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The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.
Sequence analysis of a human immunodeficiency virus type 1 env gene PCR amplified from a Brazilian woman's peripheral blood mononuclear cell DNA (sample RJIO1) showed that it was likely to have been derived from a double recombination event between human immunodeficiency virus type 1 subtypes B and F. The major portion of the gp120 coding sequence belonged to the B lineage, but a segment of the C2 to V3 region (approximately 135 nucleotides) clearly associated with sequences of the F lineage. The subtype F-like segment had 15 noncontiguous signature nucleotides in common with Brazilian subtype F sequences that were not found, or were rare, in subtype B sequences. In contrast, this same segment had only 3 signature nucleotides shared with subtype B sequences and not present in the Brazilian subtype F sequences. Phylogenetic analysis, amino acid signature pattern analysis, and the pattern of synonymous mutations all supported the hypothesis of a recombinational origin of the RJIO1 sequence. Related recombinant genes were also detected in peripheral blood mononuclear cell DNA obtained from the woman's recent sexual partner, indicating that the recombination event probably occurred at some previous time in the chain of virus transmission. Divergent viral sequences in the V3 region were found in the male sexual partner, while a relatively homogeneous viral population was detected in the woman, consistent with her recent infection.
BACKGROUND: NPC1L1 encodes a putative intestinal sterol transporter which is the likely target for ezetimibe, a new type of lipid-lowering medication. We previously reported rare non-synonymous mutations in NPC1L1 in an individual who had no plasma lipoprotein response to ezetimibe. We next hypothesized that common variants in NPC1L1 would underlie less extreme inter-individual variations in the plasma LDL cholesterol response to ezetimibe. RESULTS: In 101 dyslipidemic subjects, we found that NPC1L1 haplotype was significantly associated with inter-individual variation in the response of plasma LDL cholesterol to treatment with ezetimibe for 12 weeks. Specifically, about one subject in eight lacked the common NPC1L1 haplotype 1735C-25342A-27677T and these subjects had a significantly greater reduction in plasma LDL cholesterol with ezetimibe than subjects with at least one copy of this haplotype (-35.9+4.0 versus -23.6+1.6 percent reduction, P = 0.0054). This was paralleled by a similar non-significant trend of between-haplotype difference in reduction of total cholesterol. CONCLUSION: These preliminary pharmacogenetic results suggest that NPC1L1 variation is associated with inter-individual variation in response to ezetimibe treatment.
The NAD(P)H:quinone acceptor oxidoreductase (NQO) gene family belongs to the flavoprotein clan and, in the human genome, consists of two genes (NQO1 and NQO2). These two genes encode cytosolic flavoenzymes that catalyse the beneficial two-electron reduction of quinones to hydroquinones. This reaction prevents the unwanted one-electron reduction of quinones by other quinone reductases; one-electron reduction results in the formation of reactive oxygen species, generated by redox cycling of semiquinones in the presence of molecular oxygen. Both the mammalian NQO1 and NQO2 genes are upregulated as a part of the oxidative stress response and are inexplicably overexpressed in particular types of tumours. A non-synonymous mutation in the NQO1 gene, leading to absence of enzyme activity, has been associated with an increased risk of myeloid leukaemia and other types of blood dyscrasia in workers exposed to benzene. NQO2 has a melatonin-binding site, which may explain the anti-oxidant role of melatonin. An ancient NQO3 subfamily exists in eubacteria and the authors suggest that there should be additional divisions of the NQO family to include the NQO4 subfamily in fungi and NQO5 subfamily in archaebacteria. Interestingly, no NQO genes could be identified in the worm, fly, sea squirt or plants; because these taxa carry quinone reductases capable of one- and two-electron reductions, there has been either convergent evolution or redundancy to account for the appearance of these enzyme functions whenever they have been needed during evolution.
Balantioides coli is a zoonotic ciliated protozoan that infects humans and other mammals. Conventional and ITS-based genotyping approaches have limitations that hinder precise molecular epidemiological investigations. The objective of this study was to develop a new β-tubulin gene-based approach to enhance the detection and genotyping of B. coli. We performed single-cell isolation and whole-genome sequencing on two B. coli isolates from pigs and two from guinea pigs. We then used the β-tubulin gene sequences to design PCR primers for the new genotyping assay. We validated the assay using 56 ITS-confirmed B. coli-positive fecal DNA samples from pigs, cattle, sheep, and guinea pigs. Phylogenetic analyses were conducted using both β-tubulin and ITS sequences. The β-tubulin-based nested PCR assay exhibited 100% detection efficiency and greater specificity than ITS-based methods. Phylogenetic analysis of the β-tubulin gene sequences classified B. coli into three genotypes (I-III). Genotype III appears to be specific to guinea pigs. Genotypes I and II were found across multiple hosts, indicating potential cross-species transmission. Of the five full-length B. coli β-tubulin sequences obtained in this study, 264 polymorphic sites (19.8%) were identified, including both synonymous and non-synonymous mutations. Frequent recombination events within the β-tubulin locus were detected, indicating substantial genetic diversity. Therefore, the β-tubulin gene is a robust marker for genotyping and epidemiological studies of B. coli. The novel nested PCR assay overcomes the limitations of ITS-based methods and has produced data revealing previously unrecognized genetic diversity and host specificity patterns of B. coli.
We have investigated patterns of within-species polymorphism and between-species divergence for synonymous and nonsynonymous variants at a set of autosomal and X-linked loci of Drosophila miranda. D. pseudoobscura and D. affinis were used for the between-species comparisons. The results suggest the action of purifying selection on nonsynonymous, polymorphic variants. Among synonymous polymorphisms, there is a significant excess of synonymous mutations from preferred to unpreferred codons and of GC to AT mutations. There was no excess of GC to AT mutations among polymorphisms at noncoding sites. This suggests that selection is acting to maintain the use of preferred codons. Indirect evidence suggests that biased gene conversion in favor of GC base pairs may also be operating. The joint intensity of selection and biased gene conversion, in terms of the product of effective population size and the sum of the selection and conversion coefficients, was estimated to be approximately 0.65.
An analysis of sequence variation of 250 bp of the mitochondrial cytochrome b gene of 1278 Atlantic cod Gadus morhua ranging from Newfoundland to the Baltic shows four high-frequency (>8%) haplotypes and a number of rare and singleton haplotypes. Variation is primarily synonymous mutations. Natural selection acting directly on these variants is either absent or very weak. Common haplotypes show regular trans-Atlantic clines in frequencies and each of them reaches its highest frequency in a particular country. A shallow multifurcating constellation gene genealogy implies young age and recent turnover of polymorphism. Haplotypes characterizing populations at opposite ends of the geographic distribution in Newfoundland and the Baltic are mutationally closest together. The haplotypes are young and have risen rapidly in frequency. Observed differentiation among countries is due primarily to clinal variation. Hypotheses of historical isolation and polymorphisms balanced by local selection and gene flow are unlikely. Instead the results are explained by demic selection of mitochondria carried by highly fit females winning reproductive sweepstakes. By inference the Atlantic cod, a very high-fecundity vertebrate, is characterized by a high variance of offspring number and strong natural selection that leads to very low effective to actual population sizes.
In winter 2003-04, large numbers of budgerigars (Mellopsitacus undulatus) and cockatiels (Nymphicus hollandicus) fell ill and died in a large parrot-breeding aviary in Slovakia. In budgerigars, the disease outbreak occurred at the age of 2-3 weeks; cockatiels died within their first 7 days of life. In budgerigars, symptoms of the disease included delayed growth, tremor, darkish discoloration of skin, quill bleeding, and feathering defects. cockatiels often died without any symptoms and with a full crop; feathering defects occurred sporadically. Electron microscopy with negative staining of aqueous lysates of the affected skin and of bleeding quills showed isolated or clustered polyomavirus particles 45-50 nm in size. Long filamentous forms of the virus were also found in virion clusters of skin lysates from the budgerigars. In ultrathin sections through the pathologically altered skin tissue of budgerigars, virus particles were present in both nuclei and cytoplasm of epidermal cells, often in crystalline form. In infected cells, enlarged nuclei showed an extensive chromatin margination. On the DNA level, presence of a polyomavirus infection was conclusively proved by the polymerase chain reaction using avian polyomavirus (APV)-specific primers. A sequence analysis of the gene encoding viral protein (VP)1 and of the combined region for VP2 and VP3 proteins revealed a previously undescribed synonymous mutation in this isolate. This report extended the knowledge of the area of APV occurrence and of the spectrum of hosts in the context of genomic and morphologic variability of APV isolates.
Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -coefficient of variance below 10%- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with approximately 3-fold higher kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and one beneficial mutation S510G (TCG/GGG) that can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.
A bioinformatics analysis was conducted on the four members of the uterine serpin (US) family of serpins. Evolutionary analysis of the protein sequences and 86 homologous serpins by maximum parsimony and distance methods indicated that the uterine serpins proteins form a clade distinct from other serpins. Ancestral sequences were reconstructed throughout the evolutionary tree by parsimony. These suggested that some branches suffered a high ratio of nonsynonymous to synonymous mutations, suggesting episodes of adaptive evolution within the serpin family. Analysis of the sequences by neutral evolutionary distance methods suggested that the uterine serpins diverged from other serpins prior to the divergence of the mammals from other vertebrates. The porcine uterine serpins are paralogs that diverged from a single common ancestor within the Sus genus after pigs separated from other artiodactyls. The uterine serpins contain several protein kinase C and tyrosine kinase phosphorylation sites. These sites may be important for the lymphocyte-inhibitory activity of OvUS if, like other basic proteins, OvUS can cross the cell membrane of an activated lymphocyte. Internalized OvUS could serve as an alternative target to protein kinases important for the mitogenic response to antigens.
The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.
OBJECTIVE: To determine whether the FHIT gene at 3p14.2 is abnormal in Chinese lung cancers. METHODS: Matched normal and cancerous tissues from 21 cases of primary pulmonary squamous cell carcinoma (SQC) and 10 cases of adenocarcinoma (ADC) were obtained immediately after surgery. Total RNA was extracted and the FHIT gene was detected by RT-PCR and DNA sequencing technology. RESULTS: Normal-sized FHIT transcript was detected in all 31 cases of normal matched tissues. Aberrant transcripts were observed in 15 (48%) of 31 cases of cancerous tissues, 57% (12/21) in SQC and 33% (3/10) in ADC, respectively. All the abnormal transcripts observed lacked exon 5, the first coding exon. The sequence analyses of the aberrant cDNAs revealed deletions of various regions between exon 4 and 10. A synonymous mutation in E8, codon98CAT(H)-->CAC(H), was found in a SQC. CONCLUSION: The high deletion rate of the FHIT gene in Chinese lung cancer supports the hypothesis that the FHIT gene alteration is involved in tumorigenic development of human neoplasm.
OBJECTIVE: To study the polymorphism of human immunodeficiency virus (HIV)-1 coreceptor CXCR4 in Chinese Han ethnic group for AIDS prevention and treatment. METHODS: Totally 48 individuals were enrolled into the study. CXCR4 (cDNA No-AF147204) was cloned by PCR amplification using 2 pairs of primers, then sequenced using sequencing primers. The results of the same sequencing primers were analyzed by DNAstar software to find and identify single nucleotide polymorphism (SNP) sites. RESULTS: Totally 7 SNPs were found in the coding region of CXCR4, among them 3 were synonymous mutation (C-->T at loci 129, 426 and 968), 3 were missense mutation (C-->T at locus 38, A-->T at locus 90, and A-->C at locus 712) and 1 was stop mutation (C-->T at 106, which converted the codon for glutamic acid into stop codon). CONCLUSIONS: The polymorphism of CXCR4 coding region in Chinese Han is probably different from that of the other ethnic groups. Six of the 7 SNPs were discovered for the first time. Their influences on AIDS progression are worthy of studying.
To investigate the origin and genetic diversity of domestic pigs, the porcine TYR exon 1 in 36 individuals from 12 Chinese indigenous breeds, three European breeds, eight Chinese wild boars and two Vietnamese wild boars was sequenced. Sequence analysis revealed six synonymous mutations, and all the sequences could be sorted into 4 haplotypes. Combining with the published sequences, we constructed a reduced median network (RM network), in which TYR*2 was a haplotype dominated by European domestic pigs and wild boars, plus only three chromosomes from Asian pigs. Most Asian domestic pigs and wild boars shared haplotype TYR*1, demonstrating that TYR*1 was an Asian specific haplotype. Meanwhile, some European domestic pigs and wild boars carried the haplotype TYR*1. TYR*3 and TYR*4 were two haplotypes with low frequencies, containing mainly Chinese indigenous pigs and Asian wild boars, plus some European domestic pigs. Independent domestication of pigs from Asia and Europe was supported by the pattern of RM network. The European commercial breeds had been suffered from introgression from Chinese pigs, and a few Chinese indigenous breeds and Japanese wild boars were also suffered from introgression from European pigs as well.
The GC (G + C, or G or C)-contents of codon silent positions in all two-codon sets and three codons AUY/A (IIe), and in most of the family boxes of Micrococcus luteus (genomic GC-content: 74%) are 95% to 100% in both the highly and weakly expressed genes. In some family boxes, there is a decrease in NNC codons and an increase in NNG codons from the highly expressed to weakly expressed genes without apparent involvement of NNU and NNA codons. From these observations, we conclude that the selective use of synonymous codons in M. luteus may be largely determined by GC-biased mutation pressure and that in the highly expressed genes tRNAs would act as a weak selection pressure in some family boxes. Available data suggest that the effect of selection pressure by tRNAs on the synonymous codon choice becomes more apparent in the highly expressed genes in eubacteria with intermediate GC-contents such as Escherichia coli and Bacillus subtilis, and that the U/C ratio of the codon third positions in NNU/C-type two-codon sets in the weakly expressed genes would represent the approximate magnitude of directional mutation pressure throughout eubacteria.
In species where actin genes exist as single copies, analysis of their synonymous codon usage and of the substitutions occurring between the genes of closely related species shows that there is a positive selection for codons that do not have highly mutable CpG dinucleotides in codon positions 2 and 3 when the GC content of these genes is less than 57%.
The genetic diversity of Cymbidium mosaic virus (CymMV, family Flexiviridae) was assessed by analysing the nucleotide sequences of coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes. Thirty CymMV sequences from vanilla isolates, obtained in this work by direct sequencing of RT-PCR products, were compared to the sequences from ornamental orchid isolates available in GenBank. The CymMV population exhibited overall low genetic diversity (pi=0.054 and pi=0.053 for CP and RdRp genes, respectively). Phylogenetic analyses of the 85 CP and 37 RdRp sequences revealed the segregation of the isolates into two congruent monophyletic clusters; however these two subgroups did not cluster in amino sequence analysis because most of the nt mutations were synonymous. Nevertheless, the subgrouping was confirmed by highly significant Kst tests for the CP and RdRp genes. Analysis of population genetic parameters and distribution of synonymous and nonsynonymous mutations revealed that both genes were under negative selection with no recombination events. These results suggested that the CymMV isolates found in cultivated orchids worldwide have a dual origin and are expanding as if following bottlenecks.
Statistical analysis of the distribution of 156 kinds of human hemoglobin beta (Hbbeta) chain variants suggests that mutations are essentially random in their location. Thus differential fitness, not differential mutability, is the principal source of nonrandom distribution of interspecies differences in Hbbeta amino acid sequence. Similar analyses of both the location and the kind of interspecies differences detected among primates support this viewpoint and lead us to estimate that at least 95% of all amino acid subsitutions,i.e., nonsynonymous mutations, in Hbbeta are functionally unacceptable in homozygous state. Through the combined use of this estimate and the number of nonsynonymous and synonymous substitutions per nucleotide site inferred from comparisons of entire human and rabbit HbbetamRNA nucleotide sequences, we calculate (a) approximately 70% of synonymous Hbbeta mutations are adaptively undersirable and (b) the mutation rate underlying all changes is lesser than or equal to 10(-8) nucleotide substitutions per nucleotide site per year. Apart from such calculations, analyses of nucleotide patterns in HbbetamRNA as well as in rat preproinsulin mRNA reinforce the notion that a large portion of synonymous mutations are functionally unacceptable and rendered so by selective constraint, at a pretranslational level, of the abundance of particular nucleotide doublets such as CpG.