Heterodera glycines (soybean cyst nematode [SCN]), the major pathogen of Glycine max (soybean), undergoes muscle degradation (sarcopenia) as it becomes sedentary inside the root. Many genes encoding muscular and neuromuscular components belong to the uncoordinated (unc) family of genes originally identified in Caenorhabditis elegans. Previously, we reported a substantial decrease in transcript abundance for Hg-unc-87, the H. glycines homolog of unc-87  during the adult sedentary phase of SCN. These observations implied that changes in the expression of specific muscle genes occurred during sarcopenia.C. elegans and H. glycines (CeHg database). We identify H. glycines homologs of C. elegans unc genes whose protein products are involved in muscle composition and regulation. RT-PCR reveals the transcript abundance of H. glycines unc homologs at mobile and sedentary stages of its lifecycle. A prominent reduction in transcript abundance occurs in samples from sedentary nematodes for homologs of actin, unc-60B (cofilin), unc-89, unc-15 (paromyosin), unc-27 (troponin I), unc-54 (myosin), and the potassium channel unc-110 (twk-18). Less reduction is observed for the focal adhesion complex gene Hg-unc-97.We developed a bioinformatics database that compares expressed sequence tag (est) and genomic data of H. glycines muscle biology. Our bioinformatics comparison of C. elegans and H. glycines genomic data and our Hg-unc-87 expression experiments demonstrate that the transcript abundance of specific H. glycines homologs of muscle gene decreases as the nematode becomes sedentary inside the root during its parasitic feeding stages.The CeHg bioinformatics database is shown to be useful in identifying homologs of genes whose protein products perform roles in specific aspects of  C. elegans [C. elegans focus on the location of the proteins or examine their genetic and biochemical nature. There is less work on determining what happens to these muscle proteins (and hence changes in muscle composition) over the course of normal development [Many aspects of muscle development and maintenance were elucidated through genetic screens in the free-living nematode  elegans -3. Subseelopment ,5.C. elegans, contraction-related injury of pharynx muscles causes sarcopenia [The formation, maintenance and degradation (wasting) of muscles involve a suite of proteins, many that are highly conserved -12. The rcopenia . Sarcopercopenia . In thesrcopenia . The decHeterodera glycines and Glycine max [H. glycines is the major parasite of G. max and is responsible for causing losses approaching a billion dollars annually for the agricultural industry in the U.S. [C. elegans-H. glycines database (CeHg database) allows us to assign function and better understand H. glycines genes [C. elegans gene function with H. glycines expressed sequence tags (ests) to rapidly identify essential H. glycines genes that could be attributed to a specific defect  genes perform many functions in C. elegans. The protein products of the unc genes are involved in muscle focal adhesion, architecture and stimulation (via neuromuscular connections). However, null alleles of unc genes can exhibit Paralyzed Arrested at Two-fold stage (pat) phenotypes. The unc mutants all display uncoordinated motion, slow movement, or paralysis [unc family of mutants contains 114 different members [H. glycines would likely involve transcriptional regulation of H. glycines homologs of unc genes whose protein products are involved in (1) the acto-myosin complex, (2) muscle focal adhesion or (3) other aspects of muscle composition and regulation.The genetically-defined aralysis . The unc members ,28. We bH. glycines homologs of unc genes. We identify H. glycines homologs of genes composing (1) acto-myosin complex, (2) muscle focal adhesion and (3) other aspects of muscle composition and regulation. We determine the transcript abundance of these H. glycines unc homologs using RT-PCR. Gene expression for many of these Hg-unc homologs is high during the mobile phase of H. glycines development and is lower during the sedentary phase of H. glycines life cycle.In this paper, we use an in-house bioinformatics database  to identUnc gene products compose various parts of the body wall muscle   whose mutants exhibit unc phenotypes or whose protein products interact with UNC proteins in C. elegans. However, the original unc mutant screens did not identify them  Figs.  and 3. Wem Figs.  and 3.Hg-unc-87 during the transition from the mobile to the sedentary phase of the H. glycines lifecycle [H. glycines ests that are homologous to C. elegans thin filament genes, including Hg-act-1, Hg-unc-27, Hg-unc-60A, Hg-unc-60B and Hg-unc-78  and Hg-unc-78. Hg-unc-60A is the non-muscle unc-60 isoform, while Hg-unc-60B is the muscle-specific isoform) RT-PCR of Hg-unc-60A reveals little change in transcript abundance occurring throughout the H. glycines lifecycle , Hg-unc-31(CAPS), Hg-unc-52 (perlecan), Hg-unc-101, Hg-unc-115, Hg-unc-110 (Hg-twk-18), Hg-dys-1, and Hg-nep-1. RT-PCR analysis indicated that modest changes in transcript abundance occur for Hg-unc-9, Hg-unc-22, Hg-unc-31, Hg-unc-52, Hg-unc-101, Hg-unc-115, Hg-dys-1, and Hg-nep-1 between the J2 stage and 15 dpi nematodes  are primary components of the thin filaments. Actin is not classified as an unc gene. However, the unc-92 mutant of C. elegans maps to the actin locus and may actually be actin. Recently, Willis et al. [C. elegans is composed of five highly conserved isoforms (act-1–5) and yields an unc phenotype [H. glycines [Unc-27 is involved in thin filament maintenance. UNC-27 forms a complex with troponin C (PAT-10) and troponin T [unc-27 disorganizes dense body positioning. Mutant unc-27 causes less well-defined sarcomeres with small regions of thin filaments interspersing within the overlap of A-bands [Hg-act-1 and Hg-unc-27 experience a substantial decrease in expression between J2 and 15 dpi nematodesThe acto-myosin complex is composed of interdigitating thin and thick filaments that are bundled by UNC-87 -31. Prevg-unc-87 . This des et al. , found thenotype . Only onglycines . Unc-27 oponin T -35 to acoponin T . Mutant  A-bands . We founactin depolymerizing factor (ADF) cofilin. Mutations in unc-60 cause disorganization in muscles by preventing bundling of thin filaments with myosin into functional contractile units [C. elegans the unc-60 gene actually encodes two completely different protein products. UNC-60A and UNC-60B are products of SUP-12-dependent alternative splicing [C. elegans [H. glycines has orthologous mRNA sequences for both unc-60A and unc-60B. UNC-78 is the muscle-specific actin interacting protein (AIP). UNC-78 works in concert with UNC-60B to depolymerize microfilaments into actin monomers [unc-60, unc-78 does not appear to have multiple splice variants that perform distinct muscle and non-muscle functions. Our examination of unc-60, indicates that the muscle-specific unc-60 isoform, Hg-unc-60B, exhibits a substantial decrease in transcript abundance between J2 and 15 dpi nematodes. This is consistent with its important role in muscle organization. As expected, the non-muscle unc-60 isoform, Hg-unc-60A, does not exhibit changes in transcript abundance during the H. glycines lifecycle. Hg-unc-78, a gene whose protein product regulates actin polymerization does not experience a substantial change in gene expression during the transition from the J2 stage to the sedentary phase. These observations, taken together with the substantial decrease in transcript abundance of the actin bundling muscle gene Hg-unc-87 [Hg-act-1, Hg-unc-27 and the protein products (i.e. Hg-UNC-60B) that regulate actin in the body wall muscles.The dynamic nature of actin filaments is under control of the actin interacting proteins UNC-60 and UNC-78. UNC-60 is the le units . Howeversplicing . UNC-60Asplicing . UNC-60A elegans , H. glycmonomers -43. Unlig-unc-87 , indicatC. elegans, myosin (UNC-54), paromyosin (UNC-15) and myosin heavy chain A (MYO3) compose thick filaments. Thick filaments are anchored to the M-line on one side and bound to the dense body on the side by the protein titin [C. elegans [Hg-unc-15, Hg-unc-54 and Hg-unc-89 occurring during muscle wasting. Thus, a decrease in transcript abundance for actin and myosin gene products occurs during muscle wasting as nematodes are becoming sedentary during their parasitic feeding stages. Loss of muscle mass occurs in mutants for muscle genes. For example, loss of muscle mass is a characteristic of DMD, caused by dys-1 mutants. However, in C. elegans, DMD-like muscle defects also require dystrobrevin (DYB-1). A microarray experiment explored the complexities of the dys-1 mutant background [dys-1 revealed 44 total probe sets are induced while 71, including unc-89, are suppressed [unc-89 is involved in DMD. Differential expression of myosin transcripts was also observed in that study [Thick filaments are major components of muscles. In in titin . UNC-89 in titin . UNC-89 in titin . There a elegans . Our RT-ckground . Microarppressed . It is nat study .C. elegans, UNC-97 is part of the PINCH family of proteins that are composed of five lin-11 isl-1 mec-3 [2CX16–23HX2CX2CX2CX16–23CX2–3 and are putative structural motifs for binding zinc [C. elegans UNC-97 does this by positioning itself with the β-integrin PAT-3 of muscles [unc-97 displays an unc phenotype, while the phenotype displayed by RNAi is pat and is embryonic lethal. Thus, UNC-97 is necessary for assembly and stability of muscular adherens junctions [C. elegans, the structural components that secure myofibers to the extracellular matrix, such as integrin, vinculin, talin, and α-actinin are conserved. This complex is similar in organization to adherens junctions in tissue culture cells [+2-binding module of the first LIM domain through an interaction with the N-terminal-most region of ankyrin repeat 1 (ANK1). In C. elegans, a biochemical interaction occurs between the sex-linked UNC-98 and UNC-97 [+2-fingers of UNC-98 [The focal adhesion complex is composed of numerous proteins. In -1 mec-3  (LIM) doing zinc ,48. The ing zinc ,50. C. ed UNC-97 . This inf UNC-98 . The biof UNC-98 ; PAT-6, f UNC-98  and TALIf UNC-98 .Hg-unc-97, Hg-unc-112, Hg-talin, and Hg-pat-6. A modest decrease in transcript abundance occurs for these genes between the J2 and 15 dpi nematodes. These results demonstrate that the deterioration of focal adhesion sites, by depletion of Hg-UNC-97, Hg-UNC-112, Hg-TALIN, and Hg-PAT-6, may not be a major contributor to body wall muscle wasting.Our bioinformatics analysis identified the focal adhesion complex genes Unc gene products perform other important roles in muscle biology. For example, UNC-9, is a neuromuscular gap junction protein [unc-52 can also exhibit a pat phenotype, depending on the mutant allele [dys-1) and neprilysin (nep-1) are other genes whose mutants exhibit unc-like phenotypes. The dys-1 gene is the C. elegans Duchenne muscular dystrophy homolog. The dys-1 gene product is part of the dystrophin-glycoprotein complex that is found in the plasma membranes of muscle cells. The dystrophin-glycoprotein complex is responsible for linking the intracellular cytoskeleton to the extracellular matrix and thought to be important for organizing signal molecules [nep-1 gene product is involved in the neuronal network of pharyngeal pumping [ protein ; UNC-22  protein ,57; UNC- protein , UNC-52  protein . Mutant t allele ; UNC-101t allele ,62 and Ut allele . Dystropolecules  and mecholecules . The nep pumping .H. glycines unc gene homologs. However, the H. glycines homolog of the body wall muscle-specific potassium channel protein, UNC-110, experiences a substantial decrease in transcript abundance between the J2 and 15 dpi nematodes. This decrease in transcript abundance is similar to the decrease in transcript abundance shown for the acto-myosin genes. It is not clear how a decrease in abundance for potassium channel proteins like Hg-UNC-110 would contribute to the sedentary nature of H. glycines during later stages of parasitism. However, potassium channels do perform major roles in muscle function in C. elegans [C. elegans genome that encode two-P domain K(+) (TWK) channels. These K+ channel subunits contain four transmembrane domains and two pore regions. Unc-110 is twk-18 and in C. elegans, TWK-18 localizes to the body wall muscle. The twk-18 mutant confers both uncoordinated movement and paralysis, probably a consequence of their expression of much larger potassium currents [Hg-unc-110 transcript abundance could contribute to the lack of mobility in H. glycines during its sedentary phase.We observed only modest changes in gene expression occurring for many of the other  elegans ,67,68. Acurrents . The loccurrents ,67,68 inH. glycines homologs of unc genes. This decrease in transcript abundance correlates to the sedentary phase of the H. glycines lifecycle. We show a substantial decrease in transcript abundance of genes composing the acto-myosin complex and also for the K+ channel homolog Hg-unc-110 during muscle wasting. Deterioration of focal adhesion sites does not appear to account for much of the muscle mass lost during sarcopenia in H. glycines.Our results demonstrate a decrease in transcript abundance for a specific subset of Plant and nematode materials were grown at the United States Department of Agriculture Soybean Genomics and Improvement Laboratory as described previously  accordinG. max cv. Peking seeds were grown in a sand mix in standard greenhouse conditions. To promote hatching, eggs from the H. glycines isolate TN8 (susceptible reaction) were incubated in sterile water at room temperature on a rotary shaker at 25 rpm. After two days on the rotary shaker, the J2s were collected and concentrated by centrifugation to approximately 5,200 J2/ml. Replicate experiments were performed and completed by running one experiment to completion and then collecting data. A second experiment was then run at approximately one month later after the first experiment was completed. Thus, we isolated different isolations of H. glycines eggs, J2, 15 dpi and 30 dpi nematodes for each experiment. Four plants contained in a beaker were inoculated with 5,200 J2 nematodes. For RT-PCR experiments, recovery of the 15 and 30 dpi H. glycines samples from G. max roots was performed according to [H. glycines-infected G. max roots grown for either 15 or 30 dpi were dipped into water to remove sand. At 15 and 30 dpi, female H. glycines are partially emerged from the root, facilitating collection of pure nematode samples. Roots were lightly massaged to liberate female H. glycines into a sieve of 150 μm pore size . This filtration step would remove any additional male nematodes that were remaining in the root. To obtain egg and J2 samples, mature females were harvested at 30 dpi and crushed. The eggs were sifted through a 250 μm sieve and captured onto a 25 μm sieve. The eggs were hatched for two days in distilled water on a rotary shaker at 25 rpm. Pure J2 suspensions are made by sifting them through a 41 μm nylon mesh and collected with a low-speed centrifugation. The RT-PCR samples  were flash-frozen in liquid nitrogen and ground to a fine powder using mortar and pestle chilled in liquid nitrogen. Total RNA extraction was performed using the method of Mujer et al. [Briefly, rding to  and alsording to . Brieflyr et al. .C. elegans and 24,438 ests and 231 genomic sequences from H. glycines . These C. elegans and H. glycines sequences were used to create a local database using SQLServer2000. The sequences were imported into our local database. Subsequently we created a unigene set using the contig assembly program Seqman  resulting in 3,782 contigs of 2 or more sequences and 4,522 singletons for H. glycines. These sequences were then blasted against the local C. elegans database. Parsing of the results of the blast searches was done with customized Perl scripts. These scripts extracted the best hits from the blast results, E-value, score and identities values. The parsed results were imported back into the database. SQL scripts were written to query the CeHg database for C. elegans genes having high homology. Our data base was then linked to WormBase [H. glycines ests homologous to C. elegans unc genes. These results then were confirmed by performing manual blast searches of each C. elegans unc gene against the H. glycines sequences.The CeHg bioinformatics database  is a datWormBase  and PubMWormBase  to identHg-unc-87 PCR primers  that amplify different size fragments in the presence of genomic DNA as compared to pure cDNA. RT-PCR reactions containing no template and reactions using RNA processed in parallel but with no Superscript reverse transcriptase also served as controls for RT-PCR and produced no amplicon. After we determined that no contaminating genomic DNA existed in our cDNA, we performed RT-PCR. Relative quantities of expression using their respective primers  values were plotted against the dilution series. PCR efficiencies were equal between the target and endogenous control. Ct values and relative abundance were calculated using software supplied with the Mx3000P Real-Time PCR system. Our RT-PCR data was standardized against an est (CB380016) determined to experience no change in expression during H. glycines development. The relative abundance of mRNA was compared to that of CB380016 in the different sample types to calculate fold change. For each gene, a ratio was established between the control (CB380016) and the gene of interest (GOI) for the egg J2, 15 dpi and 30 dpi samples. To calculate fold expression, the ratio between CB380016 and the GOI at 15 dpi was set to a value of one. Other fold expression values for egg, J2 and 30 dpi were calculated using the ratio obtained at 15 dpi for GOI as the denominator. The ratio of the GOI for egg, J2 and 30 dpi, respectively, was used as the numerator. The value obtained after calculation was fold expression for those time-points. Standard error was used in the analyses.RNA was extracted from nematodes as previously described and treated with DNase I to remove genomic DNA. The cDNA was reversed transcribed from RNA using SuperScript First Strand Synthesis System for RT-PCR  with oligo d(T) as the primer according to manufacturer's instructions. All the primer sets were initially tested for specificity with a mixture of RNAs for all stages of nematodes. Genomic DNA contamination was assessed by PCR as described previously . We perflin-11, isl-1, mec-3; CeHg, C. elegans H. glycines database; uncoordinated, unc; zinc finger, Zn+2-finger; RT-PCR, real-time quantitative PCR; nt, nucleotide; bp, base pair; J2, second stage juvenile; dpi, days post inoculationest, expressed sequence tag; SCN, soybean cyst nematode; DMD, Duchenne muscular dystrophy; LIM,


Previous studies have demonstrated that BR-931, a hepatic peroxisome proliferator, can induce liver tumours in mice and rats. Since alterations in gene expression may play a critical role in multistage hepatocarcinogenesis, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor (EGF) receptor and ODC (ornithine decarboxylase) genes, as well as endogenous retrovirus-like sequences, in F344 rat liver during the first 8 weeks of feeding a 0.16% Br931 diet and in liver tumours induced by chronic feeding of this diet. Northern blot analysis of poly A + liver RNA samples showed an increase in the level of RNAs homologous to rat leukaemia virus (RaLV) but no significant change in the level of 30S-retrovirus related RNAs in the liver RNA samples obtained from rats during the first 8 weeks of feeding the diet containing BR931. An increase in the levels of c-myc, c-H-ras and ODC transcripts was also seen in the liver RNA samples from the treated rats. Of particular interest was a decrease in the abundance of EGF receptor transcripts in the liver RNA samples from rats fed the BR931 diet. Increased levels of RaLV, c-myc, and ODC RNAs were also seen in the tumours induced by BR931, but this was not the case for 30S and c-H-ras. The liver tumour samples also showed a decrease in EGF receptor RNA. These changes in cellular levels of specific RNAs resemble, in several respect, those we previously described in rodent liver during regeneration and tumour promotion, and also those seen in rodent hepatomas induced by other agents. Therefore, they may reflect a common profile of gene expression relevant to liver proliferation and carcinogenesis.


For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs.Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus, and related RNA sequences. It integrates a large number of Subviral RNA sequences, their respective RNA motifs, analysis tools, related publication links and additional pertinent information , allowing users to efficiently retrieve and analyze relevant information about these small RNA agents.The Subviral RNA database is a web-based environment that facilitates the research and analysis of viroids, satellite RNAs, satellites viruses, the human hepatitis .With its design, the Subviral RNA Database could be considered as a fundamental building block for the study of these related RNAs. It is freely available via a web browser at the URL:  The HDV genome consists of a small single-stranded, circular negative sense RNA genome  containing self-cleaving motifs (i.e. delta ribozymes), and a single open reading frame (ORF) encoding two viral proteins (HDAgs) [Viroids, satellite RNAs, satellites viruses and the human hepatitis  (HDAgs) . Viroids (HDAgs) . Double-Most of the Subviral RNA agents are composed of single-stranded RNA molecules that rely significantly on proteins from their hosts or from helper viruses for replication and propagation -3. In adThe Subviral RNA Database is hosted on a dedicated server built around a LAMP setup: Linux, Apache, MySQL, Perl (an operating system (OS), a web-server, a database and a script language, respectively). This architecture maximizes the speed and permits a superior customization of the database. In addition, it allows the Subviral server to offer several web-based tools  and web-forms. Using these forms, users can record directly their entries related to links, conferences and announcements, which are subsequently validated and dynamically displayed using the web interface.To integrate new data autonomously, automated in-house scripts search for new Subviral RNA sequences, annotations and related publications from the NCBI server. New sequences are then manually adjusted in order to standardize sequences among a specific species (i.e. same sequence origin and same polarity (genomic vs antigenomic) for single-stranded RNA species).From a simple sequence database , the Subin vivo sequence fitness. In addition, links to known RNA motifs specific for each variant are indicated. From this section, users can select sequences, individually or in groups, and display them under various formats for further analysis using users' software. These entries can also be processed directly on the Subviral server using various bioinformatics tools (see below), which results are either displayed in HTML format or saved in raw text to facilitate transfer to another program.The four sections related to Subviral RNA sequences form the main part of the database . All the RNA variants are indexed according to their families, genus and species in a classification scheme based on the criteria established by the International Committee on Taxonomy of Viruses (ICTV) . When noPospiviroidae [Because most Subviral RNAs are mosaics of functional RNA motifs, an RNA motif database presenting the known motifs associated with the indexed species is now included. Most of these RNA motifs were determined by sequence comparison using experimentaly derived motifs as models. The secondary structures of the motifs without pseudoknots were also supported thermodynamically using the Mfold software . Primaryviroidae . This anVarious bioinformatics web tools such as ClustalW, WebLogo and Blast  are now accessible directly on the Subviral RNA Database server. In addition, in-house tools such as QuickFasta  and a Species Identification Service have been implemented. The latter uses a queried sequence and attempts to align it, using ClustalW, to a set of preselected sequences for each species and produces a report including a phylogenetic tree. Finally, sections related to new sequences, new publications, various related links, conferences and announcements can also be accessed through the database.delta ribozymes and 260 RY motifs. As soon as they will become available, secondary structure predictions of new RNA motifs will also be implemented.At the time of this writing, the Subviral RNA database contains 1949 sequences indexed in 83 species. It comprises information on 1046 viroid sequences, 83 complete and 508 partial HDV sequences, 299 satellites RNA sequences and 13 related RNA sequences. Users have also access to information on 189 type I hammerhead ribozymes, 320 type III hammerhead ribozymes, 8 hairpin ribozymes, 203 mpelchat@uottawa.ca.The Subviral RNA Database is designed as a robust and relational database system for these unusual species and their RNA motifs, and also to function as a resource for the entire scientific community by providing full public access to recent sequences, along with many tools to explore and analyze those data. Users of this database are encouraged to provide corrections, or other information, for inclusion in the database via electronic mail , is OS independent , do not require user account and do not need installation of plug-ins.This database is publicly accessible by web browser at LR and MP designed the database concept and participated in its design and construction, including the server. MP acted as the final editorial authority. All authors have read and approved the final manuscript.


The origin of the translation system is, arguably, the central and the hardest problem in the study of the origin of life, and one of the hardest in all evolutionary biology. The problem has a clear catch-22 aspect: high translation fidelity hardly can be achieved without a complex, highly evolved set of RNAs and proteins but an elaborate protein machinery could not evolve without an accurate translation system. The origin of the genetic code and whether it evolved on the basis of a stereochemical correspondence between amino acids and their cognate codons (or anticodons), through selectional optimization of the code vocabulary, as a "frozen accident" or via a combination of all these routes is another wide open problem despite extensive theoretical and experimental studies. Here we combine the results of comparative genomics of translation system components, data on interaction of amino acids with their cognate codons and anticodons, and data on catalytic activities of ribozymes to develop conceptual models for the origins of the translation system and the genetic code.Our main guide in constructing the models is the Darwinian Continuity Principle whereby a scenario for the evolution of a complex system must consist of plausible elementary steps, each conferring a distinct advantage on the evolving ensemble of genetic elements. Evolution of the translation system is envisaged to occur in a compartmentalized ensemble of replicating, co-selected RNA segments, i.e., in a RNA World containing ribozymes with versatile activities. Since evolution has no foresight, the translation system could not evolve in the RNA World as the result of selection for protein synthesis and must have been a by-product of evolution drive by selection for another function, i.e., the translation system evolved via the exaptation route. It is proposed that the evolutionary process that eventually led to the emergence of translation started with the selection for ribozymes binding abiogenic amino acids that stimulated ribozyme-catalyzed reactions. The proposed scenario for the evolution of translation consists of the following steps: binding of amino acids to a ribozyme resulting in an enhancement of its catalytic activity; evolution of the amino-acid-stimulated ribozyme into a peptide ligase  yielding, initially, a unique peptide activating the original ribozyme and, possibly, other ribozymes in the ensemble; evolution of self-charging proto-tRNAs that were selected, initially, for accumulation of amino acids, and subsequently, for delivery of amino acids to the peptide ligase; joining of the peptide ligase with a distinct RNA molecule  carrying a built-in template for more efficient, complementary binding of charged proto-tRNAs; evolution of the ability of the peptide ligase to assemble peptides using exogenous RNAs as template for complementary binding of charged proteo-tRNAs, yielding peptides with the potential to activate different ribozymes; evolution of the translocation function of the protoribosome leading to the production of increasingly longer peptides (the first proteins), i.e., the origin of translation. The specifics of the recognition of amino acids by proto-tRNAs and the origin of the genetic code depend on whether or not there is a physical affinity between amino acids and their cognate codons or anticodons, a problem that remains unresolved.We describe a stepwise model for the origin of the translation system in the ancient RNA world such that each step confers a distinct advantage onto an ensemble of co-evolving genetic elements. Under this scenario, the primary cause for the emergence of translation was the ability of amino acids and peptides to stimulate reactions catalyzed by ribozymes. Thus, the translation system might have evolved as the result of selection for ribozymes capable of, initially, efficient amino acid binding, and subsequently, synthesis of increasingly versatile peptides. Several aspects of this scenario are amenable to experimental testing.This article was reviewed by Rob Knight, Doron Lancet, Alexander Mankin (nominated by Arcady Mushegian), and Arcady Mushegian. This article was reviewed by Rob Knight, Doron Lancet, Alexander Mankin (nominated by Arcady Mushegian), and Arcady Mushegian....there is no logical impossibility in the acquirement of any conceivable degree of perfection through natural selection.Ch. Darwin [. Darwin ...the origin of protein synthesis is a notoriously difficult problem.F.H.C. Crick et al. [k et al. As first outlined by Darwin , the evoTaking the replication process over the Eigen threshold is required for sustainable replication and is, per force, a pre-requisite for the start of biological evolution Fig. . Indeed,-3, replication is catalyzed by a complex protein replicase [The crucial question on the origin of life is how did the Darwin-Eigen cycle start, i.e., how was the minimal complexity attained that is required to achieve the minimally acceptable replication fidelity. In even the simplest modern systems, such as RNA viruses with the replication fidelity of only ~10Origin, the one on the evolution of the vertebrate eye [Continuity Principle, following the recent insightful discussion of this issue by Penny [The origin of complex biological systems is a classical topic in evolutionary biology and, probably, the principal object of attacks of anti-darwinists of all ilk, including the notorious Intelligent Design movement. The gist of the criticisms is that many biological systems are not just complex but "irreducibly complex" and, as such, could never evolve via the Darwinian mechanism of gradual, stepwise adaptive change because intermediate stages of evolution would have no selective value and so could not be fixed. Darwin himself was perfectly aware of the problem and its dimensions and addressed it in one of the most famous passages of the rate eye . The solrate eye  and emphrate eye ,12. In eby Penny . The devby Penny . Furtherby Penny ,14. NeveWe discussed the principles of evolution of complex biological systems at some length because they are most pertinent to the fundamental problem we wish to address here: the origin of the translation system and the genetic code. Indeed, the translation system might appear to be the epitome of irreducible complexity because, although some elaborations of this machinery could be readily explainable by incremental evolution, the emergence of the basic principle of translation is not. Indeed, we are unaware of translation being possible without the involvement of ribosomes, the complete sets of tRNA and aminoacyl-tRNA synthetases (aaRS), and  several translation factors. In other words, staggering complexity is inherent even in the minimally functional translation system. Thus, as outlined above, it appears that the evolutionary origin of translation is to be sought along the exaptation route, i.e., by retrodiction of the ancestral functions of various components of the translation system that would allow them to evolve functionalities enabling their recruitment for translation.Even this, however, does not do the full justice to the difficulty of the problem. The origin of translation appears to be truly unique among all innovations in the history of life in that it involves the invention of a basic and highly non-trivial molecular-biological principle, the encoding of amino acid sequences in the sequences of nucleic acid bases via the triplet code,16. ThisThe obvious common wisdom is that a system as complex as the translation machinery, even in its primitive state , could not have emerged in one sweep. Such an abrupt emergence would appear an outright miracle and an obvious violation of the Continuity Principle. Elsewhere, one of us considers a different worldview that might bring the chance emergence of complex (pre)biological systems, in particular, translation and replication, within the realm of the possible . Here, hCarsonella, Mycoplasma, or Nanoarchaeon) is extremely complex. At the heart of the system is the ribosome, a large complex of at least three RNA molecules and 60–80 proteins arranged in a precise spatial architecture and interacting with other components of the translation system in the most finely choreographed fashion [The design of the translation system in even the simplest modern cells , in which a primitive version of the Darwin-Eigen cycle was already operating, can be conjectured from the comparative analysis of the translation system components .This is not all the comparative analysis can do: comparison of RNAs themselves also yields important information and startling puzzles. The conservation of the structure, some sequence elements , and even modification sites of the tRNAs of all specificities  leaves no doubt that they all evolved from a single common ancestor -34. HencThe famous central dogma of molecular biology  states tDNA⇔RNA→proteinObviously, when considering the origin of first life forms, one faces the proverbial chicken-and-egg problem: what came first, DNA or protein, the gene or the product? In that form, the problem might be outright unsolvable. Indeed, there is a crucial feedback in this system: to replicate and transcribe DNA, functionally active proteins are required, but production of these proteins requires accurate replication, transcription, and translation of nucleic acids. If one sticks to the triad of the Central Dogma, it is impossible to envisage what could serve as the starting material for the Darwin-Eigen cycle. Even removing DNA from the triad and postulating that the original genetic material consisted of RNA, while an important idea (see below), is not going to help much because the feedback remains as crucial as it is elusive. In order for evolution toward greater complexity to take off, the system needs to somehow get started on the Darwin-Eigen cycle prior to establishing this feedback.Tetrahymena rRNA intron [The brilliantly ingenious and, perhaps, the only possible solution has been independently proposed by Woese , Crick , and OrgA intron , and by A intron . Since tA intron -43).The discovery of ribozymes made the idea that the first replicating systems consisted solely of RNA molecules, which catalyzed their own replication, extremely attractive. In 1986, Gilbert coined the term "RNA world" to designate this hypothetical stage in life's evolution , and thein vitro selection that, at least conceptually, mimics the Darwinian evolution of ribozymes thought to occur in the primeval RNA world [The popularity of the RNA World hypothesis has, in turn, further stimulated ribozyme research, aimed, in large part, at testing the feasibility of various RNA-based catalytic activities, above all, perhaps, an RNA replicase. It is noteworthy that the main approach to developing ribozymes with desired activities is NA world ,50. EsseNA world .conditio sine qua non for the evolution of the hypothetical RNA World.Understandably, major effort has focused on the demonstration of nucleotide polymerization and, ultimately, RNA replication catalyzed by ribozymes, the key processes for the hypothetical, primordial RNA World. While these reactions are not directly involved in translation, they are highly relevant to the problem considered here inasmuch as replication with a fidelity above the Eigen threshold is a pre-requisite of biological evolution (see above). The outcome of the experiments aimed at the creation of ribozyme replicases so far has been somewhat mixed. Ribozymes have been obtained capable of extending a primer annealed to a template by 10–14 nucleotides; initially, the ribozymes with this activity could function only by specific base-pairing to the template but, subsequently, general ribozyme polymerases of this class have been evolved through additional selection -56. HoweIt is often noted that the RNA World is not just a concept supported by the catalytic prowess of ribozymes: while overshadowed by the multitude of proteins with catalytic and structural functions, the RNA World still lurks within modern life forms ,58. ReacTo recapitulate, three independent lines of evidence converge in support of a major role of RNA, and in particular, RNA catalysis at the earliest stages of life's history, and are compatible with the reality of a complex, ancient RNA world that was first postulated by Woese, Crick, and Orgel on purely logical grounds. First, comparative analysis of the protein components of the translation machinery and their homologs involved in other functions strongly suggests that extensive diversification of the protein world took place at the time when the translation system was comprised, primarily, of RNA. Second, several classes of ribozymes operate within modern cells, and their properties are compatible with the notion that they are relicts of the ancient RNA world. Third, while limited in scope and, obviously, inferior in catalytic activity compared to protein enzymes , ribozymin vitro evolved ribozymes remain (relatively) poor catalysts; the lack of efficient ribozyme polymerases seems particularly troubling. Admittedly, it might be unrealistic to expect that experiments on in vitro evolution of ribozymes could easily mimic the actual complexity of the primordial RNA world. Indeed, although these experiments harness the power of selection, they are, obviously, performed on a totally different time scale and conditions that cannot possibly reproduce those of life's origin. The latter, of course, are not known but it seems reasonable to surmise that, if there was a complex RNA World at the brink of the Translation Breakthrough, it was brought about by millions of years of evolution of ensembles of replicating RNAs in a compartmentalized environment similar, at least, in principle, to the networks of iron sulfide compartments existing at hydrothermal vents [All these arguments in favor of the reality of the RNA World notwithstanding, there are two major sources of doubts. First, despite all invested effort, the al vents -74. The al vents ,76, the -3 errors per nucleotide per replicase cycle, an RNA "organism" with ~100 "genes" the size of a tRNA (~80 nucleotides) would be sustainable. This level of fidelity would require only an order of magnitude improvement over the most accurate ribozyme polymerases obtained by in vitro selection [A recent study of Szathmary and coworkers puts some important numbers on the complexity that, potentially, might be attainable in the RNA World and the replication fidelity required to reach this level of complexity . An estielection ,78. Concelection . As aptlelection . Of courEven under the best case scenario, the RNA world does not appear to have potential to evolve beyond very simple "organisms". To attain greater complexity, invention of translation and the Protein Breakthrough were required. However, the selective forces underlying the emergence of the translation system in the RNA World remain obscure, and tracing the path to translation is extremely hard. This lack of clarity with regard to the continuity of evolution from the RNA World to an RNA-protein world can be construed as a second major objection against the RNA World as a crucial stage of life's evolution, an objection, perhaps, even more prohibitive than the first one, dealing with the imperfection of ribozymes. A radical alternative, "no RNA World" hypothesis, is considered elsewhere . In the To understand how translation might have emerged, the nature and origin of the codon assignments in the universal genetic code are crucial. The problem of code evolution fascinated researchers even before the code was fully deciphered, and the earliest treatises on the subject already clearly recognized three, not necessarily mutually exclusive models: i) steric complementarity resulting in specific interactions between amino acids and the cognate codon  or anticodon triplets , ii) "frozen accident" – fixation of a random code that would have been virtually impossible to significantly change afterwards , and iii) adaptive evolution of the code starting from an initially random codon assignment ,36,80-86The principal dilemma is whether or not a stereochemical correspondence between amino acids and cognate triplets (in the form of either CRM or ARM) exists or not. The answer to this straightforward question proved to be surprisingly elusive. The early attempts to establish specificity in interactions of (poly)amino acids and polynucleotides have been inconclusive, indicating that, if a correspondence exists, it must be much less than precise, and the interactions involved would be weak and dependent on extraneous factors -96. AlthA recent resurgence of the stereochemical hypothesis was brought about by the application of the selection amplification (SELEX) methodology for isolation of oligonucleotides (aptamers) that specifically bind amino acids ,104. TheNevertheless, serious questions remain as to the ultimate validity and relevance of these results. The presence of both codons and antidocons in aptamers binding several amino acids is hard to interpret in terms of stereochemical complementarity. Furthermore, the amino acids for which detailed aptamer data is available are those that have complex side chains  and are thought to be late recruitments to the genetic code . At leasA different, and elegant version of the stereochemical correspondence hypothesis has been proposed by Copley and coworkers. This scThus, the jury is still out with regard to any role direct interactions between amino acids and cognate triplets might have played in the origin of the code. Accordingly, in what follows, we strive to be objective and consider the origin of the code in three distinct contexts: i) specific interaction between amino acids and the cognate codons (CRM), ii) specific interactions between amino acids and the cognate anticodons (ARM), and iii) frozen accident (FAM) as the starting point for the evolution of the code.During the 40 years since the discovery of the translation mechanism and deciphering of the genetic code, numerous theoretical  models of the origin and evolution of various components of the translation apparatus and aspects of the process itself have been proposed. A comprehensive, critical review of this literature would be a truly daunting task and will not be attempted here. We outline only a few of the more straightforward and, in our opinion, more plausible, evolutionary schemes and then discuss in somewhat greater detail the only published coherent scenario for the evolution of the translation system we are aware of.One popular and potentially important idea on the origin of the genetic code is the hypothesis of Szathmary on the role of so-called coding coenzyme handles (CCH), i.e., oligonucleotides with various ribozyme activities using amino acids as cofactors, as evolutionary progenitors of tRNAs -117. ThiThese and other hypotheses tackle important aspects of the origin and evolution of the translation system. However, they all stop short of proposing a complete, coherent scenario for the transition from the RNA world to the modern mode of translation. We believe that the reason for the near lack of such scenarios in the current literature is the formidable difficulty of breaking this transition into incremental steps associated with a biologically plausible selective advantage, thus making the entire transition compatible with the Continuity Principle.We are aware of two proposals that come closest to such a complete scenario, and it seems to be more than a remarkable coincidence that the two present essentially the same model, differences in detail notwithstanding. The essence of this model, originally sketched by Altstein -122, andLet us examine in some detail the model of Poole and coworkers, which is better reconciled with various facets of the RNA World than the original proposal of Altstein . Comparative analysis of translation system components points in the same direction, i.e., indicates that the primordial translation system consisted (predominantly) of RNA. The RNA World is a conjecture not a proven fact but, for the purpose of this paper, we assume that it existed.2. Evolution has no foresight – thus, before there were functional proteins facilitating replication, production of proteins could not be the driving force behind the evolution of the translation system. Translation must have evolved as a by-product of selection for some other function, i.e., via the exaptation route.3. Fidelity of translation in the late RNA world was comparable to that of the modern translation. Counter-intuitively but undeniably, the fidelity of the primitive translation system that evolved within the ancient RNA world could not have been dramatically lower than that of the modern translation system, with all its numerous, essential proteins. This is the logical conclusion from the results of protein sequence and structure comparisons which reveal extensive diversification of at least several protein folds antedating the emergence of the protein components of the modern translation system . A corollary is that, already within the confines of the RNA world, the translation machinery, in its principal features, resembled the modern one. In particular, it is impossible to imagine a high-fidelity translation system functioning without a set of tRNAs for many, probably, most of the 20 amino acids found in modern proteins.4. Specific interactions (or lack thereof) between amino acids and codons or anticodons. We believe that the jury is still out on the reality and relevance of putative specific interactions between amino acids and cognate triplets – either codons or anticodons. Accordingly, we formulate and explore three alternative models for translation origin depending on whether or not amino acids specifically recognize cognate triplets: i) interaction of amino acids with codons (CRM), ii) interaction of amino acids with anticodons (ARM), and iii) no specific interactions between amino acids and any of the cognate triplets – the frozen-accident model (FAM).5. Adaptors must have been part of the emerging translation system from the start because peptide formation via direct binding of amino acids to a RNA template is stereochemically implausible. Indeed, a simple 3D model shows that, even if there was a specific affinity between amino acids and cognate codons, the distances between amino acid assembled on an RNA template would be far too long for the formation of peptide bonds , the three models – CRM, ARM, and FAM – have many features and steps in common. As we will point out, it seems that these steps are, in effect, logically inevitable in any model of the evolutionary origin of translation. Therefore, in the presentation of these models that follows, the common steps are outlined just once, and forking paths are taken consecutively as they emerge .As outlined above, the starting point of all scenarios for the origin of translation is a replicating ensemble of selfish cooperators consisting of RNA molecules with various ribozyme activities and existing within a network of inorganic compartments ,74. One R  at this stage is possible but would not substantially affect the proposed scenario. It is postulated that the bound amino acids enhance the X→Y reaction catalyzed by R . An interesting example could be a peptide containing a pair of acidic amino acids and coordinating a magnesium ion as seen in a great variety of unrelated modern nucleic acid metabolism enzymes  [P boosts the catalytic activity of E, it again increases the fitness of the entire ensemble. Consequently, selection will favor appropriate adjustments to E, strengthening its association with P, and further increase of the selective pressure on R to keep producing P.3. Spontaneous disassembly or decay of ent Fig. . If P hand more) -135. If E relying on the presence of P, a copy of R (RL) might lose the original X→Y activity, with a concomitant enhancement of the amino acid ligase activity, whereas other copies (R0) would retain the original activity, still enhanced by the peptide P .4. With the activity of e P Fig. . This woT) evolve under the pressure of selection for amion acid accumulation ; these molecules would be analogous to amino-acid-binding aptamers that have been studied in a variety of experiments [T evolves, resulting in an increase in affinity and specificity of amino acid binding .T RNAs specifically binding different amino acids evolve by duplication and diversification, with the retention of variants driven by selection for efficient accumulation of a broad repertoire of amino acids.6. Different species of T would be mediated, in a concerted fashion, by two distinct moieties, the amino-acid-specific anticodon loop and the stem region with a non-specific affinity to the amino acid backbone (NH3-Cα-COOH)  Fig. . The steids Fig. .T, one can envisage a folding flip between two conformations (one of them involving a complementary pairing of codon and anticodon), induced by the interaction with the cognate amino acid  in RNA T is chosen by chance , this is the critical step where the correspondence between amino acids and cognate triplets is established, directly or indirectly, creating the basis of the genetic code.RL evolves the capacity to bind aminoacyl-T RNA complexes instead of individual amino acids  interaction between RNA T and RL to the specific base-pairing between the exposed (now anticodon) loop of T and an extended RNA strand of RS. The transpeptidation activity remains the function of RL. This is the crucial step of the emergence of bona fide translation, a mechanism based on the adaptors  combining amino acids with the cognate codons [8. An accessory RNA subunit  RL Fig. . The bure codons .T RNAs evolved along, roughly, parallel (convergent) paths. However, the common origin of tRNAs implies a subsequent bottleneck through which only a single winner has passed, an L-shaped molecule with the acceptor CCA 3'-end. Selection for spatial complemtarity and efficient interaction between the aminoacylated T RNAs and the peptidyl-transferase RL could be the driving force behind the selection for this structure. This selection originally would affect only one T RNA, perhaps, the one chargeable with the most abundant primordial amino acid. Since a relatively minor modification (a concerted change in the amino-acid-binding site and the anticodon loop) would switch the specificity of the proto-tRNA, a sweep by a single proto-tRNA species, taking over the function of other, unrelated and unevolved, T RNAs one by one, seems to be plausible. We tentatively place this sweep in an early stage in the evolution of the translation system; however, an alternative possibility is that it took place at a later stage, concomitantly with the evolution of aaRS and their takeover of the key role in the pairing of amino acids with the cognate anticodons.The evolutionary path from the set of primitive T RNAs Fig.  to the mR – with the binding/recognition of aminoacyl-T RNAs now relegated to RS and the transpeptidation reaction remaining the function of RL – provides for the separate evolution and elaboration of the respective activities . In particular, new forms of RS would evolve by duplication and diversification, increasing the repertoire of peptides produced by the RSRL complex  with different oligo/polynucleotides present in the compartment would lead to the production of an increasing variety of peptides, thus enhancing the catalytic potential of the ensemble. Furthermore, this step would enable the selection for improved replication potential  of those species of M that encode useful peptides, leading to enrichment of these RNA species in the compartment. Thus, an RNA-peptide Eigen- Schuster hypercycle [10. As the next step, we envision the physical separation of the template strand sis Fig. . At thispercycle , a distiRSRL upon trans-peptidation triggered the trinucleotide shift, the crucial movement performed by modern ribosomes [11. Conceivably, the release of a discharged (proto)tRNA from ibosomes ,124 and The evolutionary path from the breakthrough stage outlined above to the modern-type translation system was, largely, a story of takeover of the primordial ribozyme functions by evolving proteins. Proteins have an incomparably greater potential for evolution of diverse binding and catalytic capacities than peptides or RNA and, accordingly, they soon began to gradually supplant the ribozymes. Given the greater chemical versatility and efficiency of proteins as catalysts, each such displacement is irreversible, as insightfully stressed by Penny .The rest, as they say, is history.there to here. There being the early, cooling earth with no complex organic molecules, and here being a minimally complex genetic system with modern-type translation, transcription, and replication machineries, a system that would be subject to biological evolution much like modern organisms. The replication and transcription problems are, at least, logically relatively straightforward, even if hard from the chemical point of view, inasmuch as no new principles, beyond base complementarity, and enzymatic catalysis need to be invented. Thus, plausible, even if conflicting, accounts of the emergence of these systems have been derived from comparative-genomic data and evolutionary reasoning [a priori unconnected sequences of nucleotides and amino acids. It might not be much of an exaggeration to note that, at least, at first glance, the origin of the translation system evokes the scary specter of irreducible complexity.The scenarios for the origin of the translation system and the genetic code outlined here are both sketchy and highly speculative. Why, then, bother building such conceptual, qualitative models at all? The justification for this kind of theorizing can be succinctly put in the short phrase: we have to get from easoning ,140-144.Thus, our main incentive with the present analysis was to deconstruct the formidable problem of the emergence of translation into a series of plausible and manageable steps, in accordance with the Continuity Principle. We believe that, in doing so, we achieved a somewhat greater level of detail and coherence than any of the previous models we are aware of. Importantly, in constructing this model, we were both constrained and driven by: i) comparative-genomic data, ii) experimental data on amino-acid-codon recognition, iii) experimental data on the diverse catalytic activities of ribozymes.Comparative-genomic analysis indicates that an elaborate translation system, comparable to the modern one in terms of fidelity and efficiency, has evolved within the RNA world. Indeed, extensive diversification of many protein folds occurred before the advent of some of the essential components of the modern translation system, such as aaRS and translation factors. Before the emergence of these dedicated proteins, the translation system must have been a machine comprised primarily, if not exclusively, of RNA. The only conceivable alternative, that the primordial translation system employed a different, currently, extinct complement of essential protein factors, inevitably leads to infinite regression. Thus, it seems to be a virtually inevitable conclusion that the ancient, RNA-only translation system was comparable in efficiency to the modern one. This might seem paradoxical and even not credible at a superficial glance. However, a quick reflection suggests that: i) the skeleton of the modern translation system actually consists of RNA, with the proteins being elaborations, however numerous and important, and ii) logically, it hardly could have been otherwise: indeed, in order to switch to a new type of constituents (proteins), biological systems needed the means to produce them accurately. It is conceivable and, indeed, likely that peptides produced by the first, RNA-based proto-translation systems provided positive feedback leading to hypercycle formation Figs. , 6. HoweIn our description of the model, the alternative scenarios based on CRM, ARM, and FAM are considered on equal footing. As discussed above, the currently available data are too ambiguous to conclude which of these models for the origin of coding is most likely. However, it should be noted that, important as they are in terms of the actual physico-chemical underpinning of the code, the differences between CRM, ARM, and FAM do not translate into major modifications of the evolutionary scenario. Indeed, the central principles remains the same, i.e., specific recognition of amino acids by proto-tRNAs such that an amino acid is paired with the cognate anticodon with sufficient reliability.The models presented here were deliberately constructed at the level of considerable detail -at the risk of getting many, perhaps, most aspects wrong – in order to provide a proof of principle, i.e., to illustrate a plausible sequence of selectively advantageous steps along the path from the RNA world to the modern-type translation system. This being said, there seem to be several underlying principles that are likely to stand regardless of further developments. We briefly recapitulate these:per se is not feasible.1. Evolution having no foresight, selection for translation Translation must have evolved as a by-product of selection for some other function, i.e., via the exaptation route.2. Given that the essence of translation is the intimate link between RNA and proteins, it seems most likely that, in some form, this connection existed from the very beginning of the evolutionary path from the RNA World to translation. Thus, the proposed starting point, i.e., stimulation of ribozymes by amino acids and peptides seems to be a strong, almost, logically required, candidate for this role .3. Synthesis of peptides directly on an RNA template is stereochemically unfeasible. Hence adaptors must have been part of the primordial translation system from the start. Accordingly, from the very onset of translation, adaptors have been key to the establishment of the genetic code. These ancestral adaptors, although, in all likelihood, smaller and simpler than modern tRNAs, must have been endowed with catalytic capacities lacking in the latter, i.e., they would have to catalyze specific self-aminoacylation with the cognate amino acids.4. The primordial translation system was dominated by RNA although peptides might facilitate its functioning. However, the fidelity of this primordial, (nearly) RNA-only translation system must have been comparable to that of modern translation systems, considering that extensive protein evolution took place prior to the diversification of the proteins that are essential for the modern translation.RL), and at a subsequent stage, recognition of a specific region in the progenitor of the small subunit (RS). With regard to RL and RS themselves, ribozyme stimulation by amino acids and peptides has been demonstrated but, beyond that, the postulated properties of these molecules remain hypothetical. It seems that a focused experimental effort aimed at the construction/selection of ribozymes with the properties of the postulated T RNAs, in particular, their postulated interaction with other, more complex ribozymes, could provide crucial evidence in support of this or a similar scenario for the evolution of translation.The current scenario for the evolution of translation in the RNA World faces formidable difficulties because, although the ribozyme catalysis of the elementary reactions required for translation has been demonstrated experimentally Table , the reqAlthough the individual ribozyme-catalyzed reactions involved in the postulated scheme are feasible, the succession of multiple evolutionary steps that appear to be required for the emergence of translation might be legitimately viewed as far fetched, particularly, considering the inevitably inefficient ribozyme-mediated replication that must have been prevalent in the RNA World. Be as it may, this is, at present, our best effort to develop a conceptual model for the origin of translation. Elsewhere, one of us (EVK) examines a radical alternative .Reviewer 1: Rob Knight (University of Colorado)In this intriguing manuscript, Wolf & Koonin combine comparative genomics with Eigen's (1978) concept of the error threshold to provide a new, comprehensive model for the origins of translation. Specifically, they build on Szathmary's (1993) model of amino acids as coenzymes in an RNA metabolism as a starting point for the genetic code. As pointed out by Knight & Landweber (2000), there are three pathways to a protein-based genetic code from the RNA world that preserves continuity of features of the genetic code: the RNAs that bind directly could have played the roles of tRNAs, mRNAs, or aminoacyl-tRNA synthetases. Wolf & Koonin favor a model along the lines of the latter role, suggesting that cofactor-enhanced catalysis, and then nonribosomal synthesis of short peptides, were the original driving force for RNA-catalyzed translation. They present an intriguing new overall model of the evolution of the translation system, and highlight aspects of this model that could be tested in the laboratory. The main weakness of the manuscript in its current form is its endorsement of the frozen accident model (FAM) of the genetic code's evolution without the presentation of alternative explanations of the evidence in favor of the optimality of the genetic code relative to random codes, and the coding triplet/binding site associations that have been observed through SELEX and in the Group I intron. However, as the authors themselves point out, the resurrection of the frozen accident model is not an important feature of their overall model for the emergence of translation, and this discussion could be omitted without diminishing the manuscript's contribution.The manuscript presents some interesting ideas that I have not seen elsewhere and that appear to shed substantial new light on the difficult problem of the origin of translation.For example, the discussion on p. 13 that shows that the domains in the aaRS are highly derived relative to domains in other proteins is extremely interesting, because we might have expected the aaRS to be among the earliest proteins. If they are not, the likelihood that they displaced some other system for coded translation increases dramatically . One point that should be specifically noted in this context is that not only do these relationships imply that the aaRS are relatively late arrivals, but also that coded translation must have predated the aaRS so that the sequence information that allows us to determine the phylogenetic relationships among these folds could be transmitted to the present. In other words, if comparable folds were once produced by a different synthesis mechanism, either we would need either a system of reverse translation to copy the sequence information into nucleic acids, or all of the proteins produced by that mechanism would have been lost when coded translation took over.Similarly, the discussion on pp. 33–39 of a plausible scenario for the evolution of the modern translation system seems plausible and is more detailed than most such scenarios to be found in the literature.A couple of areas of the manuscript could potentially be supported by drawing on additional literature. For example, on p. 8, Dennett has an excellent discussion in "Darwin's Dangerous Idea"  of the production of apparently irreducibly complex phenomena through simplification of an even more complex system, e.g. building an arch by taking away stones from a pile of rubble. The complexity of the system of peptide- specific synthetases that would be required for the model proposed here might make this an appropriate metaphor. Similarly, Yarus's (2001) article "On translation by RNAs alone", and Yarus & Welch's (2000) article "Peptidyl transferase: ancient and exiguous" contain some thoughts that would be relevant here and later in the manuscript.Author response: Dennett's metaphor of the Roman arch is, indeed, excellent and might be relevant, even if not directly, because, here, we are talking more of stepwise displacement than selective elimination, and do not really postulate an initial state that was more complex than the final one. In any case, one of the strengths of the Biology Direct model is that the review is published, so the reader can read about this metaphor here. Ditto for the reviews by Yarus: the reader now knows of them and may turn to them if desirable (other work from Yarus' laboratory is cited extensively).The discussion of ribozymes on p. 18 could possibly benefit from a discussion of riboswitches and their implications for control mechanisms in the cell, and/or for the other roles or RNA that suggest the RNA World  However, the manuscript is fairly long as it is, and most of these points have been raised many times in the cited literature already.Author response: Yes, the paper is fairly long, and we believe that riboswtiches are of no direct relevance.Finally, some of the specific contentions could benefit from more elaboration. For example, on pp. 11–12, we find the statement:"Put another way, the conservation of the core of the translation machinery is the strongest available evidence that some form of LUCA actually existed ."Given that the present manuscript already proposes the evolution of an entire suite of RNA-based aminoacyl-tRNA synthetases that no longer exist, and given that some authors such as Carl Woese propose that the division of life into distinct phylogenetic lineages was a relatively late event (e.g. Woese 2002), it is unclear why horizontal gene transfer should be dismissed in this context.Author response: Upon more careful consideration , we have deleted this whole claim. Suffice it to say, in this context, that the conservation of the translation machinery is evidence of some form of LUCA.Similarly, on p. 20, the authors seem to be strongly in favor of the hydrothermal vent scenario for the origin of life. A few words of caution to the effect that this is one of many hypotheses for life's origin, and that data are still far from conclusive, might be in order.Author response: we have included a few words to that effect but also cite new references that, we believe, add credibility to the hydrothermal vent scenario .The discussion of the current evidence relating to the hypothesis that the genetic code arose through direct interactions between RNA and amino acids on p. 23 is good, but on p. 41 we read that "these affinities are weak, only manifest as a statistical trend, and worst of all, are seen, mostly, for chemically complex amino acids like arginine or histidine, rather than simple ones, such as glycine or alanine, that would be readily produced abiogenically." This statement requires some elaboration. Many of the potentially prebiotic amino acids, such as glycine, are difficult to evaluate with the affinity chromatography paradigm for technical reasons. It is possible that other methodologies, such as the allosteric selections pioneered by Tang & Breaker (1997), will allow us to see interactions in these cases, but for now absence of evidence should not be taken as evidence of absence. It is also far from certain that the biosynthesis of complex amino acids such as arginine would have been beyond the capabilities of RNA World organisms, so the primordial genetic code need not have been confined to simple amino acids. Second, the physical interactions involved are often far from weak: some amino acid aptamers, such as the best of Famulok's (1996) arginine aptamers, have sub-micromolar dissociation constants. It is true that the inconsistency between codon and anticodon modes of recognition remains to be resolved, but I do not agree with the assertion that "objectively, we should accept FAM as the most likely model for the emergence and evolution of translation". To accept FAM given what we know now about the optimality of the genetic code relative to random genetic codes, and the relationships between amino acid binding sites and cognate triplets, requires an alternative explanation for the strong statistical evidence that supports these hypotheses. In the absence of such an alternative explanation for why we see these patterns, which would be extremely unlikely under the FAM, I would recommend that the discussion be confined to pointing out where these processes would most likely be able to act in the model . It is possible that FAM is not an optimal description of what is actually meant in the discussion in the text – really, the claim seems to be that there is no necessary relationship between triplets of RNA and amino acids, rather than that there is in fact no pattern. However, in my opinion, the discussion of FAM vs. ARM vs. CRM as presented is likely to be a distraction from the overall value of the new ideas presented in the manuscript.Author response: We cannot agree that this description is a distraction; we think it is part and parcel of the paper, even if the choice between ARM, CRM, and FAM has a limited effect on the actual model considered here. However, this discussion has been shortened and modified to make it more neutral with regard to the choice between the model of amino acid- T RNA recognition. The statement regarding weak interactions between amino acids and aptamers has been dropped along with the over-assertive statement regarding FAM as "the most likely model". It seems like in the text we clearly explain what we mean by FAM – indeed, it is about a lack of any direct connection between amino acids and cognate triplet. Also, we consider the amended version of FAM where subsequent adaptation of the code is deemed likely.Finally, the description of experimental tests on p44 could benefit from more detail. Which properties of the postulated T RNAs are in doubt, and which steps would, if experimentally confirmed, best support the model? More specific guidance might increase the probability that supporting laboratory work would be carried out.Author response: A brief discussion has been added.Reviewer 2: Doron Lancet (Weizmann Institute of Science)This reviewer made no comments.Reviewer 3: Alexander Mankin, University of Illinois at Chicago (nominated by Arcady Mushegian)It is a fairly straightforward task to evaluate an experimental paper driven by the data. It is a much more fuzzy assignment to evaluate a theoretical paper discussing a possible evolutionary scenario of the origin of protein synthesis. It is very tempting to buy into all of the authors' arguments. It is equally tempting to criticize them all.The main postulate of Wolf and Koonin is that they are trying to build a model based on the Continuity Principle. In lay language, this means they are trying to put little solid rocks into the vast swamp that separates the evolutionary island of the RNA World, where most of the biochemical reactions are catalyzed by ribozymes, from the island of the modern nucleic acid-protein world, where biochemistry is carried out primarily by protein enzymes whilst nucleic acids are involved mostly in storage and expression of genetic information. Trying to bridge this gap, the authors envision the intermediate steps on the evolutionary path to the genetic code and coded protein synthesis, where innovations that arose at each of the steps could be selected for. In this approach, Wolf and Koonin strive to allow for the fewest number of evolutionary gaps that would require a significant leap rather than a small jump. Not that this is a new approach – most of the previous attempts to delineate the origin of protein synthesis were based on a generally similar idea. However, in the prior works, it was probably more of an intuitive attempt to build a plausible scenario than a formulated goal as in the essay of Wolf and Koonin.The question is how closely those rocks of Wolf and Koonin are spaced and how solid they are. Some of them appear to be nicely positioned and are fairly solid, whereas the others, in my view, are either shaky or missing.It seems to be a very reasonable idea that some of the RNA World ribozymes could benefit from a bound amino acid cofactor or even cofactors. It appears to be a much more far-fetched speculation that two or even more of these cofactors would bind in such close proximity of each other that the formation of a peptide bond between them would be possible and beneficial. Furthermore, it is not entirely clear from where a hypothetical peptide ligase would derive the energy that is required for peptide bond formation. In the modern ribosome, the energy that powers peptide bond formation is conserved in the high-energy ester bond that links the C-terminal amino acid of a nascent peptide to tRNA. The energy of this ester bond is derived from ATP consumed by an aminoacyl-tRNA synthetase – a source hardly available in the RNA world.Author response: Yes, the issue of the energy source is important. One would have to propose that one of the substrates of the primordial peptide ligase was an activated amino acid, perhaps, even an aminoacyl adenylate. In the RNA world, such derivatives would have to be produced by other ribozymes, and ribozymes with such an activity, indeed, have been described  EMBO Rep., 7, 699–703). From the point of view of this reviewer, it is more reasonable to root the origin of the small subunit in one of the pre-existing ribozymes that could operate with RNA templates. The extant activities of the modern small ribosomal subunit, including its interaction with an RNA template (mRNA) and ability to assemble on it the complementary sequences of the tRNA anticodons, bear the features expected from the ancestral RNA replicase/RNA ligase. Such a ribozyme could be viewed as an ancestor of the ribosome decoding center. The suspected ability of the modern 30 S subunit to cleave mRNA during ribosome stalling or under the influence of specific protein factors argues that the putative ancient catalytic center capable of breaking (and thus forming) phosphodiester bonds may still exist in the ribosome.The origin of the coded protein synthesis is based on availability of three main players: the adaptor aminoacyl-RNA molecules with a strict amino acid-anticodon correlation, an enzyme that can polymerize the activated amino acids , and a precursor of the small ribosomal subunit, a "reading head" that selects the adaptor aminoacyl-RNA according to the input genetic text. Wolf and Koonin derive the origin of the ancestor of the small ribosomal subunit not from a pre-existing ribozyme but from a segment of the large subunit precursor. In this 'Adam's rib' scenario, an accessory RNA subunit RAuthor response: The possibility that the small subunit of the ribosome evolved from an RNA replicase/triplicase is an interesting one, and we have considered a version of it when working on the current model. This could directly connect the model discussed here with the triplicase model of Pool-Jeffares-Penny. However...direct evidence is missing, so we decided to avoid "overfitting" the model. Let the reader learn about this idea from Mankin's comment. However, it is completely unclear to us why the work of Wohlgemuth et al. is construed as evidence against the model presented in the paper. We believe that, on the contrary, it is readily compatible with this model, and we cite it in the revision.In conclusion, the essay of Wolf and Koonin is an interesting and highly stimulating work. Inadvertently, my review sounds more critical than was intended. The reason is simple: the ideas we disagree with are more interesting for us than the points we easily accept. The majority of the points in the paper are of this latter category; the points my comments mostly focus on are of the former.Other points of critique and comments:per se starts on p. 28. It seems that an almost 30-page introduction is excessive and often repetitive. The work would strongly benefit if the first 28 pages were expressed more succinctly, possibly as bulleted points in 2 pages.1. The discussion of the model Author response: We appreciate the virtues of brevity but this paper was conceived as a specific model for the origin of translation placed against the critically examined background of the relevant general evolutionary principles and previous research in the area. We feel that it has to stay that way.Reviewer #4: Arcady MushegianThe most significant contribution of this study is in decomposing the tantalizingly complex problem of the origin of genetic code, translation, and RNA replication into a series of proposed small evolutionary transitions, each associated with its own contribution to the fitness of the genetic system that experiences these transitions. I whole-heartedly recommend this manuscript for publication and expect that this series of transitions will be further scrutinized, perhaps along the lines of necessity and sufficiency.My only scientific complain is about the half-haphazard conclusion that the frozen-accident model of adaptor recognition by amino acids is the most likely one. It might be, or it might be not: the fact that current direct experiments fail to establish specific recognition of cognate (anti)codons for evolutionarily more primitive amino acids does not make a "frozen accident" mechanistically attractive. Moreover, if, for example, primitive nucleobases were abiotically derivatized (see the work from S.Benner's lab that seems to point in this direction), then the experiments with the present-day codons or anticodons are not even answering the right question. The authors should mention that work or at least stay even more agnostic about the recognition model.Author response: we infused considerable extra agnosticism, also, in response to Knight's comments (see above).Other, minor, comments:"The Continuity Principle" has connections with Anton Dorn's change-of-function principle  – perhaps this is worth acknowledging.Author response: In truth, the principle really goes back to Darwin, the rest are reformulations and explanations. We jump to a modern version immediately, leaving Dorn out.As discussed by the authors, should Darwin-Eigen cycle be renamed Darvin-Eigen-Lynch-Conery cycle?Author response: If one wants to be really fair, then, maybe, Darwin-Eigen-Penny-Lynch-Conery -(Wolf-Koonin)? For the time being, we are sticking with the original name, after Penny.The study is well-written, but perhaps it can be edited a bit more. For example, the notion that "evolution has no foresight", however important, is seen at least five times, including two times within one bulleted list on pg 29.YIW incepted the study, developed the initial version of the model, and wrote the first draft of the model description; YIW and EVK jointly finalized the model; EVK wrote the manuscript. All authors have read and approved the final version of the manuscript.


Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode  Like Olympic swimmers crouched on their starting blocks, synaptic vesicles prepare for fusion with the neuronal plasma membrane long before the starting gun fires. This preparation enables vesicles to fuse rapidly, synchronously, and in the correct place when the signal finally arrives. A well-known but poorly understood part of vesicle preparation is docking, in which vesicles prepare for release by attaching to the plasma membrane at the eventual site of release. Here, we outline a molecular mechanism for docking. Using a combination of genetics and electron microscopy, we find that docking requires two proteins: the cytoplasmic protein UNC-13 and the plasma membrane protein syntaxin. Syntaxin is known to form two configurations, closed and open. We find that the open form of syntaxin can bypass the docking function of UNC-13, while the closed form cannot. These experiments suggest that docking is the attachment of synaptic vesicles to syntaxin; that syntaxin must be open for this attachment to occur; and that UNC-13′s role in docking is to promote open syntaxin. C. elegans support a model for synaptic vesicle docking in which the active zone protein UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane.Experiments in  Fusion of synaptic vesicles with the plasma membrane is thought to occur in three ordered steps: docking, priming, and fusion . The bioPriming describes a molecular state in which a four-helix SNARE complex has formed between SNARE proteins on a synaptic vesicle and those on the plasma membrane . It is bDocking precedes priming and at this point is defined solely by morphological criteria. Synaptic vesicle docking is observed in electron micrographs of the synapse and is defined as the attachment of vesicles to their target membranes –28. HoweIn addition, the molecular basis for docking is unknown. It is recognized that protein interactions must specifically associate a vesicle to the correct target membrane. In the original SNARE hypothesis, contacts between the SNARE proteins were proposed to confer specificity during docking . HoweverC. elegans using a new fixation method that reduces artifacts ; EG1983, unc-13(s69), unc-64(js115), and oxIs34; EG1985, unc-64(js115) and oxIs34; EG2279, unc-49(e407); EG2466, unc-64(js115) and oxIs33; EG3278, unc-64(js115) and oxEx536; EG3817, unc-64(js115) and oxEx705; EN560, krIs1 and lin-15(n765); MT8247, lin-15(n765) and nIs52; and NM959, unc-64(js115)/bli-5(e518).Wild type was Bristol N2. All strains were obtained from the unc-64(js115) and oxEx536, the strain NM959 unc-64(js115)/bli-5(e518) was injected using standard techniques  was generated in a similar way, except the injection mix contained pMH441, pMH417, and pMH427 . Multiple stable lines were obtained, and homozygous unc-64 animals were recovered from each line and found to have similar phenotypes.The GABA(−) syntaxin mosaic strain EG3817, H427 see , as wellunc-64(js115)/+, oxIs12, and oxEx536; EG3349, unc-64(js115)/+, nIs52, and oxEx536; and EG3299, unc-64(js115)/+, krIs1, and oxEx536. Homozygous unc-64 animals were recovered from these strains, allowed to self, and their progeny used for analysis.For fluorescence analysis of neuroanatomy in the syntaxin mosaic, strains carrying the appropriate fluorescent marker were crossed with EG3278 to generate the three strains EG3301, http://www.lemkesoft.com) and manually aligned using Midas . The VA/VD relationship was used as a fiduciary mark during the alignment. Image segmentation was performed in 3dmod (Boulder Laboratory for 3-D Electron Microscopy of Cells) by manually tracing neuronal profiles and presynaptic specializations at 200% magnification. Synaptic vesicles were modeled as spheres with a diameter of 28 nm, and section thickness was set to 33 nm.Reconstruction was performed on a VA synapse from a wild-type animal. We converted 16-bit TIFFs to 8-bit using Graphic Converter , oxIs12, oxEx536, and wild-type oxIs12) were imaged on a confocal microscope and scored blind to genotype for the number of commissures. oxIs12 expresses GFP in the GABA neurons under the control of the unc-47 promoter. For synapse density, five young adult animals of each genotype (unc-64(js115), nIs52, oxEx536, and wild-type nIs52) were imaged on a confocal microscope. nIs52 expresses synaptobrevin-GFP in the GABA neurons under the control of the unc-25 promoter. For each animal, ImageJ was used to measure a region of the dorsal nerve cord, and puncta within the region were counted. For pre- and postsynaptic colocalization, ten young adult animals of each genotype (unc-64(js115), krIs1, oxEx536, and wild-type krIs1) were imaged on a confocal microscope. krIs1 expresses synaptobrevin-CFP in the GABA neurons under the control of the unc-47 promoter and expresses GABAA-receptor-YFP in muscles under the unc-49 promoter. Colocalization of CFP and YFP was observed in all cases.For overall neuronal morphology, ten young adult animals of each genotype (C. elegans synapses (our observations). Finally, changes in membrane trafficking in the coelomocytes can be observed using the slow fixation method (our observations). For these reasons we defined docked vesicles in our previous study as those within 30 nm of the plasma membrane since identifying vesicles docked at the surface was unreliable. No docking defect was observed in unc-13 mutants using this definition [unc-13(e1091): acetylcholine = 4.45 ± 1.17, 33 profiles, GABA = 4.53 ± 1.07, 28 profiles; unc-13(s69): acetylcholine = 3.35 ± 1.30, 34 profiles, GABA = 4.16 ± 1.37, 32 profiles). Using high-pressure freezing we can now subdivide pools of docked vesicles and reliably determine if vesicles are touching the membrane; using this definition we see differences in docking in unc-13 mutants compared to the wild type.Previously we used ice-cold glutaraldehyde fixations for electron microscopy . We havefinition . Our curhttp://www.bal-tec.com). Frozen animals were fixed in a Leica EM AFS system (http://www.leica.com) with 0.5% glutaraldehyde and 0.1% tannic acid in anhydrous acetone for 4 d at −90 °C, followed by 2% osmium tetroxide in anhydrous acetone for 38.9 h with gradual temperature increases . Fixed animals were embedded in araldite resin . Mutant and control blocks were blinded. Ribbons of ultrathin (33 nm) serial sections were collected using an ultracut E microtome. Images were obtained on a Hitachi H-7100 electron microscope (http://www.hitachi.com) using a Gatan (http://www.gatan.com) slow=scan digital camera. A total of 250 ultrathin contiguous sections were cut and the ventral nerve cord reconstructed from two animals representing each genotype. Image analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij).Worms were prepared for transmission electron microscopy essentially as described ,93. BrieAll morphometry was conducted blind to genotype and included a matched wild-type worm that was fixed in parallel. The number of synaptic vesicles (∼30 nm in diameter) in each synapse was counted, and their diameters and distances from the dense projection and plasma membrane were measured. Analysis included the acetylcholine neurons VA and VB and the GABA neuron VD.n = 28 profiles; GABA 27.7 SV, n = 16 profiles) compared to freeze-substituted samples .To compare freeze-substitution fixations with our previous methods using ice-cold glutaraldehyde , we analhttp://www.aesculapusa.com), and a lateral incision was made to expose the ventral medial body wall muscles. The preparation was then treated with collagenase  for 15 s at a concentration of 0.5 mg/ml. The muscle was then voltage clamped using the whole cell configuration at a holding potential of −60 mV. See d-tubocurare  from a perfusion system. Pressure ejection of GABA from pipets of 4–5 MΩ resistance was computer triggered. Evoked responses were elicited using a fire-polished electrode positioned along the ventral nerve cord. The stimulating electrode was placed at least half a muscle length away from the patched muscle to cleanly separate the stimulus artifact from the evoked response. A square wave depolarizing current of 1 ms was then delivered from an SIU5 stimulation isolation unit driven from an S48 stimulator . All recordings were made at room temperature (21 °C) using an EPC-9 patch-clamp amplifier  run on an ITC-16 interface . Data were acquired using Pulse software (HEKA). All data analysis and graph preparation were performed using Pulsefit (HEKA), Mini Analysis , and Igor Pro . Bar graph data are presented as the mean ± S.E.M.Electrophysiological methods were performed as previously described ,73 with d-tubocurare provided a complete block. d-tubocurare block was tested daily on unc-49(e407) to insure that the solution aliquot completely blocked acetylcholine neurotransmission. d-tubocurare was added after 2 min of recording; recordings in d-tubocurare were done for 1 min for each animal. Mini analysis was performed on the traces beginning 10 s after d-tubocurare application and on traces both before d-tubocurare application and after washout. Only those animals with full recovery after d-tubocurare washout were used. From the matched unc-49 controls, no minis were observed in unc-49 in 4 min of total recordings from four animals. Thus, the probability of seeing a rogue acetylcholine mini from the matched controls is less than 0.0041 fusions per second. In addition we have recorded from 103 nonmatched unc-49 animals covering greater than an hour in d-tubocurare without seeing a single fusion event. The 0.06 fusions per second observed in the syntaxin mosaic (six minis observed) are therefore likely to be fusions from the GABA neurons. However, we cannot claim that these six minis are syntaxin-independent, since we cannot exclude the possibility that there is a low level of syntaxin expression in the GABA neurons from our transgenic array.To be confident about low mini rates we needed to be certain that Figure S1unc-64 locus. The plasmid pTX21 (gift of M. Nonet) rescues unc-64. At the 3′ end of the gene are three splice variants that encode alternative transmembrane domains (gray exons), which produce transcripts UNC-64C, UNC-64A, and UNC-64B.(A) Syntaxin is encoded by the unc-64 cDNA-genomic hybrid was constructed that removes the 5′ introns but keeps the alternatively spliced 3′ exons.(B) An unc-64 null mutant background. Each finished construct contains promoter sequence up to the ATG, an SphI site not present in wild-type syntaxin that encodes Ala-Cys and the syntaxin minigene. The oxEx536 transgene array does not express syntaxin in the GABA motor neurons or the acetylcholine motor neurons. The oxEx705 transgene array does not express syntaxin in the GABA motor neurons but does express it in the acetylcholine motor neurons. See (C) The minigene was placed under the control of various promoters to drive expression of UNC-64 in subsets of cells in the (105 KB PDF)Click here for additional data file.Figure S2C. elegans [(A) Organization of motor neuron commissures on the right side of  elegans ; there aoxEx536 transgene, which lacks syntaxin in GABA and acetylcholine motor neurons. The right side of the animal is shown. There are no commissures that are syntaxin positive, consistent with lack of syntaxin in all motor neurons. Syntaxin is expressed only in the nerve ring and the head neurons that project into the ventral nerve cord. See (B) Shown is antisyntaxin antibody staining of EG3278 with the oxEx705 transgene, which lacks syntaxin in GABA neurons. The right side of the animal is shown. The position and number of commissures that are syntaxin positive is consistent with syntaxin expression in acetylcholine, but not GABA, motor neurons. Syntaxin is also expressed in the nerve ring and the sublateral cords. The green pharynx is due to GFP expression under the myo-2 promoter (see (C) Shown is antisyntaxin antibody staining of EG3817 with the oter see .(753 KB PDF)Click here for additional data file.Figure S3The distribution of synaptic vesicles was determined for fully reconstructed GABA synapses. Lateral axon profiles flanking the center of each dense projection (DP) were analyzed until the cloud of synaptic vesicles disappeared. These data include vesicles in the cytoplasm and docked at the plasma membrane. Because docked vesicles represent a minor portion of the total number of vesicles, this distribution largely reflects the reserve pool of vesicles.(A) In the wild type, vesicles are most concentrated in sections containing a dense projection and form a cloud of vesicles about 700 nm in diameter.n = 10 synapses, two animals; mosaic, n = 5 synapses, one animal.(B) In syntaxin(−) neurons (EG3817), synaptic vesicles are distributed in a similar pattern. Distributions were centered on the center of the dense projection, and the average number of synaptic vesicles in each section determined. The average number of synaptic vesicles per axon profile is shown. Section thickness was 33 nm, so the distance from the center of the dense projection is sorted into 33-nm bins. Wild type, (225 KB PDF)Click here for additional data file.Protocol S1(43 KB DOC)Click here for additional data file.Table S1(380 KB DOC)Click here for additional data file.