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Infectious laryngotracheitis virus (ILTV) is a highly contagious acute respiratory poultry pathogen. Modified live ILTV vaccines are the only control against ILT infections. Reversions and establishment of latent infections are the major concerns imparting the need to develop safer vaccines against ILTV infection. ILTV glycoprotein B and D (gB and gD) are major protective immunogens. The factors shaping synonymous codon usage bias and nucleotide composition in ILTV glycoprotein genes have not yet been reported. In the present study, we have analyzed the synonymous codon usage indices of ILTV gB and gD genes. Variation in the codon usage was seen in both the glycoproteins majorly by mutational pressure. The pattern was determined using the correspondence analysis, effective number of codon (Nc), GC3 plot and correlation analyses among different indices. The study is a comprehensive analysis of the codon usage patterns of ILTV glycoprotein genes. This will be helpful in understanding the codon usage bias of ILTV and related DNA viruses which could further explore its biology., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Since the elucidation of the genetic code almost 50 years ago, many nonrandom aspects of its codon organization remain only partly resolved. Here, we investigate the recent hypothesis of 'dual-use' codons which proposes that in addition to allowing adjustment of codon optimization to tRNA abundance, the degeneracy in the triplet-based genetic code also multiplexes information regarding DNA's helical shape and protein-binding dynamics while avoiding interference with other protein-level characteristics determined by amino acid properties. How such structural optimization of the code within eukaryotic chromatin could have arisen from an RNA world is a mystery, but would imply some preadaptation in an RNA context. We analyzed synonymous (protein-silent) and nonsynonymous (protein-altering) mutational impacts on molecular dynamics in 13823 identically degenerate alternative codon reorganizations, defined by codon transitions in 7680 GPU-accelerated molecular dynamic simulations of implicitly and explicitly solvated double-stranded aRNA and bDNA structures. When compared to all possible alternative codon assignments, the standard genetic code minimized the impact of synonymous mutations on the random atomic fluctuations and correlations of carbon backbone vector trajectories while facilitating the specific movements that contribute to DNA polymer flexibility. This trend was notably stronger in the context of RNA supporting the idea that dual-use codon optimization and informational multiplexing in DNA resulted from the preadaptation of the RNA duplex to resist changes to thermostability. The nonrandom and divergent molecular dynamics of synonymous mutations also imply that the triplet-based code may have resulted from adaptive functional expansion enabling a primordial doublet code to multiplex gene regulatory information via the shape and charge of the minor groove.

Recent outbreaks of Zika virus (ZIKV) in Africa, Latin America, Europe, and Southeast Asia have resulted in serious health concerns. To understand more about evolution and transmission of ZIKV, detailed codon usage analysis was performed for all available strains. A high effective number of codons (ENC) value indicated the presence of low codon usage bias in ZIKV. The effect of mutational pressure on codon usage bias was confirmed by significant correlations between nucleotide compositions at third codon positions and ENCs. Correlation analysis between Gravy values, Aroma values and nucleotide compositions at third codon positions also indicated some influence of natural selection. However, the low codon adaptation index (CAI) value of ZIKV with reference to human and mosquito indicated poor adaptation of ZIKV codon usage towards its hosts, signifying that natural selection has a weaker influence than mutational pressure. Additionally, relative dinucleotide frequencies, geographical distribution, and evolutionary processes also influenced the codon usage pattern to some extent.

The polymerase (P) and surface (S) genes of hepatitis B virus (HBV) show the longest gene overlap in animal viruses. Gene overlaps originate by the overprinting of a novel frame onto an ancestral pre-existing frame. Identifying which frame is ancestral and which frame is de novo (the genealogy of the overlap) is an appealing topic. However, the P/S overlap of HBV is an intriguing paradox, because both genes are indispensable for virus survival. Thus, the hypothesis of a primordial virus without the surface protein or without the polymerase makes no biological sense. With the aim to determine the genealogy of the overlap, the codon usage of the overlapping frames P and S was compared to that of the non-overlapping region. It was found that the overlap of human HBV had two patterns of codon usage. One was localized in the 59 one-third of the overlap and the other in the 39 two-thirds. By extending the analysis to non-human HBVs, it was found that this feature occurred in all hepadnaviruses. Under the assumption that the ancestral frame has a codon usage significantly closer to that of the non-overlapping region than the de novo frame, the ancestral frames in the 59 and 39 region of the overlap could be predicted. They were, respectively, frame S and frame P. These results suggest that the spacer domain of the polymerase and the S domain of the surface protein originated de novo by overprinting. They support a modular evolution hypothesis for the origin of the overlap.

The non-universality of the genetic code is now widely appreciated. Codes differ between organisms, and certain genes are known to alter the decoding rules in a site-specific manner. Recently discovered examples of decoding plasticity are particularly spectacular. These examples include organisms and organelles with disruptions of triplet continuity during the translation of many genes, viruses that alter the entire genetic code of their hosts and organisms that adjust their genetic code in response to changing environments. In this Review, we outline various modes of alternative genetic decoding and expand existing terminology to accommodate recently discovered manifestations of this seemingly sophisticated phenomenon.

The reading frame coding (RFC) of codes (sets) of trinucleotides is a genetic concept which has been largely ignored during the last 50 years. A first objective is the definition of a new and simple statistical parameter PrRFC for analysing the probability (efficiency) of reading frame coding (RFC) of any trinucleotide code. A second objective is to reveal different classes and subclasses of trinucleotide codes involved in reading frame coding: the circular codes of 20 trinucleotides and the bijective genetic codes of 20 trinucleotides coding the 20 amino acids. This approach allows us to propose a genetic scale of reading frame coding which ranges from 1/3 with the random codes (RFC probability identical in the three frames) to 1 with the comma-free circular codes (RFC probability maximal in the reading frame and null in the two shifted frames). This genetic scale shows, in particular, the reading frame coding probabilities of the 12,964,440 circular codes (PrRFC=83.2% in average), the 216 C(3) self-complementary circular codes (PrRFC=84.1% in average) including the code X identified in eukaryotic and prokaryotic genes (PrRFC=81.3%) and the 339,738,624 bijective genetic codes (PrRFC=61.5% in average) including the 52 codes without permuted trinucleotides (PrRFC=66.0% in average). Otherwise, the reading frame coding probabilities of each trinucleotide code coding an amino acid with the universal genetic code are also determined. The four amino acids Gly, Lys, Phe and Pro are coded by codes (not circular) with RFC probabilities equal to 2/3, 1/2, 1/2 and 2/3, respectively. The amino acid Leu is coded by a circular code (not comma-free) with a RFC probability equal to 18/19. The 15 other amino acids are coded by comma-free circular codes, i.e. with RFC probabilities equal to 1. The identification of coding properties in some classes of trinucleotide codes studied here may bring new insights in the origin and evolution of the genetic code., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Codons expanded by a silent position (quadruplet or tetracodons) may solve the conundrum that at life's origins, the weak tricodon-anticodon interactions could not promote translation in the absence of complex ribosomes. Modern genomes have isolated tetracodons resulting from insertion mutations. Some bioinformatic analyses suggest that tetracoding stretches overlap with regular mitochondrial protein coding genes. These tetragenes are probably decoded by (antisense) tRNAs with expanded anticodons. They are GC-rich, which produce stronger basepairs than A:T interactions, suggesting expression at high temperatures. The hypothesis that tetracoding is an adaptation to high temperatures is tested here by comparing predicted mitochondrial tetracoding in Lepidosauria (lizards, amphisbaenia, and Sphenodon), in relation to body temperature, expecting more tetracoding in species with high body temperature. The association between tRNAs with expanded anticodons and tetracoding previously described for mammals and Drosophila is confirmed for Lepidosauria. Independent evidence indicates that tetracoding increases with body temperature, supporting the hypothesis that tetracoding is an adaptation for efficient translation when conditions (temperature) make triplet codon-anticodons too unstable to allow efficient protein elongation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Elongator is required for the synthesis of the mcm(5)s(2) modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.

The second codon of a transcript, besides encoding for an amino acid, is now known to also have multiple molecular functions and is involved in translation efficiency and protein turn-over and maturation processing. These multiple purposes therefore make the selection constraints on this codon's composition more complex. To examine the biological significance of various permutations of the second codon, we conducted a systematic survey of second codon composition from 442 selected genomes across three domains. The amino acid bias of the second codon is associated with specific protein functions. The most common amino acids (S, A, K and T) are significantly avoided in Cell Envelope-related genes but preferred in Translation or Energy Metabolism-related genes, suggesting that the function of a gene product is a significant factor influencing the composition of the second codon., (2010 Elsevier Inc. All rights reserved.)

We have evaluated the effect of mammalian codon optimization on the immunogenicity and protective efficacy of plasmid DNA vaccines encoding pre-erythrocytic stage Plasmodium falciparum and Plasmodium yoelii antigens in mice. Codon optimization significantly enhanced in vitro expression and in vivo antibody responses for P. falciparum circumsporozoite protein (PfCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) but not for P. yoelii circumsporozoite protein (PyCSP). Unexpectedly, more robust CD4+ and CD8+ T cell responses as measured by IFN-gamma ELIspot, lymphoproliferation, and cytotoxic T lymphocyte assays were noted with native as compared with codon optimization constructs. Codon optimization also failed to enhance CD8+ T cell dependent protection against P. yoelii sporozoite challenge as measured by liver-stage parasite burden. These data demonstrate that the effect of mammalian codon optimization is antigen-dependent and may not be beneficial for vaccines designed to induce T cell dependent protective immunity in this malaria model.

The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni-NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.

In Streptomyces coelicolor, bldA encodes the only tRNA for a rare leucine codon, UUA. This tRNA is unnecessary for growth, but is required for some aspects of secondary metabolism and morphological development, as revealed by the phenotypes of bldA mutants in diverse streptomycetes. This article is a comprehensive review of out understanding of this unusual situation. Based on information from four sequenced genomes it now appears that, typically, about 2 approximately 3% of genes in any one streptomycete contain a TTA codon, most having been acquired through species-specific horizontal gene transfer. Among the few widely conserved TTA-containing genes, mutations in just one, the pleiotropic regulatory gene adpA, give an obvious phenotype: such mutants are defective in aerial growth and sporulation, but vary in the extent of their impairment in secondary metabolism in different streptomycetes. The TTA codon in adpA is largely responsible for the morphological phenotype of a bldA mutant of S. coelicolor. AdpA-dependent targets include several genes involved in the integrated action of extracellular proteases that, at least in some species, are involved in the conversion of primary biomass into spores. The effects of bldA mutations on secondary metabolism are mostly attributable to the presence of TTA codons in pathway-specific genes, particularly in transcriptional activator genes. This is not confined to S. coelicolor-it is true for about half of all known antibiotic biosynthetic gene sets from streptomycetes. Combined microarray and proteomic analysis of liquid (and therefore non-sporulating) S. coelicolor bldA mutant cultures revealed effects of the mutation during rapid growth, during transition phase, and in stationary phase. Some of these effects may be secondary consequences of changes in the pattern of ppGpp accumulation. It is argued that the preferential accumulation of the bldA tRNA under conditions in which growth is significantly constrained has evolved to favour the expression of genes that confer adaptive benefits in intermittently encountered sub-optimal environments. The evolution of this system may have been a secondary consequence of the selective pressure exerted by bacteriophage attack. Some biotechnological implications of bldA phenomenology are considered.

Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

Codon usage in Clonorchis sinensis was analyzed using 12,515 codons from 38 coding sequences. Total GC content was 49.83%, and GC1, GC2 and GC3 contents were 56.32%, 43.15% and 50.00%, respectively. The effective number of codons converged at 51-53 codons. When plotted against total GC content or GC3, codon usage was distributed in relation to GC3 biases. Relative synonymous codon usage for each codon revealed a single major trend, which was highly correlated with GC content at the third position when codons began with A or U at the first two positions. In codons beginning with G or C base at the first two positions, the G or C base rarely occurred at the third position. These results suggest that codon usage is shaped by a bias towards G or C at the third base, and that this is affected by the first and second bases.

Members of the genus Xanthomonas are significant phytopathogens, which cause diseases in several economically important crops including rice, canola, tomato, citrus, etc. We have analyzed the genomes of six recently sequenced Xanthomonas strains for their synonymous codon usage patterns for all of protein coding genes and specific genes associated with pathogenesis, and determined the predicted highly expressed (PHX) genes by the use of the codon adaptation index (CAI). Our results show considerable heterogeneity among the genes of these moderately G+C rich genomes. Most of the genes were moderate to highly biased in their codon usage. However, unlike ribosomal protein genes, which were governed by translational selection, those genes associated with pathogenesis (GAP) were affected by mutational pressure and were predicted to have moderate to low expression levels. Only two out of 339 GAP genes were in the PHX category. PHX genes present in clusters of orthologous groups of proteins (COGs) were identified. Genes in the plasmids present in two strains showed moderate to low expression level and only a couple of genes featured in the PHX list. Common genes present in the top-20 PHX gene-list were identified and their possible functions are discussed. Correspondence analysis showed that genes are highly confined to a core in the plot.

In all, 238 and 155 transfer (t)RNA genes were predicted from the genomes of Phytophthora sojae and P. ramorum, respectively. After omitting pseudogenes and undetermined types of tRNA genes, there remained 208 P. sojae tRNA genes and 140 P. ramorum tRNA genes. There were 45 types of tRNA genes, with distinct anticodons, in each species. Fourteen common anticodon types of tRNAs are missing altogether from the genome in the two species; however, these appear to be compensated by wobbling of other tRNA anticodons in a manner which is tied to the codon bias in Phytophthora genes. The most abundant tRNA class was arginine in both P. sojae and P. ramorum. A codon usage table was generated for these two organisms from a total of 9,803,525 codons in P. sojae and 7,496,598 codons in P. ramorum. The most abundant codon type detected from the codon usage tables was GAG (encoding glutamic acid), whereas the most numerous tRNA gene had a methionine anticodon (CAT). The correlation between the frequencies of tRNA genes and the codon frequencies in protein-coding genes was very low (0.12 in P. sojae and 0.19 in P. ramorum); however, the correlation between amino acid tRNA gene frequency and the corresponding amino acid codon frequency in P. sojae and P. ramorum was substantially higher (0.53 in P. sojae and 0.77 in P. ramorum). The codon usage frequencies of P. sojae and P ramorum were very strongly correlated (0.99), as were tRNA gene frequencies (0.77). Approximately 60% of orthologous tRNA gene pairs in P sojae and P. ramorum are located in regions that have conserved synteny in the two species.

The novel heterodimeric glycoprotein hormone thyrostimulin consists of two unique subunits, A2 and B5. To understand its yet unknown transcriptional regulation, we characterised the 3.1-kb immediate 5'-flanking region of the human A2 gene localised on chromosome 11q13. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -506 to -347 relative to the ATG start codon. Interestingly, this minimal promoter appeared to be non-tissue-specific. Deletional, mutational and gel shift analyses revealed regulatory elements that are essential for the regulation of the A2 gene expression. Another noteworthy feature of this gene is the presence of silencer elements upstream and downstream of the promoter. To surmise, our results provide an initial step toward a detailed analysis of the underlying molecular mechanisms of the human thyrostimulin gene expression.

Codon usage studies have been carried out on the coding sequences of Thermoplasma acidophilum, which is an archaeon and grows at very low pH and high temperature. Overall codon usage data analysis indicates that all the four bases are almost equifrequent at the third position of codons, which is expected (since genomic GC % of this genome is about 46%). However, multivariate statistical analysis indicates that there are two major trends in the codon usage variation among the genes in this organism. In the first major trend it is observed that genes having G and C ending codons are clustered at one end while, A and T ending ones are clustered at the other end. We have also found a significant positive correlation between the expressivities of genes and GC contents at the synonymous third codon positions. In the second major trend, it is seen that the genes are clustered into three distinct parts. A comparative analyses of codon usage data of T. acidophilum and Sulfolobus solfataricus reveals that one of the three clusters of genes of T. acidophilum is very similar to a considerable number of S. solfataricus genes, suggesting possible occurrences of lateral gene transfer between these two microorganisms as reported by earlier workers., ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We found that the diauxic shift causes no overall change in translational accuracy but does cause a strong reduction in the frequency of one type of programmed error: Ty +1 frameshifting. Genetic data suggest that this effect may be due to changes in the relative amounts of tRNA participating in translation elongation. We discuss possible implications for expression strategies that use recoding.

The association between codon usage and gene function was analyzed in the complete genomes of Eschericia coli, Bacillus subtilis, Lactococcus lactis and Campylobacter jejuni, using the functional annotation provided by NCBI. Two distinctly different ways of quantifying codon usage were used in the analysis. By using contingency tables it was found that for most amino acids a highly significant association with gene function exists for all species, indicating that codon usage at the level of individual amino acids is generally closely coordinated with gene function. By computing the effective number of codons in the annotated genes and comparing the median values in groups of different gene functions it was shown for all species that codon bias gene by gene also differs.

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

It becomes increasingly clear that therapeutic gene delivery should provide not only for the sustained high level of gene expression but also, in most cases, for the regulated expression of transgenes as much as it occurs under natural conditions. Over the past few years a variety of different systems have been developed in order to regulate the amounts of transcribed RNA upon administration of exogenous agents, or in autoregulated manner. While efforts were focused on optimizing gene expression at the transcriptional level, other levels are still overlooked. In the meantime, regulation of gene expression is not restricted to transcription, but is also executed at the post-transcriptional level, i.e. mRNA stability, processing, transport, translation, protein stability, and modification. Codon usage is considered to be one of the critical factors that limit the expression rate of heterologous genes in different organisms at the posttranscriptional level. HIV-1 structural genes gag, pol, and env represent one of the most extensively utilized models for studying codon usage-mediated effects on transgene expression. In the current work we demonstrate that the codon content affects not only CMV-driven HIV-1 gag expression but also the expression of luciferase reporter gene transcribed independently from the SV40 promoter. The expression levels of both transgenes co-transfected into the human H1299 were inversely co-dependent. The observed phenomenon may be described as sequence-independent post-transcriptional gene silencing, which reflects the existing limitation of transgene expression in mammalian cells at the post-transcriptional level. Optimization of the codon usage may provide for the additional level of regulation of transgene expression in gene transfer experiments in order to maintain the concentration of the protein at the therapeutic levels.

We have measured, by reverse transcription and real-time quantitative PCR, the steady-state levels of the mitochondrial and nuclear transcripts encoding several subunits of the human oxidative phosphorylation (OXPHOS) system, in different normal tissues (muscle, liver, trachea, and kidney) and in cultured cells (normal fibroblasts, 143B osteosarcoma cells, 143B206 rho(0) cells). Five mitochondrial transcripts and nine nuclear transcripts were assessed. The measured amounts of these OXPHOS transcripts in muscle samples corroborated data obtained by others using the serial analysis of gene expression (SAGE) method to appraise gene expression in the same type of tissue. Steady-state levels for all the transcripts were found to range over more than two orders of magnitude. Most of the time, the mitochondrial H-strand transcripts were present at higher levels than the nuclear transcripts. The mitochondrial L-strand transcript ND6 was usually present at a low level. Cultured 143B cells contained significantly reduced amounts of mitochondrial transcripts in comparison with the tissue samples. In 143B206 rho(0) cells, fully depleted of mitochondrial DNA, the levels of nuclear OXPHOS transcripts were not modified in comparison with the parental cells. This observation indicated that nuclear transcription is not coordinated with mitochondrial transcription. We also observed that in the different tissues and cells, there is a transcriptional coregulation of all the investigated nuclear genes. Nuclear OXPHOS gene expression seems to be finely regulated.

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.

The opal mutation (UGA) at nucleotide 2670-2672 in the replicase gene of the attenuated tomato mosaic virus K (ToMV-K) mainly contributes to the virus attenuation based on a series of studies on the viral attenuation mechanism. From analysis and comparison between the replicase gene mutation point of ToMV-K and the related regions of all plant viruses containing the leaky UGA, we have found that some characters, including the CGG motif, alpha-helix structure and some specific amino acids, are, presumably, able to helpfully confer the readthrough mechanism. Finally, some other ssRNA plant viruses like PVX, PVY, CMV have been analyzed. We found that their genomic modifications and viral attenuations could be explored according to the mutation mode of the ToMV-K replicase.

Cellular SRC (c-SRC), which is the human homolog of the Rous sarcoma viral oncogene, v-src, is highly activated in colorectal cancer. Recently, a subset of colon cancers have shown a nonsense mutation at codon 531, which truncates c-SRC directly C-terminal to the c-SRC kinase regulatory domain. This specific mutation has been demonstrated to be activating, transforming, and tumorigenic and to promote metastasis. We investigated 100 rectal cancers, half of which had tumor spread outside the rectum, for this mutation by using direct sequencing. None of these tumors displayed any genetic alteration at this locus, and we thus conclude that the codon 531 mutation is a rare cause of c-SRC activation in rectal cancer.

Most amino acids are specified by more than one trinucleotide codon. Here we show that amino acids of differing functional importance may be distinguished by the pattern of synonymous codon usage. GC-rich genes tend to be of a greater transcriptional (p<0.01) and mitogenic (p<0.0001) significance than AT-rich genes, consistent with GC-->AT mutational drift in methylated genomic regions. Third-base GC retention also identifies critical amino acids within individual proteins, as indicated by non-random patterns of codon variation between gene homologs and also by differential sequelae of site-directed mutagenesis. Sequence analysis of human receptor tyrosine kinase genes confirms that functionally important transmembrane hydrophobic amino acids are specified by codons containing GC third bases more often than are transmembrane neutral amino acids (chi(2)=134.2). Amino acids encoded by GC third bases thus appear more tightly linked to cell function and survival than are those encoded by AT third bases.

A lambdaZAP Express cDNA library was constructed with mRNA obtained from immature miracidia within eggs, hatched miracidia, and sporocysts of Echinostoma paraensei. This cDNA library was amplified and 213 expressed sequence tag (EST) sequences (averaging 466 nucleotides in length) were obtained. The mean percentage of unresolved bases within the EST sequences was 0.4%, ranging from 0 to 4.6%. The 213 ESTs represent 151 unique messages. BLAST (version 2.0.8) analysis disclosed that 64 unique E. paraensei messages (42.4%) had significant similarities (BLAST score < or =e-5), at deduced amino acid or nucleotide levels, with known sequences in the nonredundant GenBank databases or the dbEST database (NCBI). The remainder, 57.6% of the unique EST-encoded messages, scored nonsignificant hits. Most of the E. paraensei messages that could be assigned a cellular role based on sequence similarities were involved in gene/protein expression. Several ESTs scored highest similarities with sequences obtained from trematode species. A total of 22,560 nucleotides present in open reading frames from ESTs that aligned with known sequences was used to determine codon usage for E. paraensei. Analysis of a subset of eight ESTs that contained full-length open reading frames did not reveal a bias in codon usage. Also, EST sequences were found to contain 3' untranslated regions with an average length of 69.9 +/- 88.4 nucleotides (n = 46). The EST sequences were submitted to GenBank/dbEST, adding to the 51 available Echinostoma-derived sequences, to provide reference information for both phylogenetic analysis and study of general trematode biology.

At the end of 1996 we approximated the total number of protein coding ORFs in the Saccharomyces cerevisiae genome, based on their properties, as 4700-4800. The number is much smaller than the 5800 which is widely accepted. According to our calculations, there remain about 200-300 orphans-ORFs without known function or homology to already discovered genes, which is only about 5% of the total number of genes. Our results would be questionable if the analysed set of known genes was not a statistically representative sample of the whole set of protein coding genes in the S. cerevisiae genome. Therefore, we repeated our estimation using recently updated databases. In the course of the last 18 months, previously unknown functions of about 500 genes have been found. We have used these to check our method, former results and conclusions. Our previous estimation of the total number of coding ORFs was confirmed., (Copyright 1999 John Wiley & Sons, Ltd.)

Computational modeling was performed to determine the potential function of the queuosine modification of tRNA found in wobble position 34 of tRNAasp, tRNAasn, tRNAhis, and tRNAtyr. Using the crystal structure of tRNAasp and a tRNA-tRNA-mRNA complex model, we show that the queuosine modification serves as a structurally restrictive base for tRNA anticodon loop flexibility. An extended intraresidue and intramolecular hydrogen bonding network is established by queuosine. The quaternary amine of the 7-aminomethyl side chain hydrogen bonds with the base's carbonyl oxygen. This positions the dihydroxycyclopentenediol ring of queuosine in proper orientation for hydrogen bonding with the backbone of the neighboring uridine 33 residue. The interresidue association stabilizes the formation of a cross-loop hydrogen bond between the uridine 33 base and the phosphoribosyl backbone of the cytosine at position 36. Additional interactions between RNAs in the translation complex were studied with regard to potential codon context and codon bias effects. Neither steric nor electrostatic interaction occurs between aminoacyl- and peptidyl-site tRNA anticodon loops that are modified with queuosine. However, there is a difference in the strength of anticodon/codon associations (codon bias) based on the presence or lack of queuosine in the wobble position of the tRNA. Unmodified (guanosine-containing) tRNAasp forms a very stable association with cytosine (GAC), but is much less stable in complex with a uridine-containing codon (GAU). Queuosine-modified tRNAasp exhibits no bias for either of cognate codons GAC or GAU and demonstrates a lower binding energy similar to the wobble pairing of guanosine-containing tRNA with a GAU codon. This is proposed to be due to the inflexibility of the queuosine-modified anticodon loop to accommodate proper positioning for optimal Watson-Crick type associations. A preliminary survey of codon usage patterns in oncodevelopmental versus housekeeping gene transcripts suggests a significant difference in bias for the queuosine-associated codons. Therefore, the queuosine modification may have the potential to influence cellular growth and differentiation by codon bias-based regulation of protein synthesis for discrete mRNA transcripts.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

The sequenced genomes of the two closely related bacteria Mycoplasma genitalium and Mycoplasma pneumoniae were compared with emphasis on genome organization and coding capacity. All the 470 proposed open reading frames (ORFs) of the smaller M.genitalium genome (580 kb) were contained in the larger genome (816 kb) of M.pneumoniae. There were some discrepancies in annotation, but inspection of the DNA sequences showed that the corresponding DNA was always present in M. pneumoniae. The two genomes could be subdivided into six segments. The order of orthologous genes was well conserved within individual segments but the order of these segments in both bacteria was different. We explain the different organization of the segments by translocation via homologous recombination. The translocations did not disturb the continuous bidirectional course of transcription in both genomes, starting at the proposed origin of replication. The additional 236 kb in M.pneumoniae,compared with theM.genitalium genome, were coding for 209 proposed ORFs not identified in M.genitalium. Of these ORFs, 110 were specific to M.pneumoniae exhibiting no significant similarity to M.genitalium ORFs, while 76 ORFs were amplifications of ORFs existing mainly as single copies in M. genitalium. In addition, 23 ORFs containing a copy of either one of the three repetitive DNA sequences RepMP2/3, RepMP4 and RepMP5 were annotated in M.pneumoniae but not in M.genitalium,although similar DNA sequences were present. TheM.pneumoniae-specific genes included a restriction-modification system, two transport systems for carbohydrates, the complete set of three genes coding for the arginine dihydrolase pathway and 14 copies of the repetitive DNA sequence RepMP1 which were part of several different translated genes with unknown function.

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the beta subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.

There is no easy way to selectively introduce mixtures of codon triplets into mutagenesis libraries. Solid-phase-supported DNA synthesis using successive coupling of mixtures of mononucleotides can be made to supply 32 codons, which gives redundancies in coding for 20 natural amino acids, as well as an often unwanted stop codon. Resin-splitting methods have been described, but the representation of all permutations is limited by mechanical factors for a large library, and the method is experimentally cumbersome. To demonstrate a third, improved method, the 3'-cyanoethyl phosphoramidite codon triplets dATA, dCTT, dATC, dATG and dAGC were made by solution-phase methods, with protecting groups fully compatible with modern automated phosphoramidite DNA synthesis chemistry. The reagents were then used to synthesize a 54-mer DNA fragment, wherein 15 internal base pairs were randomized by coupling a mixture of the five codons five times. The fragment was amplified as a cDNA pool, which was subcloned into a phagemid vector, and 16 randomly selected recombinants from this mini-library were sequenced. These clones showed random incorporation of the proper transcribed codon sequences at the correct location. Other functional tests involving the trinucleotide phosphoramidites showed modest (ca. 70%) coupling efficiencies and structural integrity of the DNA produced.

In some families with early-onset Alzheimer's disease (AD) pathogenic mutations have been found in exons 16 and 17 of the amyloid precursor protein (APP) gene. One case of schizophrenia has been described with a mutation at codon 713. We have developed a single strand conformation polymorphism (SSCP) method that detects mutations in these exons and investigated 98 AD cases and 56 elderly healthy controls. An earlier reported mutation at codon 713 in a healthy control and a previously undescribed polymorphism at codon 705 in a sporadic case of AD were found. These mutations are probably not related to disease pathogenesis.

Conformational transitions of Phe-tRNA(Phe) that take place during elongation factor Tu (EF-Tu)-dependent binding to the A site of Escherichia coli ribosomes were followed by transient fluorescence measurements. The fluorescence signal of proflavin replacing dihydrouracil at position 16 or 17 in yeast tRNA(Phe) was utilized to monitor changes of the conformation of the D loop. The ternary complex EF-Tu.GTP.Phe-TRNA(Phe)(Pf16/17) was purified by gel filtration. Upon binding of the complex to the A site of poly(U)-programmed, P-site-blocked ribosomes, the fluorescence changes in several steps. First, the rapid formation of an initial complex gives rise to a small fluorescence increase. Subsequent codon-anticodon recognition leads to a conformational rearrangement of the D loop of the tRNA that is reflected in a major fluorescence increase. Fluorescence-quenching data indicate an unfolding of the D loop in this state. The latter conformational state is short-lived, and the aminoacyl-tRNA refolds during the following rearrangement that occurs after GTP hydrolysis and accompanies the release of the aminoacyl-tRNA from EF-Tu.GDP and/or its accommodation in the A site. Further experiments show that the status of the P site influences the binding to the A site in that the two rearrangement steps are slowed down when the P site is unoccupied and even more so when it is occupied with the near-cognate tRNA(Leu2). In contrast, the occupancy of the E site has no influence on A-site binding, and vice versa, thus excluding any coupling between the two sites.

The phytopathogenic fungus Sclerotinia sclerotiorum produces a number of extra-cellular pectin-degrading enzymes. We have cloned and determined the complete sequence of a gene (pg1) encoding an endopolygalacturonase (PG1). The coding region consists of a non-interrupted 1143-bp open reading frame. S. sclerotiorum pg1 was compared to other fungal PG-encoding genes. Basic transcription control sequences were identified in the 5' non-coding region. The deduced amino acid (aa) sequence (380 aa) of the enzyme is compared to seven fungal PG sequences and shows a high level of identity (41.5 to 59.8%). Predicted secondary structures were compared, revealing a similar protein organization most probably in antiparallel beta sheets. Hybridization analysis using a pg1 0.65-kb BamHI fragment as a probe allowed the identification of seven different recombinant phages from a genomic library. Analysis of the hybridizing restriction fragments suggests that PG-encoding genes are organized as a family.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in the H-ras gene are predominantly single-base substitutions and are most frequently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for ras mutation with respect to abnormalities in copying by purified DNA polymerase alpha, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mp19, single-stranded DNA constructs were isolated, and the progression of synthesis by polymerase alpha was measured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the middle base of codon 12, the same alteration that converts H-ras into an activated oncogene. Resistance of codon 12 in double-stranded constructs to digestion with restriction enzymes and computer investigation of the ras sequence suggest that these termination sites are in a region of secondary structure. The frequency of sequence alterations within DNA chains that have been extended past codons 12 and 13 was found to be < 0.01. We consider a variety of mechanisms by which the potential secondary structure involving codons 12 and 13 may contribute to the pausing of DNA polymerase alpha and to the generation of clustered mutations at this site.

Codon usage data of bacteriophage T4 genes were compiled and synonymous codon preferences were investigated in comparison with tRNA availabilities in an infected cell. Since the genome of T4 is highly AT rich and its codon usage pattern is significantly different from that of its host Escherichia coli, certain codons of T4 genes need to be translated by appropriate host transfer RNAs present in minor amounts. To avoid this predicament, T4 phage seems to direct the synthesis of its own tRNA molecules and these phage tRNAs are suggested to supplement the host tRNA population with isoacceptors that are normally present in minor amounts. A positive correlation was found in that the frequency of E. coli optimal codons in T4 genes increases as the number of protein monomers per phage particle increases. A negative correlation was also found between the number of protein monomers per phage and the frequency of "T4 optimal codons", which are defined as those codons that are efficiently recognized by T4 tRNAs. From these observations it was proposed that tRNAs from the host are predominantly used for translation of highly expressed T4 genes while tRNAs from T4 tend to be used for translation of weakly expressed T4 genes. This distinctive tRNA-usage in T4 may be an optimization of translational efficiency, and an adjustment of T4-encoded tRNAs to the synonymous codon preferences, which are largely influenced by the high genomic AT-content, would have occurred during evolution.

The codon usage of 10 E. histolytica genes comprising 4455 codons was analysed. The codon usage revealed an extremely biased use of synonymous codons with a preference for NNU (44%) and NNA (41.4%) codons. Codons CGG (arg), AGG (arg) and CCG (pro) were absent in the E. histolytica genes examined. The codon usage of E. histolytica resembled that of Plasmodium falciparum.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.

The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied. The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined. Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E. coli elongation system. Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid. It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre. The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin. Cephalotaxine did not exert a significant influence on the polypeptide chain elongation.

The relative reactivity of the chemical carcinogen (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to the guanine bases of the first two coding exons of the human c-Ha-ras1 protooncogene is determined to test if (+/-)-anti-BPDE reactivity is correlated with mutations reported for human c-Ha-ras1 protooncogene activation. Plasmid DNA containing the sequence for the human c-Ha-ras1 gene is modified with (+/-)-anti-BPDE to provide approximately 1 covalent adduct per 250 bp. High-resolution mapping of the covalent adducts is achieved by laser-induced photolysis of 32P-labeled restriction fragments of the BPDE-modified plasmid DNA. The (+/-)-anti-BPDE binding profiles to exons 1 and 2 of the human c-Ha-ras1 protooncogene show enhanced reactivity to guanine-rich regions. The guanine bases of oncogene-activating codons 12 (GGC) and 13 (GGT) are 5 times more reactive than the least reactive guanine analyzed within this region of the gene. The guanine base of oncogene-activating codon 61 (CAG) exhibits intermediate reactivity relative to the guanines analyzed within this region of the gene. Although preferential chemical reactivity plays a role in the activation of the c-Ha-ras1 protooncogene, the in vivo activation of the c-Ha-ras1 protooncogene by (+/-)-anti-BPDE is a complex process, with other important factors involved in the chemically induced activation.

We examined mutational activation of ras genes in rat liver preneoplasias and tumours induced by diethylnitrosamine and N-methyl-N-nitrosourea (NMU). In accordance with previous reports on H- and K-ras genes, no mutations were detected in the investigated hepatic tumours and prestages suggesting that neither mutations at codons 12, 13 and 61 of H- and N-ras nor a mutation in the last intron of the H-ras gene are involved in initiation and progression of rat hepatocellular carcinomas. In the course of this investigation we found two N-ras genes (N-rasA, N-rasB). Surprisingly N-rasC, which is present in the germ line of Fischer rats, is missing in Wistar rats. This suggests different numbers of germline N-ras genes in members of one species. Two out of eight NMU-induced liver tumours exhibited additional N-ras-related sequences of unknown origin.