNA copying process, are left unresolved the chromosomes cannot be separated and sent to the two nascent daughter cells. Like in the repair process, the Smc5/6 complex appears to resolve these intertwines by direct interaction with the DNA molecules, but this process is differently regulated as compared to the function in repair.”

The press release starts with a “wow” factoid: “Every second, the cells constituting our bodies are replaced through cell division. An adult human consists of about 50,000 billion cells, 1% of which die and are replaced by cell division every day.” Machines like the Smc5/6 complex are essential to maintaining our genomic integrity.

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Cell Backup Systems Challenge Evolution, Show Design Principles 07/21/2006
Has an intelligent design paper been published in the Proceedings of the National Academy of Sciences?1 Read the abstract and decide whether this research supports Darwinism or design:

“Functional redundancies, generated by gene duplications, are highly widespread throughout all known genomes. One consequence of these redundancies is a tremendous increase to the robustness of organisms to mutations and other stresses. Yet, this very robustness also renders redundancy evolutionarily unstable, and it is, thus, predicted to have only a transient lifetime. In contrast, numerous reports describe instances of functional overlaps that have been conserved throughout extended evolutionary periods. More interestingly, many such backed-up genes were shown to be transcriptionally responsive to the intactness of their redundant partner and are up-regulated if the latter is mutationally inactivated. By manual inspection of the literature, we have compiled a list of such “responsive backup circuits” in a diverse list of species. Reviewing these responsive backup circuits, we extract recurring principles characterizing their regulation. We then apply modeling approaches to explore further their dynamic properties. Our results demonstrate that responsive backup circuits may function as ideal devices for filtering nongenetic noise from transcriptional pathways and obtaining regulatory precision. We thus challenge the view that such redundancies are simply leftovers of ancient duplications and suggest they are an additional component to the sophisticated machinery of cellular regulation. In this respect, we suggest that compensation for gene loss is merely a side effect of sophisticated design principles using functional redundancy.”

The three authors, all from the Weizmann Institute in Rehovot, Israel, speak freely of the evolution of this phenomenon in their paper; they also, interestingly, refer to design and design principles just as often:

“In particular, we suggest the existence of regulatory designs that exploit redundancy to achieve functionalities such as control of noise in gene expression or extreme flexibility in gene regulation. In this respect, we suggest that compensation for gene loss is merely a side effect of sophisticated design principles using functional redundancy.
Clues for regulatory designs controlling redundancy were obtained first in a recent study...”

They call these cases of functional redundancy responsive backup circuits (RBCs). Interestingly, they found some cases where one RBC is regulated by another RBC. Though often the two backup copies were differently regulated, they could become coregulated under certain environmental conditions. The team also found that some of these functionally redundant genes are found all the way from yeast to mammals; this is sometimes called “evolutionary conservation” but actually describes stasis, not evolution.
The authors do not deny that these backup systems evolved somehow: “For a single cell, the ability to quickly and efficiently respond to fluctuating environments is crucial and offers an obvious evolutionary advantage,” they postulate, suggesting that accidental duplication of genes was co-opted for this purpose. They do not get into any details of how this might have happened, however, and their analysis seems more interested on the complexity and design benefit of the systems.
Their criteria for functional backups were stated thus: “Two lines of evidence could indicate a function’s direct benefit from existing redundancy: first is the evolutionary conservation of the functional overlap, and second is a nontrivial regulatory design that utilizes it.” How many such systems exist in nature they could not say, because there have not been enough studies. Many functionally equivalent copies of enzymes (isozymes) are known. The genes that produce them are often regulated by different pathways. Under stress, however, some can become coregulated to provide robustness against environmental irregularities or damaging mutations.

“The model that emerges is that although many isozymes are specialized for different environmental regimes, alarm signals induced by particular stress stimuli may call for their synergistic coexpression. Here, RBCs provide functional specialization together with extreme flexibility in gene control that could be activated when sufficient stress has been applied. For example, in yeast, glucose serves as a regulatory input for alternating between aerobic and anaerobic growth. Its presence is detected by two separate and independent signaling pathways, one probing intracellular glucose concentrations and the other probing extracellular concentrations.”

They searched the literature and found several interesting ones that are described in detail in the paper. “In all these cases, the common denominator is that one of the two duplicates is under repression in wild type and that that repression is relieved upon its partner’s mutation.”
This raises an interesting question – one that could have been asked by someone in the intelligent design movement. They even answer a possible objection with a design principle:

“The extent to which genomic functional redundancies have influenced the way we think about biology can be appreciated simply by inspecting the vast number of times the word “redundancy” is specifically referred to in the biomedical literature (Fig. 5, which is published as supporting information on the PNAS web site). Particularly interesting is the abundance with which it is addressed in studies of developmental biology (Fig. 5). In fact, it is here that concepts such as “genetic buffering” and “canalization” first had been suggested. Furthermore, the robustness of the developmental phenotypes such as body morphologies and patterning have been repeatedly demonstrated. So the question is, are these redundancies simply leftovers of ancient duplications, or are they an additional component to the sophisticated machinery of cellular regulation?
In criticism, one may argue that many of the reported redundancies do not actually represent functionally equivalent genes but rather reflect only partial functional overlap. In fact, knockout phenotypes have been described for a number of developmental genes that have redundant partners. For these reasons, it has been suggested to define redundancy as a measure of correlated, rather than degenerate, gene functions. Although these facts may suggest that redundancies have not evolved for the sake of buffering mutations, it has, in our opinion, little relevance to the question of whether they serve a functional role. The interesting question is, then, can such a functional role for the duplicated state be inferred from the way the two genes are regulated?”

Along that line, they found that the amount of upregulation of one gene was often dependent on the regulation of the other. This suggested to them that the sum of the expression of the two copies is nearly constant as a buffer against noise in the system. When one line gets noisy, due to a mutation, the other responds with more signal. They call this “dosage-dependent linear response.” In some cases during development, the responsive overlap decreases as the organism grows. In short, “The abundance of redundancies occurring in genes related to developmental processes, and their functional role as master regulators (Fig. 5) may be taken to suggest their utilization in either the flexibility or robustness of regulatory control.”
Some examples they give are even more complex. RBCs may also be implicated in the resistance of some organisms to multiple drugs. In some cases, each isoform can compensate equally for the other; in others, one of the forms is the main (the controller) and the other acts as the backup (the responder), only coming into play when the primary goes sour. “One of the most profound and insightful of these recurring regulatory themes,” they exclaim, “is that, although both genes are capable of some functional compensation, disruption of the responder produces a significantly less deleterious phenotype than disruption of the controller”. In evolutionary terms, why would the backup copy be better?

“A simple potential interpretation may suggest that although the controller is the key player performing some essential biological role, the responder is merely a less efficient substitute. Yet, accepting the notion that redundancy could not have evolved for the sake of buffering mutations, this interpretation still is severely lacking.
A different, and more biologically reasonable, hypothesis accounting these asymmetries is that one of the functions of the responder is to buffer dosage fluctuations of the controller. This buffering capacity requires a functional overlap that also manifests itself in compensations against the more rare event of gene loss. Other models accounting for this assymetry are discussed further in this work, but our main point of argument is that this complex regulation of functionally redundant, yet evolutionarily conserved genes, strongly indicates utilization of redundancy.”

Their next subsection is called “Regulatory Designs.” What emerges from their discussion of how each gene can regulate its partner is a complex picture: in one case, “redundancy is embedded within a more complex interaction network that includes a unidirectional responsive circuit in which the controller (dlx3) also represses its own transcription, whereas the responder (dlx7) is a positive autoregulator.” More examples like this are described. They predicted, and found, that RBCs could also regulate “downstream processes from variation and fluctuations arising from nongenetic noise.” The net result is that by using these functional backup systems, the organism has more robustness against perturbations, yet more flexibility in a dynamic environment.
What is the fruit of this research? Why should scientists look for these “regulatory designs” in the cell? They offer an intriguing example. It is known that one form of human muscular dystrophy occurs when a member of an RBC suffers a mutation. Studies of this pair in mice, however, shows that the other member can respond by upregulating its expression. It is thought a similar response might occur in humans. “Inspired by the compensatory effect demonstrated by this RBC in mice, its artificial induction in humans by means of gene therapy has been suggested. Although such modalities have not yet been realized, they suggest a fruitful possibility.”
1Kafri, Levy and Pilpel, “The regulatory utilization of genetic redundancy through responsive backup circuits,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0604883103, published online before print July 21, 2006

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Self-Correcting RNA: Is It a Missing Link? 07/28/2006
A team of Russian scientists at Rutgers discovered a remarkable phenomenon: RNA that proofreads itself during its own synthesis. The work was reported in Science1: “We show that during transcription elongation, the hydrolytic reaction stimulated by misincorporated nucleotides proofreads most of the misincorporation events and thus serves as an intrinsic mechanism of transcription fidelity.” It has already been known that DNA transcription and translation includes a whole suite of error-correcting mechanisms, but this is the first instance of RNA self-correction.
The researchers did not comment on the evolution of this capability except to state that it “is likely evolutionarily conserved” (i.e., unevolved in all living organisms), and that in an RNA-protein world, a “proofreading and repair mechanism similar to the one described here could have allowed a large RNA genome of the last common universal ancestor to exist.” This is because without an accurate proofreading mechanism even in an RNA world, duplication fidelity would have been too low for evolution: “the relatively low fidelity of RNAP-catalyzed synthesis could not have been sufficient for stable maintenance of large RNA genomes in the absence of cleavage factors.”
Patrick Cramer (Gene Center Munich), however, writing in the same issue of Science,2 launched their final, speculative paragraph into a story of how this RNA must be a missing link. Starting with the admission that “Precision can be vital,” Cramer immediately invoked the E word: “cells have evolved processes for proofreading and correction to shut down the propagation of errors” in the DNA-to-protein pathway. Referring to the work by Zenkin et al., he said, “This finding helps to explain the fidelity of gene transcription and suggests that self-correcting RNA was the genetic material during early evolution.”
But how, exactly, could that have come about? In his missing-link story, notice how many times Cramer used speculation words like could, probably and suggests compared to the hard requirements of reality:

“The discovery of self-correcting RNA transcripts suggests a previously missing link in molecular evolution. One prerequisite of an early RNA world (devoid of DNA) is that RNA-based genomes were stable. Genome stability required a mechanism for RNA replication and error correction during replication, which could have been similar to the newly described RNA proofreading mechanism described by Zenkin et al. If self-correcting replicating RNAs coexisted with an RNA-based protein synthesis activity, then an early RNA-based replicase could have been replaced by a protein-based RNA replicase. This ancient protein-based RNA replicase could have evolved to accept DNA as a template, instead of RNA, allowing the transition from RNA to DNA genomes. In this scenario, the resulting DNA-dependent RNA polymerase retained the ancient RNA-based RNA proofreading mechanism.
Whereas an understanding of RNA proofreading is only now emerging, DNA proofreading had long been characterized. DNA polymerases cleave misincorporated nucleotides from the growing DNA chain, but the cleavage activity resides in a protein domain distinct from the domain for synthesis. The spatial separation of the two activities probably allowed optimization of two dedicated active sites during evolution, whereas RNA polymerase retained a single tunable active site. This could explain how some DNA polymerases achieve very high fidelity, which is required for efficient error correction during replication of large DNA genomes.”

Of course, being only a “scenario” for how proofreading “could” have evolved, Cramer offered no evidence, lab or otherwise, for such a self-correcting RNA “missing link.” For a discussion of problems with the RNA-world scenario, see the 07/11/2002 entry.
1Zenkin, Yuzenkova and Severinov, “Transcript-Assisted Transcriptional Proofreading,” Science, 28 July 2006: Vol. 313. no. 5786, pp. 518 - 520, DOI: 10.1126/science.1127422.
2Patrick Cramer, “Perspectives: Molecular Biology: Self-Correcting Messages,” Science, 28 July 2006: Vol. 313. no. 5786, pp. 447 - 448, DOI: 10.1126/science.1131205.

bob b

Science Lover
Hall of Fame
Bacteria Rule the World – Benevolently 08/02/2006
We should love bacteria, not annihilate them. Bacteria are our friends, according to Dianne K. Newman of Caltech:1

“As a microbiologist, I’m appalled when I go to buy soap or dishwashing detergent, because these days it’s hard to find anything that doesn’t say ‘antibacterial’ on it.... It’s a commonly held fallacy that all bacteria are germs, but it’s been estimated that out of more than 30 million microbial species, only 70 are known to be pathogens. That’s a trivial number. The vast majority are actually doing remarkable things, both for the quality of our life and for the quality of the planet.”

We couldn’t annihilate them, anyway, if we wanted to. They are the most widespread and hardiest organisms on earth. Maybe you heard on the news today that there are more bacteria on your cell phone than on a toilet seat. Better to get used to it; they’re everywhere.
The realization that bacteria rule the world began when Leeuwenhoek found more organisms on his teeth than men in a kingdom. Newman continues:

“Leeuwenhoek underestimated. Not only do they exceed the number of men and women in a kingdom, they go far beyond that. We have anywhere from 5 million to 50 million bacteria per square inch on our teeth, and over 700 microbial species living in our mouths. Most of them are aiding us in our digestion—as are the 300 billion bacteria living in each gram of our colon. The palms of our hands have between 5,000 and 50,000 organisms per square inch, although that’s nothing compared to the skin of our groin and armpit areas, which as at least 5 million per square inch.
The grand total per person is about 70 trillion (70 x 10exp12), so we’re really walking vats of bacteria. There are 10 times the number of microbial cells in an adult body than there are human cells, and the gut microbiome alone is estimated to contain more than a hundred times the number of genes that we have in our own genome—so there’s a remarkable amount of metabolic diversity living within us. We shouldn’t be alarmed by this, however, because most of these bacteria are our friends.”

If you are sufficiently grossed out by the revelation that you are a zoo, consider that the animals in a zoo represent just a tiny fraction of life on earth:

“As well as living on and within animals, microbes live in plants, oceans, rivers, lakes, aquatic sediments, soils, subsoils, and air. The total number of microbes on the planet has been estimated at 5 x 10exp30, which is an enormous number. If they were all lined up end to end in a chain, it would stretch to the sun and back 200 x 10exp12 times.

A related article on BBC News noted the remarkable diversity of microbes. “One litre of seawater can contain more than 20,000 different types of bacteria,” the article begins, suggesting that microbial diversity is much greater than imagined.
Most of Dianne Newman’s delightful article is concerned with her Caltech team’s research into the amazing metabolic properties of certain bacteria that can live on rust as well as oxygen. She talks about bacteria that can generate light, orient by magnetic fields, and help larger organisms in numerous ways. Her colorful prose, unfortunately, is marred here and there by evolutionary stories that qualify for Stupid Evolution Quote of the Week:

• They invented oxygenic photosynthesis...
• Over the course of time, these types of cyanobacteria became engulfed by other organisms that then evolved into plants...
• ...the chloroplast, is nothing more than an ancient cyanobacterium.
• Moreover, we can only breathe this oxygen because our mitochondria—the little organelles in our cells that produce energy—are vestigial microorganisms descended from another ancient bacterium.
• Microbes are very, very old. They’ve been on our planet for at least 3.8 billion years, appearing just 800 million years after the planet formed. for the first 1.6 billion years or so of their existence, they had the place to themselves, and it was only after the oxygenation of the air and oceans by the cyanobacteria that the forerunners of plants and animals came along.
• The reason we find microbes almost everywhere we look is because, over the billions of years of Earth’s history they’ve been around, they’ve figured out how to be fantastic chemists.

Perhaps this is one of the reasons that Kansas school board member Connie Morris, who was just voted out of office (see yesterday’s entry), often described evolution as “a nice bedtime story.”
1Dianne K. Newman, “Bacteria Are Beautiful,” Caltech Engineering & Science (LXIX:2), Aug. 2006, pp. 8-15.

bob b

Science Lover
Hall of Fame
How Useful Is Evolutionary Theory to Biology? 08/04/2006
A favorite quote by evolutionists is the line by Theodosius Dobzhansky, “Nothing in biology makes sense except in the light of evolution.” Why, then, do so many biological papers fail to mention evolution at all? Indeed, many employ design language, sometimes with a sense of awe. Here are more recent examples in which the E word was missing (or inconsequential) in the glare of amazement over complex design:

Charged with pain: Wounds generate electric fields that guide repair crews to the site. Science Now got a charge out of this: “Talk about healing energy,” reporter Laura Blackburn challenged the faith healers. “Every wound, from the tiniest scratch to the nastiest gash, generates an electric field that pulls in cells that help repair the damage.”

Rotary switch: A team publishing in PNAS1 discussed the ID Movement’s favorite biological toy, the bacterial flagellum. They considered the switching mechanism that allows the propeller to go into reverse. Their paper sounds like something out of Popular Mechanics: “Structure of FliM provides insight into assembly of the switch complex in the bacterial flagella motor.”

Checkpoint, no Charlie: M. Andrew Hoyt appreciates even more the way the cell uses checkpoints to make sure division occurs without error. In Science2 he examined a new answer to how the cell switches this control on and off:

“Paradoxically, the mechanism responsible for separation of the chromosomes at anaphase itself creates chromosome attachments that the checkpoint would normally recognize in metaphase as improper. Yet, the cell cycle proceeds naturally unimpeded; these improper chromosome attachments fail to activate the cycle-blocking activity of the spindle checkpoint after anaphase onset. From a clever series of experiments reported on page 680 of this issue by Palframan et al., we now know why. In anaphase cells, the actions of the spindle checkpoint are extinguished by the very same protein complex that previously was the target of its anaphase-inhibitory activity.”

Hoyt did also speak of “conserved” (i.e., unevolved) proteins of the spindle checkpoint, but had no other references to evolution.
Stretchy Clots: Another paper in Science3 examined the properties of fibrin, one of the principle ingredients in blood clots, and found that they have “extraordinary extensibility and elasticity.”

“Blood clots perform an essential mechanical task, yet the mechanical behavior of fibrin fibers, which form the structural framework of a clot, is largely unknown. By using combined atomic force-fluorescence microscopy, we determined the elastic limit and extensibility of individual fibers. Fibrin fibers can be strained 180% (2.8-fold extension) without sustaining permanent lengthening, and they can be strained up to 525% (average 330%) before rupturing. This is the largest extensibility observed for protein fibers. The data imply that fibrin monomers must be able to undergo sizeable, reversible structural changes and that deformations in clots can be accommodated by individual fiber stretching.”

Readers of the primary intelligent design book Darwin’s Black Box might remember the blood clotting system as one example Michael Behe used of irreducible complexity.

When evolution is mentioned in papers dealing with complex, interacting systems in biology, the references often seem imprecise and incidental to the work that went into the research, as if tacked on as an afterthought. For instance, R. John Ellis, writing in Nature July 27,4 described the details of the protein-folding chaperone complex, Gro-EL and Gro-ES. After describing in some detail the specifications of these versatile molecular machines, noting that “both the size and surface charge of the cage are optimized to speed up the folding of several different types of chain,” he referred to evolution on only two places, both speculative, and both personifying natural selection as the wizard of technology:

“The size and surface properties of the cage represent an evolutionary compromise that helps the bacterial cell to produce functional proteins fast enough to survive in a competitive microbial world.....
It is a testament to the ingenuity of natural selection that the chaperonin cage not only combats aggregation caused by crowding outside the cage but also uses crowding to accelerate protein folding inside the cage. Nanoengineers trying to improve the yield of therapeutic proteins could profit from studying the tricks of the chaperonin nanocage.”

Go figure.
1Park et al., “Structure of FliM provides insight into assembly of the switch complex in the bacterial flagella motor,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0602811103, published online before print August 1, 2006.
2Palframan et al., “Anaphase Inactivation of the Spindle Checkpoint,” Science, 4 August 2006: Vol. 313. no. 5787, pp. 680 - 684, DOI: 10.1126/science.1127205.
3Liu et al., “Fibrin Fibers Have Extraordinary Extensibility and Elasticity,” Science, August 2006: Vol. 313. no. 5787, p. 634, DOI: 10.1126/science.1127317.
4R. John Ellis, “Protein folding: Inside the cage,” Nature 442, 360-362(27 July 2006) | doi:10.1038/442360a; Published online 26 July 2006.

bob b

Science Lover
Hall of Fame
Another Flagellum Excites Scientists 09/01/2006
“The bacterial flagellar motor excites considerable interest because of the ordered expression of its genes, its regulated self-assembly, the complex interactions of its many proteins, and its startling mechanical abilities,” begins a paper in Nature by three Caltech scientists.1 They performed electron cryotomography imaging on the flagella of Triponema primita, a different critter with a different model from the flagellum found in E. coli, the favorite toy of microbiologists (with an outboard motor that is an icon of the intelligent design movement). Treponema is a little spirochete that lives in the hindgut of termites. It has two flagella, one at each end, that apparently rotate on its inside and make the organism gyrate rather than swim through liquid.
The Caltech team got good images of the stator for the first time. Their exterior and cross-section illustrations show a multi-part circular structure with 16-fold symmetry and complex contours, with rings and other parts of unknown function. This particular motor apparently operates in low gear. It is larger than the E. coli or Salmonella flagella and apparently runs at much higher torque.

“These differences have important implications for current models of the functional and architectural relationships of the components. Whereas the Salmonella motor spins just the flagellum, because Treponema flagella are periplasmic, it is thought that they cause the whole cell to gyrate. Thus, each rotation may be much slower and require greater torque. The unusually large stud ring, C ring and rotor in Treponema may serve to increase torque by increasing the length of the effective lever arm through which each stator stud acts. These larger rings may also accommodate more stator studs and FliG molecules around the ring, in effect ‘gearing down’ the Treponema motor so that the passage of each proton across the membrane produces a smaller angular rotation.”

The paper includes a link to an animation video that shows the motor in operation from different angles. The authors talk a lot about machine specs, but don’t mention anything about evolution.
1Murphy, Leadbetter and Jensen, “In situ structure of the complete Treponema primitia flagellar motor,” Nature 442, 1062-1064(31 August 2006) | doi:10.1038/nature05015.

Yoke Up Those Bacteria [/i] 09/06/2006
My, how history repeats itself – often in unexpected ways. In ancient times, our ancestors got the heavy work done by hitching oxen, horses or slaves (like Samson, see pictures 1 and 2) to a harness and making them turn a grinding wheel. The same principle is now on the cutting edge of modern applied biological engineering – only now, the movement is measured in micrometers, and the beasts of burden are bacteria. Scientists in Japan, publishing in PNAS,1 have successfully hitched their harnesses to multi-legged crawlers named Mycoplasma mobile and made them turn a gear 20 micrometers wide, many times their size.
In the contraption rigged by Hiratsuka et al., the bacteria walk inside a circular track, pushing a six-petal rotor made of silicon dioxide above them. The inventors (slavedrivers?) developed a surface that would ensure the majority of the cell “microtransporters” would move in one direction with the right amount of friction. The cooperative workers achieved forces of 2 to 5 x 10exp-16 newton-meters, with rotation rates of 1.5 to 2.6 rpm. “To the best of our knowledge,” they boasted with some merit, “a micromechanical device that integrates inorganic materials with living bacteria has not succeeded until this study.” (They did, however, reference the PNAS research reported in our 08/19/2005 entry, “Saddle Up Your Algae.”)
The inventors didn’t mention evolution once in their paper. Instead, they spoke in glowing terms about their little microscopic oxen and marveled at their technology. First, they scanned the arena of biological micro-machinery with the delight of a gadget freak:

“Nature provides numerous examples of nanometer-scale molecular machines. In particular, motor proteins, which efficiently convert chemical energy into mechanical work, are fascinating examples of functional nanodevices derived from living systems. The molecular mechanism underlying the function of these motors has long been a major focus of biophysical research, and the information emerging from those studies should greatly aid in the design and fabrication of novel synthetic micro/nanomotors....
Turning an eye to higher-order biological structures reveals many examples of excellent mechanical devices, including bacterial and eukaryotic flagella and muscle sarcomeres. These motile units are tens of nanometers to several micrometers in size and consist of multiprotein complexes built up with atomic accuracy through the self-assembly and self-organization of protein molecules within cells. In general, these devices work far more efficiently and intelligently than the isolated proteins but, because the principles and mechanisms of self-assembly are only vaguely understood, we are currently unable to assemble higher order motile units from the isolated component proteins outside the cells. Consequently, research aimed at developing hybrid devices using biological motile units is rare at present.”

How about the machines employed by their chosen beast of burden? The praise service continues:

“Mycoplasma mobile, a species of gliding bacteria, is another example of a higher-order unit (cells in this case) with superb motility. M. mobile has a pear-shaped cell body ~ 1 micrometer in length and moves continuously over solid surfaces at speeds up to 2-5 micrometers per second. The mechanism by which it glides remains unknown, although a mechanical walking model that makes use of the rod-like structures protruding from the cell surface has been proposed. Although three proteins have been identified as essential for gliding, we speculate that this motile system may need a dozen additional proteins, including various cytoskeletal proteins.”

So why reinvent the wheel? Why go to all the trouble to invent walking nanorobots, when bacteria have it all figured out? The inventors list other reasons for enlisting biological beasts of burden instead of trying to start from scratch:

“As a result, it is currently impractical, if not impossible, to reconstitute fully functional motile units from the isolated proteins of M. mobile in vitro. For that reason, we have been attempting to construct micromechanical devices using intact M. mobile cells instead of the isolated proteins. A key benefit of this approach is that hybrid devices into which living cells are integrated enable us to take advantage of preassembled excellent motor units that have the potential for self-repair or self-reproduction when damaged.”

So there you go: spare parts and repairs come included with the package. Oxen must be fed, however, and they didn’t talk about that (cf. Solomon). Someone else may have to invent the nanomanger.
1Hiratsuka et al., “Applied Biological Sciences: A microrotary motor powered by bacteria,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0604122103, published online before print September 1, 2006.
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