The pathway for initiation of protein synthesis in eukaryotic cells has been defined and refined over the last 25 years using purified components and in vitro reconstituted systems. More recently, powerful genetic analysis in yeast has proved useful in unraveling aspects of translation inherently more difficult to address by strictly biochemical approaches. One area in particular is the functional analysis of multi‐subunit protein factors, termed eukaryotic initiation factors (eIFs), that play an essential role in translation initiation. eIF‐3, (...) the most structurally complex of the eIFs, has until recently eluded this approach. The identification of the yeast GCD10 gene as the structural gene for the ζ subunit of yeast eIF‐3(1) and the analysis of mutant phenotypes has opened the door to the genetic dissection of the eIF‐3 protein complex. (shrink)

Dormancy or hibernation is a non‐proliferative state of cells with low metabolic activity and gene expression. Dormant cells sequester ribosomes in a translationally inactive state, called dormant/hibernating ribosomes. These dormant ribosomes are important for the preservation of ribosomes and translation shut‐off. While recent studies attempted to elucidate their modes of formation, the regulation and roles of the diverse dormant ribosomal populations are still largely understudied. The mechanistic details of the formation of dormant ribosomes in stress and especially their disassembly during (...) recovery remain elusive. In this review, we discuss the roles of dormant ribosomes and their potential regulatory mechanisms. Furthermore, we highlight the paradigms that need to be answered in the field of ribosomal dormancy. (shrink)

Early research on the cell cycle revealed correlations between protein accumulation and cell proliferation. In this review, I describe the data showing that abnormality of cell growth and tumor development are dependent upon oncogene‐induced increases in the levels and activity of factors that determine the rate of protein synthesis. It is proposed that the establishment of a vicious circle, namely oncoproteins → increase in translation → oncoproteins, is a major biological mechanism that fuels neoplastic growth. The constitutively (...) high rates of protein synthesis and accumulation of proteins, including those necessary for DNA replication and mitosis, would drive cells to excessive proliferation. (shrink)

The final assembly of the protein synthesis machinery occurs during translation initiation. This delicate process involves both ends of eukaryotic messenger RNAs as well as multiple sequential protein–RNA and protein–protein interactions. As is expected from its critical position in the gene expression pathway between the transcriptome and the proteome, translation initiation is a selective and highly regulated process. This synopsis summarises the current status of the field and identifies intriguing open questions. BioEssays 25:1201–1211, 2003 © (...) 2003 Wiley Periodicals, Inc. (shrink)

It has been known for 20 years that the ribosomal protein S6 is rapidly phosphorylated when cells are stimulated to grow or divide. Furthermore, numerous studies have documented that there is a strong correlation between increases in S6 phosphorylation and protein synthesis, leading to the idea that S6 phosphorylation is involved in up‐regulating translation. In an attempt to define a mechanism by which S6 phosphorylation exerts translational control, other studies have focused on characterizing the sites of phosphorylation (...) of this protein and its location within the ribosome. Recent data show that S6 is a protein which may have diverse cellular functions and is essential for normal development, and that it may be involved in the translational regulation of a specific class of messages. (shrink)

In the 1950s and 1960s, an interfield interaction between molecular biologists and biochemists integrated important discoveries about the mechanism of protein synthesis. This extended discovery episode reveals two general reasoning strategies for eliminating gaps in descriptions of the productive continuity of mechanisms: schema instantiation and forward chaining/backtracking. Schema instantiation involves filling roles in an overall framework for the mechanism. Forward chaining and backtracking eliminate gaps using knowledge about types of entities and their activities. Attention to mechanisms highlights salient (...) features of this historical episode while providing general reasoning strategies for mechanism discovery. (shrink)

Applying mild methods of preparation, part of the ribosomes of rabbit reticulocytes are found in aggregates (later called polyribosomes) of up to six ribosomal units. Upon treatment with RNA-ase, they desintegrate into single ribosomes. The fast-sedimenting aggregates are found to be more active in protein synthesis in terms of incorporation of radioactive amino acids, whereas the single ribosomes are more receptive to stimulation by the artificial messenger RNA poly-U. The findings indicate that the linkage of ribosomes into aggregates (...) is due to the messenger RNA. They support a tape-reading mechanism of protein synthesis whereby growth of the peptide chain is accompanied by shifting the active site of the ribosome from one coding group of nucleotides of the messenger RNA to the next. (shrink)

In this paper, we compare the mechanisms of protein synthesis and natural selection. We identify three core elements of mechanistic explanation: functional individuation, hierarchical nestedness or decomposition, and organization. These are now well understood elements of mechanistic explanation in fields such as protein synthesis, and widely accepted in the mechanisms literature. But Skipper and Millstein have argued that natural selection is neither decomposable nor organized. This would mean that much of the current mechanisms literature does not (...) apply to the mechanism of natural selection.We take each element of mechanistic explanation in turn. Having appreciated the importance of functional individuation, we show how decomposition and organization should be better understood in these terms. We thereby show that mechanistic explanation by protein synthesis and natural selection are more closely analogous than they appear—both possess all three of these core elements of a mechanism widely recognized in the mechanisms literature. (shrink)

In a previous paper, an integrated account of Rosen’s relational biology and Peirce’s semiosis has been proposed. Both theories have been compared and basic concepts have been posited for the definition of a unified framework for the study of biology, as well as a method for the identification and analysis of the presence of signs in an organism. The analysis of the existence of semiotic actions in an organism must, without a doubt, begin by considering each of the rules that (...) constitute the genetic code as a candidate for a semiotic relation. Transcription and translation, which constitute protein synthesis, are the basis of the specificity that the organism needs to maintain itself in its environment and reproduce, and the precondition of the existence of any other possible semiosis. Applying the concepts and method of the aforementioned work, this paper analyzes which of the biological processes involved in protein synthesis correspond to semiotic actions and the type of the signs identified, according to Peirce’s classification of icons, indices and symbols. The results of this work demonstrate the theoretical consistency and the practical utility of integrating the theories of Rosen and Peirce, offer a way to identify other signs in an organism, and support a critical analysis of code biology and protosemiosis, two accounts that deny the possibility of explaining the signs in an organism with Peirce’s semiosis. (shrink)

In biology proteins are uniquely important. They are not to be classed with polysaccharides, for example, which by comparison play a very minor role. Their nearest rivals are the nucleic acids....The main function of proteins is to act as enzymes....In the protein molecule Nature has devised a unique instrument in which an underlying simplicity is used to express great subtlety and versatility; it is impossible to see molecular biology in proper perspective until this peculiar combination of virtues has been (...) clearly grasped. (shrink)

The possibility of combining powerful genetic methods with biochemical analysis has made baker's yeast Saccharomyces cerevisiae the organism of choice to study the complex process of translation initiation in eukaryotes. Several new initiation factor genes and interactions between components of the translational machinery that were not predicted by current models have been revealed by genetic analysis of extragenic suppressors of translational initiation mutants. In addition, a yeast cell‐free translation system has been developed that allows in vivo phenotypes to be correlated (...) with in vitro biochemical activities. We summarize here the current view of yeast translational initiation obtained by these approaches. (shrink)

Insights into the role of sleep in the molecular mechanisms of memory consolidation may come from studies of activity-dependent synaptic plasticity, such as long-term potentiation (LTP). This commentary posits a specific contribution of sleep to LTP stabilization, in which mRNA transported to dendrites during wakefulness is translated during sleep. Brain-derived neurotrophic factor may drive the translation of newly transported and resident mRNA.

Barley aleurone cells have long served as a model system for studying the regulation of gene expression in plants. In this review we survey what is known about hormone‐regulated gene expression in aleurone cells. We also describe the effects of heat stress on gene expression in this system, and speculate how the aleurone cell prioritizes its response between hormone‐induced and environment‐induced programs of gene expression.

The living world is one of complexity, the result of innumerable interactions among organisms, cells, molecules. In analyzing a problem, the biologist is constrained to focus on a fragment of reality, on a piece of the universe which he arbitrarily isolates to define certain of its parameters.In biology, any study thus begins with the choice of a “system.” On this choice depend the experimenter's freedom to maneuver, the nature of the questions he is free to ask, and even, often, the (...) type of answer he can obtain. (shrink)

Replication protein A (RPA), the major single‐stranded DNA‐binding protein in eukaryotic cells, is required for processing of single‐stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high‐affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies (...) showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error‐prone repair of double‐stranded breaks and promote error‐free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. (shrink)

Over 80% of the genes in the E. coli chromosome express fewer than a hundred copies each of their protein products per cell. It is argued here that transcription of these genes is neither constitutive nor regulated by protein factors, but rather, induced by the act of replication. The utility of such replication‐induced (RI) transcription to the temporal regulation of synthesis of determinate quantities of low copy number (LCN) proteins is described. It is suggested that RI transcription (...) may be necessitated, as well as facilitated, by the folding of the bacterial chromosome into a compact nucleoid. Mechanistic aspects of the induction of transcription by replication are discussed with respect to the modulation of transcriptional initiation by negative supercoiling effects, promoter methylation status and derepression. It is shown that RI transcription offers plausible explanations for the constancy of the C period of the E. coli cell cycle and the remarkable conservation of gene order in the chromosomes of enteric bacteria. Some experimental tests of the hypothesis are proposed. (shrink)

Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone‐bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. (...) It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a “fix me” signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis. Also see the video abstract here: https://youtu.be/gkElfmqaKG4. (shrink)

The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and define protein folding on the ribosome, revealing principles that shape co‐translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on (...) nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated. (shrink)

Ribosome biogenesis includes the making and processing of ribosomal RNAs, the biosynthesis of ribosomal proteins from their mRNAs in the cytosol and their transport to the nucleolus to assemble pre‐ribosomal particles. Several stresses including cellular senescence reduce nucleolar rRNA synthesis and maturation increasing the availability of ribosome‐free ribosomal proteins. Several ribosomal proteins can activate the p53 tumor suppressor pathway but cells without p53 can still arrest their proliferation in response to an imbalance between ribosomal proteins and mature rRNA production. (...) Recent results on senescence‐associated ribogenesis defects (SARD) show that the ribosomal protein S14 (RPS14 or uS11) can act as a CDK4/6 inhibitor linking ribosome biogenesis defects to the main engine of cell cycle progression. This work offers new insights into the regulation of the cell cycle and suggests novel avenues to design anticancer drugs. (shrink)

The short paper introduces the concept of possible branches of double-stranded DNA (later sometimes called palindromes): Certain sequences of nucleotides may be followed, after a short unpaired stretch, by a complementary sequence in reversed order, such that each DNA strand can fold back on itself, and the DNA assumes a cruciform or tree-like structure. This is postulated to interact with regulatory proteins. -/- .

Studies of the G2 to M transition in amphibian oocytes, in combination with in vitro mitotic systems and yeast genetic analysis, have significantly contributed to our understanding of the mechanisms by which M‐phase is regulated. Historically, oocyte maturation has provided a number of valuable initial observations, but the biochemical elucidation of cell cycle control mechanisms has proved more tractable in cell‐free extracts of frog eggs which reproduce aspects of early embryogenic mitosis. Recent experiments examining the importance of protein (...) class='Hi'>synthesis in the maturing oocyte have highlighted some important differences between mitosis and meiosis. Additional controls found in meiosis but not embryonic mitosis, are similar to controls found in somatic cells. This suggests that understanding the differences, as well as the similarities, between meiosis in the oocyte and mitosis in the early embryo will help us to learn more about the way in which cells enter and leave mitosis. (shrink)

The synthesis of biological membranes requires the insertion of proteins into a lipid bilayer. The rough endoplasmic reticulum of eukaryotic cells is a principal site of membrane biogenesis. The insertion of proteins into the membrane of the endoplasmic reticulum is mediated by a resident proteinaceous machinery. Over the last five years several different experimental approaches have provided information about the components of the machinery and how it may function.

In this article two questions are discussed with regard to semiosis in protein biosynthesis for nano engines. (1) What kind of semiosis is involved in the construction of these proteins? and (2) How can we explain the semiotic process observed? With regard to the first issue we draw attention to comparisons between semiosis in protein biosynthesis and human natural language. The notion of normativity appears to be of great importance for both. A comparison also demonstrates differences. Nevertheless, because (...) of the normative symbolic information processing in it, we suggest to employ the term symbolic reference (employed in linguistics as a distinguishing feature of human language) to indicate the semiotic processes in protein biosynthesis. With regard to explaining semiosis in protein synthesis we compare different approaches. We conclude that a Kantian approach should be preferred. In such an approach strengths of the mechanistic and organicist approaches can be combined, and the observed symbolic information processing acknowledged. (shrink)

It is of fundamental importance to understand how the individual processes of gene expression, transcription, and translation, as well as mRNA and protein stability, act in concert to produce dynamic cellular proteomes. We use the concept of response times to illustrate the relation between degradation processes and responsiveness of the proteome to system changes and to provide supporting experimental evidence: proteins with short response times tend to be more strongly up‐regulated after 1 hour of TNFα stimulation than proteins with (...) longer response times. Furthermore, based on process‐dependent response times, we demonstrate that synthesis and degradation act in concert to enable rapid responses. Finally, by building on a previously published data set quantifying the mammalian gene expression cascade, we speculate on how combinations of stable and unstable mRNAs and proteins may be wired to transcriptional or translational regulation to support gene function. (shrink)

Earlier investigations of the hormonal control of vitellogenin (egg‐yolk protein) synthesis in mosquitoes relied on microsurgical manipulations to identify organs that secrete the various hormones that stimulate or inhibit vitellogenesis. Advances in the last 10 years, using radioimmunoassays, HPLC, mass spectrometry and tissue‐culture incubations, have shown that the control mechanism involves a complex interaction between ecdysteroids, juvenile hormones and peptide hormones.

The primary function of smooth muscle cells is to contract and alter the stiffness or diameter of hollow organs such as blood vessels, the airways and the gastrointestinal and urogenital tracts. In addition to purely structural functions, smooth muscle cells may play important metabolic roles, particularly in various inflammatory responses. In cell culture, these cells have been shown to be metabolically dynamic, synthesizing and secreting extracellular matrix proteins, glycosaminoglycans and a wide variety of cell–cell signaling proteins, such as interleukins, chemokines (...) and peptide growth factors. Secreted cell signaling proteins participate in the inflammatory response of smooth muscle‐containing organs, and some can also stimulate smooth muscle migration, proliferation and contraction. The cellular signaling pathways controlling synthesis of these signaling proteins are similar to those used by cells mediating innate immunity and may contribute to pathogenesis of diverse diseases including atherosclerosis, asthma, inflammatory bowel diseases and preterm labor. Appreciating the role of smooth muscle cells in these diseases may lead to better understanding of the beneficial effects of anti‐inflammatory drugs as well as identification of new targets for anti‐inflammatory therapy. BioEssays 26:646–655, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

The concept of the gene has been the central organizing theme of 20th century biology. Biology has become increasingly influential both for philosophers seeking a naturalized basis for epistemology, ethics, and the understanding of the mind, as well as for the human sciences generally. The central task of this work is to get the story right about genes and in so doing provide a critical and enabling resourse for use in the further pursuit of human self-understanding. ;The work begins with (...) a wide-ranging historical reconstruction and conceptual analysis of the meaning of "the gene" that results in defining and distinguishing two different "genes". Each of these can be seen as an heir to one of the two major historical trends in explaining the source of biological order---preformationism and epigenesis. The preformationist gene predicts phenotypes but only on an instrumental basis where immediate medical and/or economic benefits can be had. The gene of epigenesis , by contrast, is a developmental resource that provides templates for RNA and protein synthesis but has in itself no determinate relationship to organismal phenotypes. The seemingly prevalent idea that genes constitute information for traits is based, I argue, upon an unwarranted conflation of these two senses which is in effect held together by rhetorical glue. Beyond this historical, conceptual, and rhetorical inquiry the bulk of the dissertation then concerns itself with an empirically up-to-date analysis of the cell and molecular basis of biological order and of the pathological loss of same. In each of these chapters I structure my analysis with the idea in mind that the "conflated" view can be held empirically accountable. Major touchpoints in this work include the ideas of I. Kant, J. Blumenbach, K. E. von Baer, J. Muller, R. Virchow, W. Johannsen, R. Falk, J. Sapp, E. Schrodinger, M. Delbruick, G. Gamow, R. Doyle, L. Kay, S. Kauffman, E. Jablonka, J. Rothman, K. Yamamoto, P. Rous, H. Temin, J. M. Bishop, H. Harris, E. Stanbridge, D. L. Smithers, B. Vogelstein, E. Farber and H. Rubin. (shrink)

Mammalian AMP‐activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent (...) evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events. (shrink)

Introduction of human plasma protein genes into the mouse genome to produce transgenic mice furnishes an in vivo model for correlating chromosomal DNA sequences with developmental and tissue‐specific expression. The liver produces an array of plasma proteins that circulate throughout the body contributing to homeostasis. Non‐hepatic tissue sites of synthesis have been identified where a local provision of plasma proteins in needed. Analysis of expression of human plasma protein genes in ageing transgenic mice appears especialy promising in (...) identifying DNA sequences that respond to environmental adversities such as inflammatory factors, hormonal changes and metal toxicity. The results indicate that human genes encoding and controlling liver plasma proteins serve as useful models for studying genetic regulation in the background of development and ageing. (shrink)

Passage through the cell cycle requires the successive activation of different cyclin‐dependent protein kinases (CDKs). These enzymes are controlled by transient associations with cyclin regulatory subunits, binding of inhibitory polypeptides and reversible phosphorylation reactions. To promote progression towards DNA replication, CDK/cyclin complexes phosphorylate proteins required for the activation of genes involved in DNA synthesis, as well as components of the DNA replication machinery. Subsequently, a different set of CDK/cyclin complexes triggers the phosphorylation of numerous proteins to promote the (...) profound structural reorganizations that accompany the entry of cells into mitosis. At present, much research is focused on elucidating the links between CDK/cyclin complexes and signal transduction pathways controlling cell growth, differentiation and death. In future, a better understanding of the cell cycle machinery and its deregulation during oncogenesis may provide novel opportunities for the diagnostic and therapeutic management of cancer and other proliferation‐related diseases. (shrink)

One distinguishing feature of vertebrate oocyte meiosis is its discontinuity; oocytes are released from their prophase I arrest, usually by hormonal stimulation, only to again halt at metaphase II, where they await fertilization. The product of the c‐mos proto‐oncogene, Mos, is a key regulator of this maturation process. Mos is a serine‐threonine kinase that activates and/or stabilizes maturation‐promoting factor (MPF), the master cell cycle switch, through a pathway that involves the mitogen‐activated protein kinase (MAPK) cascade. Oocytes arrested at prophase (...) I lack detectable levels of Mos, which must be synthesized from a pool of maternal mRNAs for proper maturation. While Mos is necessary throughout maturation in Xenopus, it seems to be required only for meiosis II in the mouse. The translational activation of c‐mos mRNA at specific times during meiosis requires cytoplasmic polyadenylation. Cis‐ and trans‐acting factors for polyadenylation are, therefore, essential elements of maturation. (shrink)

The correct execution of the DNA replication process is crucially import for the maintenance of genome integrity of the cell. Several types of sources, both endogenous and exogenous, can give rise to DNA damage leading to the DNA replication fork arrest. The processes by which replication blockage is sensed by checkpoint sensors and how the pathway leading to resolution of stalled forks is activated are still not completely understood. However, recent emerging evidence suggests that one candidate for a sensor of (...) replication stress is ATR and that, together with a member of RecQ family helicases, Werner syndrome protein (WRN) and MRE11 complex, can collaborate to promote the restarting of DNA synthesis through the resolution of stalled replication forks. Here, we discuss how WRN, the MRE11 complex and the ATR kinase could work together in response to replication blockage to avoid DNA replication fork collapse and genome instability. BioEssays 26:306–313, 2004. © 2004 Wiley Periodicals, Inc. (shrink)

Neisseria gonorrhoeae, the bacterium that causes the sexually transmitted disease gonorrhea, demonstrates extensive antigenic heterogeneity in its surface components. The organism has the capacity to switch on and off the synthesis of different versions of components such as pili, outer membrane proteins, and lipopolysaccharide. Recent studies have shown that the gonococcus uses novel and complex mechanisms, of types not described previously, to store different versions of genetic information for surface proteins, and to regulate expression of those genes.

Protein secretion is conventionally viewed as taking place by either of two cellular routes, a regulated pathway, involving external stimuli and secretory granules, and a presumptive ‘constitutive’ pathway, which does not involve hormonal or neuronal stimuli or the production of secretory granules. The evidence reviewed here strongly suggests that there are post‐synthesis rate‐limiting steps for many proteins released by the ‘constitutive’ pathway and, hence, that regulation in some sense is involved here too. The nature of these rate‐limiting determinants (...) and events is discussed. (shrink)

The order of discovery can have a profound effect upon the way in which we think about the function of a gene. In E. coli, recA is nearly essential for cell survival in the presence of DNA damage. However, recA was originally identified, as a gene required to obtain recombinant DNA molecules in conjugating bacteria. As a result, it has been frequently assumed that recA promotes the survival of bacteria containing DNA damage by recombination in which DNA strand exchanges occur. (...) We now know that several of the processes that interact with or are controlled by recA, such as excision repair and translesion synthesis, operate to ensure that DNA replication occurs processively without strand exchanges. Yet the view persists in the literature that recA functions primarily to promote recombination during DNA repair. With the benefit of hindsight and more than three decades of additional research, we reexamine some of the classical experiments that established the concept of DNA repair by recombination, and we consider the possibilities that recombination is not an efficient mechanism for rescuing damaged cells, and that recA may be important for maintaining processive replication in a manner that does not generally promote recombination. BioEssays 23:463–470, 2001. © 2001 John Wiley & Sons, Inc. (shrink)

During the course of evolution, variations of a protein sequence is an ongoing phenomenon however limited by the need to maintain its structural and functional integrity. Deciphering the evolutionary path of a protein is thus of fundamental interest. With the development of new methods to visualize high dimension spaces and the improvement of phylogenetic analysis tools, it is possible to study the evolutionary trajectories of proteins in the sequence space. Using the data-driven high-dimensional scaling method, we show that (...) it is possible to predict and represent potential evolutionary trajectories by representing phylogenetic trees into a 3D projection of the sequence space. With the case of the aminodeoxychorismate synthase, an enzyme involved in folate synthesis, we show that this representation raises interesting questions about the complexity of the evolution of a given biological function, in particular concerning its capacity to explore the sequence space. (shrink)

To further comprehend how synthesis and assembly of myofibrillar components is regulated, several laboratories have undertaken genetic studies of muscle development in Drosophila melanogaster. This small fly lends itself well to classical and molecular genetic approaches, and possesses a set of muscle fibers, termed indirect flight muscles (IFM), which is particularly advantageous for such investigations. Structural and functional analyses of cloned Drosophila contractile protein genes have revealed that protein isoforms can be specified either by multigene families or (...) by differentially splicing primary transcripts of a single gene. Characterization of mutations which disrupt flight muscle development has so far revealed that profound abnormalities are caused by defective alleles of actin, tropomyosin, and myosin heavy‐chain genes. Muscle defects associated with particular alleles can be alleviated by integration of corresponding wild‐type gene copies into germ‐line chromosomes. Strategies for further applying genetic techniques to investigations of Drosophila muscle development are discussed. (shrink)

DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU‐IP‐ssSeq, strand‐specific sequencing technologies that permit analysis of (...) protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1‐Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA. (shrink)

Villin, a calcium‐regulated actin‐binding protein, modulates the structure and assembly of actin filaments in vitro. It is organized into three domains, the first two of which are homologous. Villin is mainly produced in epithelial cells that develop a brush border and which are responsible for nutrient uptake. Expression of the villin structural gene is precisely regulated during mouse embryogenesis and is restricted in adults, to certain epithelia of the gastrointestinal and urogenital tracts. The function of villin has been assessed (...) by transfecting CV1 cells with a human cDNA encoding wild‐type villin or mutant villin. Synthesis of large amounts of villin in cells which do not normally produce this protein induces the growth of microvilli on the cell surface and the redistribution of F‐actin, concomitant with the disappearance of stress fibers. The complete villin sequence is required for the morphogenic effect. These results suggest that villin plays a key role in the morphogenesis of microvilli. (shrink)

We suggest a new view of secretory and membrane protein folding that emphasizes the role of pathways of biogenesis in generating functional and conformational heterogeneity. In this view, heterogeneity results from action of accessory factors either directly binding specific sequences of the nascent chain, or indirectly, changing the environment in which a particular domain is synthesized. Entrained by signaling pathways, these variables create a combinatorial set of necessary‐but‐not‐sufficient conditions that enhance synthesis and folding of particular alternate, functional, conformational (...) forms. We therefore propose that protein conformation is productively regulated by the cell during translocation across the endoplasmic reticulum (ER), a concept that may account for currently poorly understood aspects of physiological function, natural selection, and disease pathogenesis. BioEssays 24:741–748, 2002. © 2002 Wiley Periodicals, Inc. (shrink)

Maintenance of skeletal muscle mass and strength throughout life is crucial for heathy living and longevity. Several signaling pathways have been implicated in the regulation of skeletal muscle mass in adults. TGF‐β‐activated kinase 1 (TAK1) is a key protein, which coordinates the activation of multiple signaling pathways. Recently, it was discovered that TAK1 is essential for the maintenance of skeletal muscle mass and myofiber hypertrophy following mechanical overload. Forced activation of TAK1 in skeletal muscle causes hypertrophy and attenuates denervation‐induced (...) muscle atrophy. TAK1‐mediated signaling in skeletal muscle promotes protein synthesis, redox homeostasis, mitochondrial health, and integrity of neuromuscular junctions. In this article, we have reviewed the role and potential mechanisms through which TAK1 regulates skeletal muscle mass and growth. We have also proposed future areas of research that could be instrumental in exploring TAK1 as therapeutic target for improving muscle mass in various catabolic conditions and diseases. (shrink)

The claim that glycogen contains protein was first made exactly 100 years ago and has been the subject of contention ever since. It has now been established that rabbit‐muscle glycogen contains a covalently bound protein of Mr 37,000, present in equimolar proportion to glycogen. The protein, named glycogenin, is joined to muscle glycogen via a novel linkage involving the hydroxyl group of tyrosine, a fact of possible significance in the light of insulin's message being transmitted by tyrosine (...) phosphorylation. The protein is seen as the biogenetic precursor of glycogen. Its existence suggests an additional mode of regulation of glycogen metabolism since the amount, turnover and cellular location of glycogenin will influence the corresponding parameters for glycogen. A protein “primer” is suggested for the biogenesis of storage polysaccharides in general. (shrink)

In eukaryotic cells intron sequences are usually spliced out with a high degree of precision from heterogenous nuclear RNA (hnRNA) to give functional mRNA with exons in their right order. Provided with the right substrates, cell extracts can achieve the same. With exotic substrates, on the other hand, the same extracts can cut exons from one RNA and join them to exons from another RNA, a process termed trans‐splicing. In vivo, RNA trans‐splicing could lead to faulty, but also to novel (...) proteins and, through reverse transcription, to genomic rearrangements. So far, in living cells trans‐splicing has only been invoked to occur in trypanosomes. In these unicellular organisms most if not all mRNAs are made discontinuously, so that the mature mRNAs carry, at their 5′ end, a common 35‐nucleotide sequence, called a miniexon, encoded separately in the genome. The miniexon is most likely joined to the body of the mRNA by trans‐splicing. (shrink)

According to Denis Noble, one of the greatest illusions of the Modern Synthesis is embodied in the Central Dogma, a principle first formulated by Francis Crick in 1958. The principle holds that DNA makes protein, not the other way around. For Noble, the Dogma has contributed to the illusion that genes alone are responsible for the development and evolution of an organism’s phenotype. Though I am largely sympathetic to Noble’s critique, I argue that there may be alternative grounds (...) for accepting the plausibility of the Central Dogma and also the illusion it perpetuates. (shrink)