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Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.

Background Fragile X syndrome (FXS) is a monogenic disorder and a common cause of intellectual disability (ID). Up to now, very few pathological variants other than the typical CGG-repeat expansion have been reported in the FMR1 gene. Methods A panel of 56 intellectual disability (ID) genes including the FMR1 gene was sequenced in a cohort of 300 patients with unexplained ID. To determine the effect of a new FMR1 variant, total RNA from peripheral blood cells was reverse transcribed, amplified by polymerase chain reaction and sequenced. Results We report a novel G to A point variant (c.801G > A) located at the last nucleotide of exon 8 in the FMR1 gene in one patient with ID. Direct sequencing of the RT-PCR products revealed that the transcript from the allele with G to A variant skips exon 8 entirely, resulting in a joining of exons 7 and 9. Skipping of exon 8 may result in an abnormal FMR1 protein (FMRP), which removes the highly conserved region that encoding the KH1 domain of FMRP. Conclusions This report describes for the first time that a synonymous variant in the FMR1 gene is associated with an error in mRNA processing, leading preferentially to the production of an aberrant transcript without exon 8. This splice variant was associated with an unspecific clinical presentation, suggesting the need for more detailed investigation of silent variants in ID patients with a large spectrum of phenotypes.

Background: The nuclear receptors Rev-erb alpha and Rev-erb beta are transcription factors that regulate the function of genes in glucose and lipid metabolism, and they also form a link between circadian rhythm and metabolism. We evaluated the variations in Rev-erb alpha and Rev-erb beta genes together with biochemical parameters as risk factors in type 2 diabetic (T2DM) patients. Methods: Molecular analyses of Rev-erb alpha and Rev-erb beta genes were performed on genomic DNA by using next-generation sequencing in 42 T2DM patients (21 obese and 21 non-obese) and 66 healthy controls. Results: We found 26 rare mutations in the study groups, including 13 missense mutations, 9 silent mutations, 3 5'UTR variations, and a 3'UTR variation, of which 9 were novel variations (5 missense and 3 silent and 1 5'UTR). Six common variations were also found in the Rev-erb genes; Rev-erb beta Chr3:24003765 A > G, Rev-erb beta rs924403442 (Chr3:24006717) G > T, Rev-erb alpha Chr17:38253751 T > C, Rev-erb alpha rs72836608 C > A, Rev-erb alpha rs2314339 C > T and Rev-erb alpha rs2102928 C > T. Of these, Rev-erb beta Chr3:24003765 A > G was a novel missense mutation (p.Q197R), while others were identified as intronic variants. T2DM patients with Rev-erb beta rs924403442 T allele had lower body surface area (BSA) than noncarriers (GG genotype) (p = 0.039). Rev-erb alpha rs72836608 A allele and Rev-erb alpha rs2314339 CC genotype were associated with decreased serum HDL-cholesterol levels in T2DM patients (p = 0.025 and p = 0.027, respectively). In our study, different effects of Rev-erbs polymorphisms were found according to gender and presence of obesity. Rev-erb alpha rs72836608 (C > A) and rs2314339 (C > T) and Rev-erb alpha rs2102928 (C > T) were associated with low HDL-C levels in male T2DM patients. In female patients, Rev-erb alpha rs2102928 (C > T) was associated with high microalbuminuria and Rev-erb beta rs9244403442 G > T was associated with low HDL and high BSA values. In addition, Rev-erb alpha Chr17: 38,253,751 (T > C), rs72836608 (C > A), and rs2314339 (C > T) and Rev-erb beta Chr3:24003765 (A > G) were associated with increased serum GGT levels in obese T2DM patients. In non-obese patients, Rev-erbs SNPs had no effect on serum GGT levels. Conclusion: Our findings indicate that variations in the Rev-erb alpha and Rev-erb beta genes can affect metabolic changes in T2DM and these effects may vary depending on gender and obesity. İstanbul Üniversitesi

Exonic mutations are usually classified as missense, synonymous or nonsense mutations, however, they can affect pre-mRNA splicing either by disrupting splice sites, by creating new ones or by changing splicing regulatory sequences. In this study, we examined 21 mutations of the PKD2 gene, encoding polycystin-2, previously reported as missense or synonymous for their possible effects on pre-mRNA splicing using bioinformatics tools. All these mutations except one have been identified in patients with autosomal dominant polycystic kidney disease, a common genetic disorder characterized by the development and progressive enlargement of cysts in the kidneys leading to end-stage renal disease. We selected 12 missense mutations and 1 synonymous variant for the minigene assay, and found that three, c.1532A>T (p.D511V), c.1716G>A (p.K572K) and c.2657A>G (p.D886G) caused alterations in pre-mRNA splicing. Mutation c.1532A>T resulted in skipping of exon 6 and incorporation of a defective exon lacking the 3' end, while c.1716G>A led to skipping of exon 7. Mutation c.2657A>G resulted in incorporation of an incomplete exon 14, which is in agreement with previous results obtained with the patient's lymphoblast RNA. Our findings should be taken into account with regard to the pathogenicity of these PKD2 exonic mutations. These results together with previous reports highlight the importance to evaluate the effects of exonic single nucleotide substitutions in autosomal dominant polycystic kidney disease.

Codon usage bias is an important genomic phenomenon, where highly expressed genes use optimal codons for smoother translation with high yield, facilitated by the cognate tRNAs. Here, we presented the tRNA co-adaptation index (co-AI) by correlating tRNA gene copy number and codon composition in Saccharomyces cerevisiae. We observed that this co-AI is positively correlated with protein abundance and translation rate. Considering nucleotide substitutions, co-AI influences synonymous substitutions more than gene expression and protein abundance, the most important determinants of evolutionary rate. Co-AI correlates positively with mRNA secondary structure stability and mRNA half-life, which may lead to protein accumulation under high co-AI. However, the highly expressed proteins encoded by high co-AI genes are assisted by molecular chaperones to attain their proper functional conformation and prevent accumulation.

Codon usage bias (CUB) is a universal feature of genomes, and in most species CUB of protein coding genes is positively correlated with expression level and degree of evolutionary conservation. There is mounting experimental evidence that CUB is due in part to selection for translational efficiency and/or accuracy, i.e., translational selection. However, there is a paucity of experimental data on whether and how CUB acts in trans – does the usage of preferred codons in a highly expressed gene affect the translation of other genes by freeing up more ribosomes, thereby increasing their availability to translate all mRNA transcripts in the cell? We investigated this question by creating two extreme versions of the highly expressed Escherichia coli β-lactamase (bla) gene, one comprised almost entirely of unpreferred codons, and a second comprised almost entirely of preferred codons. We monitored the fitness effects of these synonymous mutations over hundreds of generations in two selective environments that allowed us to disentangle translational effects acting in cis from those acting in trans. In a selective environment for maximizing translational efficiency in trans of a gene (tetA) encoding a tetracycline resistance protein, unpreferred synonymous mutations had a negative impact on long-term fitness, whereas preferred mutations had a positive impact on long-term fitness, providing strong experimental evidence for a pleiotropic effect of translational selection.

Genetic variations among HR-HPV types lead to altered biological functions with possible clinical significance in different geographical locations. To explore intratype genetic variations of HPV51 E6, E7, L1 and L2 genes originating from Southwest China, a total of 5204 cervical scraped cell samples were collected for DNA extraction and HPV typing. And then the E6, E7, L1 and L2 genes of HPV51 (n = 79) were sequenced and compared to the reference sequence (M62877). The ConSurf server was used for identification of conserved structural and functional amino acids of the E6 and E7 oncogenes, and the changes of the secondary structure were analyzed by PSIPred software. Phylogenetic trees were constructed by the maximum likelihood method implemented in IQ-TREE. The selection pressure acting on the E6, E7, L1 and L2 genes was estimated by Datamonkey web server. 13 nucleotide polymorphism sites were observed in E6-E7 gene and the most common mutation sites were C395T (S100L), C756T (S66L), C796T, A832G. 36 nucleotide polymorphism sites were identified in full length L1 gene and the non-synonymous mutations T6311G, A6312T (V264G), G6313A (G265S) A5674C (I52L), A6335C (N272T), A6586C (T354P) and synonymous mutations A5649T, C6147T, A6435G, G6570A, A6651G, T6774C, A6784C, A6882G, C6918A, and G6984A were the most common mutations. 53 nucleotide variation sites were identified in full-length L2 gene including four insertion sites (4418A, 4670G, 4693A, 4694C) and one deletion site (A4430). Besides, the non-synonymous mutations G4227A (V32I), A4407G (I92V), G4945A (D271N), C4985A (T284K), T5260G (L376V), A5335C (T401P) and the synonymous mutations A4166G, G4229A, G4283A, T4453C, C4566A, T4596C, C4695T, C4830T, G4839A, A5160C, and T5286G were the most common mutations. Specially, a triallelic mutation site (G4461C/A) in L2 was identified, with 26% G4461C (E109D) being non-synonymous mutation. Selective pressure analysis showed that only codon site 66 in E7 and 52 in L1 were the positively selected sites and codon sites 72, 107, 342, 412, 427 were negatively selected sites in L2 gene. Our investigation also suggests that A2 and A4 were the most frequent HPV51 lineage in Southwest China.

The distributions of secondary structural elements appear to differ between coding regions (CDS) of mRNAs compared to the untranslated regions (UTRs), presumably as a mechanism to fine-tune gene expression, including efficiency of translation. However, a systematic and comprehensive analysis of secondary structure avoidance because of potential bias in codon usage is difficult as some of the common secondary structures, such as, hairpins can be formed by numerous sequence combinations. Using G-quadruplex (GQ) as the model secondary structure we studied the impact of codon bias on GQs within the CDS. Because GQs can be predicted using specific consensus sequence motifs, they provide an excellent platform for investigation of the selectivity of such putative structures at the codon level. Using a bioinformatics approach, we calculated the frequencies of putative GQs within the CDS of a variety of species. Our results suggest that the most stable GQs appear to be significantly underrepresented within the CDS, through the use of specific synonymous codon combinations. Furthermore, we identified many peptide sequence motifs in which silent mutations can potentially alter translation via stable GQ formation. This work not only provides a comprehensive analysis on how stable secondary structures appear to be avoided within the CDS of mRNA, but also broadens the current understanding of synonymous codon usage as they relate to the structure-function relationship of RNA.

In this study, the systemic analyses of nucleotide, codon and amino acid usages for E2 gene of bovine viral diarrhea virus (BVDV) were carried out for estimating its genetic features. The nucleotide usage pattern at the first codon position was strongly influenced by the overall nucleotide composition, while the nucleotide usage patterns at the second and third codon positions seemed to have little link to the overall nucleotide composition. The result indicated that the mutation pressure from nucleotide composition constraint was not the single evolutionary force for genetic features of BVDV E2 gene. Just 18 out of 59 synonymous codons were similar with synonymous codon usage patterns for E2 gene between BVDV1 and BVDV2, while all synonymous codons which contain CpG dinucleotides were selected at the low level by E2 gene, suggesting that this gene suppressed the usages of codons containing CpG dinucleotides to regulate E2 gene replicate and transcript efficiently and avoid immune response from infected hosts. Amino acid usage patterns of E2 protein were generally different between BVDV1 and BVDV2. The patterns of synonymous codon and amino acid usages for E2 gene might be caused by the equilibrium of evolutionary forces from virus and host. Our work gave new investigations into the role of host origin in the formations of synonymous codon/amino acid usages and the evolutionary trend of BVDV E2 gene. The genetic characteristics that codon/amino acid usages of E2 gene adapted to the internal environment of individual animals might assist in understanding the changes of genetics and antigenicity for newly emerging BVDV.

NPC1 gene is an important gene closely related to the Niemann–Pick type C (NPC). Mutations in the NPC1 gene tend to cause Niemann–Pick type C, a lysosomal storage disorder. Previous studies have shown that NPC1 protein plays an important role in subcellular lipid transport, homeostasis, platelet function and formation, which are basic metabolic activities in the process of development. In this study, to explore the association between the NPC1 gene variation and body size traits in Qinchuan cattle, we detected four novel coding single nucleotide polymorphisms (cSNPs) in the bovine NPC1 gene, including one missense mutation (SNP1) and three synonymous mutations (SNP2, SNP3 and SNP4). Population genetic analyses of 518 individuals and association correlations between cSNPs and bovine body size traits were conducted in this research. A missense mutation at SNP1 locus was found to be significantly related to the heart girth, hip width and body weight (P

Genetic mutation in cytochrome c oxidase subunit III gene (MT-CO3) could influence the kinetics of cytochrome c oxidase (COX), which catalyzes oxygen transport capacity in oxidative phosphorylation. However, the potential relationship between MT-CO3 variants and high-altitude adaptation remains poorly understood in Tibetan chicken. Here, we sequenced MT-CO3 gene of 125 Tibetan chickens and 144 Chinese domestic chickens in areas at a low elevation (below 1,000 m). Eight single nucleotide polymorphisms (SNPs) were detected; and five of them (m.10081A>G, m.10115G>A, m.10270G>A, m.10336A>G and m.10447C>T) shared by Tibetan chicken and lowland chicken with the significant difference in their respective allele frequencies. Nine haplotypes (H1-H9) were finally defined. Among them, haplotype H4 was positively associated with high-altitude adaptation whereas haplotypes H6, H7 and H8 had negative association with high-altitude adaptation. The Median-joining profile suggested that haplotype H5 had the ancestral position to the other haplotypes but had no significant relationship with high-altitude adaptation. However, there was only m.10081A>G mutation differed from haplotype H4 and H5. Results also suggested that chickens with A allele at m.10081A>G, had over 2.6 times than those with G allele in the probability of the ability to adapt hypoxia. It suggests that the synonymous mutation m.10081A>G may be a prerequisite for shaping high-altitude adaptation-specific haplotypes.

5-HT (5-Hydroxy-tryptamine) or serotonin receptors are found both in central and peripheral nervous system as well as in non-neuronal tissues. In the animal and human nervous system, serotonin produces various functional effects through a variety of membrane bound receptors. In this study, we focus on 5-HT receptor family from different mammals and examined the factors that account for codon and nucleotide usage variation. A total of 110 homologous coding sequences from 11 different mammalian species were analyzed using relative synonymous codon usage (RSCU), correspondence analysis (COA) and hierarchical cluster analysis together with nucleotide base usage frequency of chemically similar amino acid codons. The mean effective number of codon (ENc) value of 37.06 for 5-HT(6) shows very high codon bias within the family and may be due to high selective translational efficiency. The COA and Spearman's rank correlation reveals that the nucleotide compositional mutation bias as the major factors influencing the codon usage in serotonin receptor genes. The hierarchical cluster analysis suggests that gene function is another dominant factor that affects the codon usage bias, while species is a minor factor. Nucleotide base usage was reported using Goldman, Engelman, Stietz (GES) scale reveals the presence of high uracil (>45%) content at functionally important hydrophobic regions. Our in silico approach will certainly help for further investigations on critical inference on evolution, structure, function and gene expression aspects of 5-HT receptors family which are potential antipsychotic drug targets.

The Chinese yakow is the offspring of yak (Bos grunniens) and Yellow cattle (Bos taurus). The melanocortin 1receptor gene (MC1R) plays a crucial role in determining coat colour of mammals. To investigate the relationship of polymorphism of the MC1R with coat colour in the Chinese yakow, the coding sequence (CDS) and the flanking region of MC1R were sequenced from 84 Chinese yakow samples and compared with the sequences of the MC1R from other bovid species. A fragment of 1134 base pair (bp) sequences including the full CDS (954bp) and parts of the 5'- and 3'-untranslated regions (162 and 18bp, respectively) of the Chineseyakow MC1R were obtained. A total of 13 single nucleotide polymorphisms (SNPs) including 4 SNPs (T-129C, A-127C, C-106T, G-1A) in the 5'-untranslated region and 9 SNPs (C201T, T206C, C340A, C375T, T663C, G714C, C870T, G871A and T890C) in the CDS were identified, revealing high genetic variability. Four novel SNPs including T206C, G714C, C870T and T890C, which have not been reported previously in bovid species, were retrieved. Within 9 coding SNPs, C201T, C375T, T663C and C870T were silent mutations, while T206C, C340A, G714C, G871A and T890C were mis-sense mutations, corresponding to amino acid changes p.L69P, p.Q114K, p.K238N, p.A291N and p.I297T, respectively. Amino acid sequences alignment showed a more than 96% similarity with other ruminates. However, three classical bovine MC1R loci the E(D), E(+) and e were not retrieved in the Chinese yakow, indicating other genes or factors could be involved in affecting coat colour in this species.

Protein translation has been elucidated to be dictated by evolutionary constraints, namely, variations in tRNA availabilities and/or variations in codon–anticodon binding that is manifested in biased codon usage. Taking advantage of publicly available mRNA expression and protein abundance data for Saccharomyces cerevisiae, we have performed a comprehensive analysis of the diverse factors guiding translation leading to desired protein levels irrespective of the corresponding high or low mRNA levels. It has been elucidated in this study that different combinations of most abundant/non abundant tRNA isoacceptors are selected for in S. cerevisiae that helps in achieving the optimum speed and accuracy in the protein translation process. This is also accompanied by the strategic location of codon pairs in coherence to mRNA secondary structure folding stability for the above mentioned combinations of tRNA isoacceptors. We thus find that codon pair contextual effects; in addition to tRNA abundance and mRNA folding stability during translation elongation process play plausible roles in maintaining translation accuracy and speed that can achieve desired protein levels.

We present a point mutation classification method that contrasts SNP databases and has the potential to illuminate the relative mutational load of genes caused by codon bias. We group point variation gleaned from public databases by their wild-type and mutant codons, e.g. codon mutation classes (CMCs, 576 possible such as ACG→ATG), whose frequencies in a database are assembled into a BLOSUM-style matrix describing the likelihood of observing all possible single base codon changes as tuned by the intertwined effects of mutation rate and selection. The rankings of the CMCs in any database are reshuffled according to the population stratification of the typical genotyping experiment producing that resource's data. Analysis of four independent databases reveals that a considerable fraction of mutation in functional genes can be described by a few CMCs regardless of gene identity or population stratification in the genotyping experiment. For example, the top 5% (29/576) of CMCs account for 27.4% of the observed variants in dbSNP while the bottom 5% account for only 0.02%. For non-synonymous disease-causing mutation, 40.8% are described by the top 5% of all possible non-silent CMCs (22/438). Overall, the most observed polymorphism is a G→A transition at CpG dinucleotides causing ACG, TCG, GCG, and CCG to frequently undergo silent mutation in any gene due to the putative lack of impact on the protein product. In order to assess how well CMC spectrums estimate the aggregate non-synonymous mutational trends of a single gene, a CMC matrix was applied to seven unrelated genes to compute the most likely point mutations. In excess of 87% of these mutation predictions are historically known to play an important role in a disease state according to published literature. CMC-based mutation prediction may aid design and execution of direct association genotyping studies.

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G>A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A>T (p.G109G) and c.11257C>A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level.

Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons. Nucleic Acids Res. 17, 5029-5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525-2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H. influenzae and M. tuberculosis are similar to those of E. coli, implying that though the composition of all three codon positions is governed by a selection-mutation balance, the mutational pressure has little influence in the choice of bases at the first two codon positions, even in organisms with highly biased base compositions.

The most frequent allele of the rabbit kappa-casein (kappa-Cas)-encoding gene (A allele) has previously been shown to possess two sequences similar to those found in the 5' end of long interspersed repeated elements (LINE). Part of an inverted rabbit LINE is present in the first intron and part of a direct rabbit LINE in the fourth intron. We describe herewith a less frequent allele (B allele) that lacks both 100bp in the first intron and 1550bp in the fourth intron. It was not possible to identify any allele exhibiting only one of the deletions in a population of 55 rabbits. The 100bp present in the first intron of the A allele, but absent from the B allele, are located at the 5' end of the inverse complementary LINE and include the poly (T) track of the LINE. The 1550bp present in the fourth intron of the A allele, but absent from the B allele, include the entire direct LINE sequence. Therefore, the B allele only possesses one partial LINE sequence that is located in the first intron and is truncated when compared to the copy found in the first intron of the A allele. The B allele might thus be more recent than the A allele. Differences between the sequences of transcripts corresponding to each allele are limited to two silent mutations and three modifications in the 3' UTR. In the mammary glands of lactating rabbits, which are homozygous for both alleles, kappa-Cas mRNA accumulate to similar levels and are translated into identical kappa-Cas that are secreted at similar concentrations into milk.

Although non-random patterns of synonymous codon usage are a prominent feature in the genomes of many organisms, the relatives roles of mutational biases and natural selection in maintaining codon bias remain a contentious issue. In some species, patterns of codon bias and empirical findings on the biology of translation suggest 'major codon preference', a balance among mutation pressure, genetic drift, and weak selection in favor of translationally superior codons. Population genetics theory makes testable predictions to distinguish such a model from a strictly mutational model of codon bias. Major codon preference predicts two fitness classes of synonymous DNA changes: 'preferred' mutations from non-major to major codons and 'unpreferred' changes in the opposite direction. An extension of current statistical methods is employed to reveal differences in the within and between species dynamics of preferred and unpreferred silent mutations in Drosophila simulans. In this lineage, codon bias appears to be maintained under roughly equal magnitudes of natural selection and genetic drift. In the sibling species, D. melanogaster, however, a reduction in N(e)s, the product of effective population size and selection coefficient, appears to have allowed a genome-wide reduction in codon bias.

To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.

Microtubules (MT) carry out several specialized morphogenetic functions in the multicellular green alga Volvox carteri (Vc), in addition to functions also executed in its closest unicellular relative, Chlamydomonas reinhardtii (Cr). To find out if these differences in morphogenetic complexity are reflected in tubulin (Tub) differences, we have compared the Vc αtub and βtub genes with their Cr counterparts. The Vc genome contains two αtub and two βtub genes. We report here the sequences of the α2tub and β2tub genes, and thus complete the set of four tub sequences. The two αtub and two βtub genes code for identical 451 (α) and 443 (β) amino acid (aa) polypeptides; they differ from the Cr homologs in two (α) and one (β) residues, respectively. Silent nucleotide (nt) exchanges between sibling genes are much more frequent in Vc than in Cr (12 vs. 2%), probably owing to a more stringent codon bias in the latter alga. Transcription of α2tub and β2tub starts with an A, 26 by (α2) or 25 by (β2) downstream from the TATA box. A 16-bp promoter element upstream and a G+C-rich sequence downstream from the TATA box are conserved in all tub of both species. Moreover, a 28-bp element of conserved sequence, and hence of possible functional significance, was found at similar locations in the 5′ untranslated region (UTR) of all four αtub. A conserved TGTAA downstream from the translation stop codon represents the algal poly(A)-addition signal (in both Vc and Cr). Northern analyses and reverse transcription (RT) followed by polymerase chain reaction have demonstrated that all four tub RNAs are present at all stages of the Vc life cycle

Nucleotide-binding oligomerization domain containing 2 (NOD2) plays a pivotal role in the host innate and adaptive immunity by recognizing the pathogenic agents. Therefore, its genetic polymorphisms and association with susceptibility to infectious diseases have been widely reported in human and farm animals. In the present study, we investigated the genetic polymorphisms in 3171 bp coding region of NOD2 gene and association with non-specific digestive disorder (NSDD) in rabbit. A total of four coding single-nucleotide polymorphisms (cSNPs) were detected. Among them, c.2961CT was further genotyped for case (n=176) and control (n=130) based on association analysis, which revealed that C allele carried the potential protective role for susceptibility to NSDD with the odds ratio (OR) values of 0.52 (95% confidence interval (CI) 0.37-0.73, P0.01). Under the dominant inheritance model, CC genotype was associated with decreased susceptibility to NSDD (OR=0.38, 95% CI 0.24-0.60, P0.01). Along with the aggravation of NSDD, we observed higher mRNA expression of NOD2 gene (P0.05). However, the mRNA expression pattern of CC genotype would be interacted by the different status of NSDD, which only showed the significantly increased level in severe NSDD group (P0.05). These results revealed by genetic association and gene expression analysis suggested that the NOD2 gene was associated with the susceptibility to NSDD in rabbit. However, the causative mutations linked to c.2961CT and corresponding functional depiction should be further explored by performing exhaustive genetic studies.

The alpha-lactalbumin (alpha LA)-encoding gene is a potential quantitative trait locus in dairy animals. In cattle, the production of alpha LA is tightly coupled to the onset of lactation and it serves as a regulatory subunit of the enzyme responsible for lactose synthesis. Lactose is the major osmole controlling water movement in the mammary gland. To better understand the control of bovine alpha LA expression, the 5'-flanking region of a Holstein alpha LA gene was cloned and sequenced. The sequenced clone contains 1952 bp of 5'-flanking region and 66-bp of the protein-coding region. Three single-bp polymorphisms were identified within this region. These polymorphisms occur at positions +15, +21 and +54 relative to the mRNA transcription start point (tsp). The +15 and +21 variations occur in the region encoding the 5'-untranslated region of the mRNA-coding sequence. The +54 polymorphism is a silent mutation in the SP-coding region of the gene. A polymerase chain reaction (PCR, Cetus)-based screening method has been employed to analyze the genotype of cattle at the +15 position. A total of 501 randomly selected cattle from seven breeds were screened for this allele. Of these animals, only the Holstein breed of cattle was found to contain the +15 variation and it occurs at a gene frequency of 32%. Sequence comparisons were conducted between the 5'-flanking regions of the bovine-milk-protein encoding genes, alpha LA, beta-casein and alpha S1-casein, which are coordinately expressed. Regions of similarity extending to 350 bp in length were observed between these sequences.

The introduction of random silent mutations into the 5−coding region of a human cDNA as the basis for successful expression in Escherichia coli is demonstrated in four steps. (1) Plasmid pUB200 containing the ϱ r ϱ l promoters of phage λ was found not to serve as an expression vector for a unchanged human glutathione reductase (hGR)-encoding cDNA. (2) When this cDNA was expressed in a two-cistron context using high-copy-number plasmids, recombinant protein was detected in low yield (0.03% of the total cell protein). (3) Silent mutations were introduced into the triplets coding for the N-terminal amino acids. When screening E. coli colonies transformed with expression plasmids containing cDNA mutants, we identified adapted clones that produced hGR in up to 70-fold higher yield than the clone containing the unchanged cDNA. Sequence analyses of adapted cDNA species revealed lower G + C contents in the modified regions, suggesting altered mRNA structures. (4) When the adapted cDNA sequences were recloned in the vector which had failed to express unchanged hGR cDNA in step 1, synthesis of recombinant protein was as high as in step 3. This means that the yield of expression for adapted cDNA was at least 1000-fold higher than for unchanged cDNA. In conclusion, random silent mutations introduced into the translation initiation region of cDNA might be a useful technique for designing sequence features which favour gene expression.

The canonical genetic code acts efficiently in minimizing the effects of mistranslations and point mutations. In the work presented we have also considered the effects of single nucleotide insertions and deletions on the optimality of the genetic code. Our results suggest that the canonical genetic code compensates for the ins/del mutations as well as mistranslations and point mutations. On the other hand, we highlighted the point that ins/del mutations have a lesser impact on the selected genes of Saccharomyces cerevisiae compared to randomly generated ones. We hypothesized that the codon usage preferences in S. cerevisiae genes are responsible for the higher efficiency of translation machinery in this organism. Our results support the conjecture that codon usage preferences render the genetic code more effective in minimizing the effects of ins/del mutations.

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.

We investigated the single nucleotide polymorphism (SNP) density across the human genome and in different genic categories using two SNP databases: Celera's CgsSNP, which includes SNPs identified by comparing genomic sequences, and Celera's RefSNP, which includes SNPs from a variety of sources and is biased toward disease-associated genes. Based on CgsSNP, the average numbers of SNPs per 10 kb was 8.33, 8.44, and 8.09 in the human genome, in intergenic regions, and in genic regions, respectively. In genic regions, the SNP density in intronic, exonic and adjoining untranslated regions was 8.21, 5.28, and 7.51 SNPs per 10 kb, respectively. The pattern of SNP density based on RefSNP was different from that based on CgsSNP, emphasizing its utility for genotype-phenotype association studies but not for most population genetic studies. The number of SNPs per chromosome was correlated with chromosome length, but the density of SNPs estimated by CgsSNP was not significantly correlated with the GC content of the chromosome. Based on CgsSNP, the ratio of nonsense to missense mutations (0.027), the ratio of missense to silent mutations (1.15), and the ratio of non-synonymous to synonymous mutations (1.18) was less than half of that expected in a human protein coding sequence under the neutral mutation theory, reflecting a role for natural selection, especially purifying selection.

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.

A simple measure is presented that quantifies how far the codon usage of a gene departs from equal usage of synonymous codons. This measure of synonymous codon usage bias, the 'effective number of codons used in a gene', Nc, can be easily calculated from codon usage data alone, and is independent of gene length and amino acid (aa) composition. Nc can take values from 20, in the case of extreme bias where one codon is exclusively used for each aa, to 61 when the use of alternative synonymous codons is equally likely. Nc thus provides an intuitively meaningful measure of the extent of codon preference in a gene. Codon usage patterns across genes can be investigated by the Nc-plot: a plot of Nc vs. G + C content at synonymous sites. Nc-plots are produced for Homo sapiens, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Dictyostelium discoideum, and Drosophila melanogaster. A FORTRAN77 program written to calculate Nc is available on request.

Part of the open reading frame of uidA, encoding beta-glucuronidase, was sequenced and two differences were found with the previously reported nucleotide sequence [Jefferson et al., Proc. Natl. Acad. Sci. USA 83 (1986) 8447-8451]. One is a silent mutation, the other results in the Glu279-->Gln substitution.

The complete genetic information contained in the influenza virus RNA segment 7 of the A/Bangkok/ 1 79 (H3N2) strain has been cloned by in vitro synthesis of the complementary dsDNA and its insertion into plasmid pBR322. The nucleotide sequence of the viral RNA segment has been determined from the cDNA insert. It is 1027 nucleotides long, and contains two open reading frames, as shown for other influenza virus strains. When compared with the previously published sequence for the A/Udorn/72 (H3N2) strain, 15 nucleotide exchanges are observed, most of them silent mutations, and only two causing amino acid changes in each of the M1 and M2 protein sequences.

The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma–carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p

We present methods for identifying a useful type of DNA site — one that can be mutated to create a new restriction site within a coding region without changing the amino acid sequence. These “latent sites” are abundant — silent mutations creating one of 44 different 6-bp or 8-bp recognition sites were found at relatively high density, roughly one latent site per 9 bp, in the eleven genes tested. Our analysis suggests that site-directed mutagenesis can be used to refashion coding sequences at will for flexible analysis.

The 7.7-kb EcoRI fragment of the resistance plasmid R1 contains the gene for the TraM protein. The sequence was identified by the presence of an open reading frame (ORF) which is preceded upstream by two promoter sequences. Both these promoters were found to be active, although the more distant one predominates, as was judged by the relative abundance of mRNA of the expected length. The TraM protein could be synthesized in an in vitro DNA-dependent protein synthesis system if the DNA of the corresponding region was supplied as template. Comparison of the traM genes of R1 and the F factor showed a high degree of similarity, although a number of mutations, especially near the 5' terminus, introduce specific amino acid (aa) changes. Two-thirds of the 3' sequences differ mainly in silent mutations; hence the aa sequence of the corresponding carboxy-terminal portion of the protein is highly conserved. The 5' and 3' untranslated regions of the mRNAs show little homology. One of the promoter regions, the ribosome-binding sequences, and the transcription termination sites are located at comparable positions but differ in details.

A method is described for the efficient saturation mutagenesis of a segment of DNA. A pool of mixed oligodeoxynucleotides (oligos) is generated in a single synthesis by deliberately contaminating each nucleotide reservoir with a low concentration of the other three monomers. The concentration at which a particular level of substitution (i.e. single, double, etc.) is favored can be calculated from simple probability. The pool of single-stranded, mixed oligos is then cloned directly into a double-stranded vector. This is achieved by designing the oligo such that it has ends which are complementary to those left by restriction enzymes generating 5' and 3' overhanging ends. The efficiency of the cloning procedure that we describe is greater than 90%. As a result, direct sequence analysis of transformant clones is justified. In the example described, 2 3 of M13 plaques containing an inserted oligo carried one or more mutations. Mutations generated by this method are randomly distributed throughout the inserted DNA and include all types of substitution. The ease and efficiency of the procedure eliminate any requirement for genetic selection of mutants and thus allow phenotypically silent mutations to be obtained.

The nucleotide sequence of the region including the viral replicase gene, the carboxy terminus of protein P18, and the 3'-extracistronic region of foot-and-mouth disease virus (FMDV) type C 1 -Santa Pau (C-S8) has been determined from previously cloned cDNA fragments [Villanueva etal., Gene 23 (1983) 185–194]. The comparison with the corresponding gene segments of FMDV of serotypes A or O shows base substitutions in 7.2–8.6% of residues in the replicase gene with no insertions or deletions. This is about fourfold lower variation than found for the region encoding capsid protein VP1 of the corresponding viruses. Intermediate variability (substitution at 16.1–23.6% positions) exists in the 3'-extracistronic region, including point mutations, insertions and deletions. The predicted amino acid sequence of the replicase gene indicates that 75.5–82.6% of mutations are silent and that 93.4% of amino acids are conserved in the four FMDV replicases. The frequency of certain types of silent mutations and of rare codon usage is significantly lower for the replicase gene than for the protein VP1 coding region.

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.