Molecular phylogenetic analysis of HIV-1 vif gene from Korean isolates

Phylogenetic studies of nef, pol, and env gene sequences of HIV-1 isolated from Koreans suggested the presence of a Korean clade in which Korean sequences are clustered to the exclusion of foreign sequences. We attempted to identify and characterize the Korean clade using all vif gene sequences isolated from Koreans registered in the NCBI GenBank database (n = 233). Most (77 %) of the Korean isolates belonged to the Korean clade as a large subcluster in subtype B, designated the Korean clade subtype B (KCB). KCB sequences were relatively homogenous compared to Korean subtype B sequences that did not belong to the KCB (non-Korean clade subtype B; NKCB). Comparison of amino acid frequencies of KCB and NKCB sequences revealed several positions where the amino acid frequencies were significantly different. These amino acid residues were critical in separating KCB from NKCB or from foreign sequences, since substitution of these amino acids in KCB with the NKCB amino acids relocated the KCB sequences to NKCB, and vice versa. Further analyses of KCB will help us to understand the origin and evolutionary history of KCB.     

Characterization and signature pattern analysis of Korean clade HIV-1 using nef gene sequences

Phylogenetic studies of the HIV-1 gene sequences isolated from Korean patients have suggested that most of Korean isolates belong to the subtype B strain. This study aims to characterize the Korean clade by molecular phylogenetic analysis using all of the Korean nef gene sequences registered in the NCBI GenBank (N=422), in addition to 41 reference strains and 94 foreign isolates. Through phylogenetic analyses, we verified that most of the Korean isolates belonged to the subtype B, where 78.8% are clustered exclusively of foreign isolates. This cluster has been named the Korean clade subtype B (KCB) in order to distinguish it from other subtype B clusters. Genetic distance analysis suggested that the KCB cluster was more homogeneous and clearly distinctive from the non-Korean clade subtype B (NKCB). Comparison of consensus amino acid sequences from KCB and NKCB revealed that characteristic KCB signature amino acid patterns composed of 11 amino acid residues, whose frequencies in the KCB were significantly higher than in the NKCB. The KCB signature amino acid residues were critical in identifying KCB from NKCB, since substitution of the NKCB sequences with KCB signature amino acids relocated them to the Koran clade, and vice versa.     

Timing and evolution of the most recent common ancestor of the Korean clade HIV subtype B based on <Emphasis Type="Italic">Nef</Emphasis> and <Emphasis Type="Italic">Vif</Emphasis> sequences

Molecular phylogenetic studies of the HIV-1 isolated from Koreans have suggested the presence of the so-called “Korean clade”, which can be defined as a cluster free of foreign isolates. The Korean clade accounts for more than 60% of Korean isolates and exerts characteristic amino acid sequences. Thus, it is merited to estimate when this Korean clade first emerged in order to understand the evolutionary pattern of the Korean clade. We analyzed and reconstructed the most recent common ancestor (MRCA) sequences from nef (n=229) and vif (n=179) Korean clade sequences. Linear regression analyses of sequence divergence estimates were plotted against sampling years to infer the year in which there was zero divergence from the MRCA sequences. MRCA sequences suggested the Korean clade was first emerged around 1984, before the first detection of HIV-1 in Korea in 1985. Further studies on synonymous and nonsynonymous substitution rates suggested positive selection event for the Korean clade, while other subtype B had undergone negative to neutral evolution.     

Tempo and mode of mitochondrial DNA evolution in vertebrates at the amino acid sequence level: Rapid evolution in warm-blooded vertebrates

Summary By using complete sequence data of mitochondrial DNAs, three Markov models (Day-hoff, Proportional, and Poisson models) for amino acid substitutions during evolution were applied in maximum likelihood analyses of mitochondrially encoded proteins to estimate a phylogenetic tree depicting human, cow, whale, and murids (mouse and rat), with chicken, frog, and carp as outgroups. A cow/whale clade was confirmed with a more than 99.8% confidence level by any of the three models, but the branching order among human, murids, and the cow/whale clade remained uncertain. It turned out that the Dayhoff model is by far the most appropriate model among the alternatives in approximating the amino acid substitutions of mitochondrially encoded proteins, which is consistent with a previous analysis of a more limited data set. It was shown that the substitution rate of mitochondrially encoded proteins has increased in the order of fishes, amphibians, birds, and mammals and that the rate in mammals is at least six times, probably an order of magnitude, higher than that in fishes. The higher evolutionary rate in birds and mammals than in amphibians and fishes was attributed to relaxation of selective constraints operating on proteins in warm-blooded vertebrates and to high mutation rate of bird and mammalian mitochondrial DNAs.Offprint requests to: M. Hasegawa     

Characterization and phylogenetic analysis of Varicella-zoster virus strains isolated from Korean patients

Varicella-zoster virus (VZV) is a causative agent of chickenpox in primary infection and shingles after its reactivation from latency. Complete or almost-complete genomic DNA sequences for various VZV strains have been reported. Recently, clinical VZV strains were isolated from Korean patients whose genome was sequenced using high-throughput sequencing technology. In this study, we analyzed single nucleotide polymorphism (SNP) of VZV strains to genetically characterize Korean clinical isolates. Phylogenetic analyses revealed that three Korean strains, YC01, YC02, and YC03, were linked to clade 2. Comprehensive SNP analysis identified 86 sites specific for the 5 VZV clades. VZV strains isolated from Korea did not form a phylogenetic cluster. Rather, YC02 and YC03 clustered strongly with Chinese strain 84-7 within clade 2, more specifically cluster 2a. Signature sequences for the cluster 2a were identified and found to play an important role in the separation of cluster 2a strains from other clade 2 strains, as shown in substitution studies. Further genetic analysis with additional strains isolated from Japan, China, and other Asian countries would provide a novel insight into the significance of two distinct subclades within clade 2.     

Phylogenetic analysis of envelope glycoprotein (E1) gene of rubella viruses prevalent in Japan in 2004

We performed a molecular epidemiological study on the envelope glycoprotein gene (E1 gene) obtained by PCR amplification from specimens of 17 rubella patients in certain areas (Gunma, Saitama, and Kagoshima prefectures, and Tokyo metropolitan area) in Japan in 2004. In these sequences of partially amplified DNAs (283 bases) within the E1 gene, no nucleotide substitution was observed. They were classified into genotype 1D of clade 1 in the constructed phylogenetic tree. One amino acid substitution was found between the amino acid sequence predicted from these DNAs and those of Japanese strains [To-336 vaccine strain (To-336 vac) and its wild progenitor (To-336 wt)]. The results suggest that the rubella viruses (RV) prevalent in certain areas of Japan in 2004 were highly homologous and were closely related with Japanese vaccine strain.     

Differences in pyrenoid morphology are correlated with differences in the rbcL genes of members of the Chloromonas lineage (volvocales,chlorophyceae)

Chloromonas is distinguished from Chlamydomonas primarily by the absence of pyrenoids, which are structures that are present in the chloroplasts of most algae and are composed primarily of the CO2-fixing enzyme Rubisco. In this study we compared sequences of the rbcL (Rubisco large subunit-encoding) genes of pyrenoid-less Chloromonas species with those of closely related pyrenoid-containing Chlamydomonas species in the "Chloromonas lineage" and with those of 45 other green algae. We found that the proteins encoded by the rbcL genes had a much higher level of amino acid substitution in members of the Chloromonas lineage than they did in other algae. This kind of elevated substitution rate was not observed, however, in the deduced proteins encoded by two other chloroplast genes that we analyzed: atpB and psaB. The rates of synonymous and nonsynonymous nucleotide substitutions in the rbcL genes indicate that the rapid evolution of these genes in members of the Chloromonas lineage is not due to relaxed selection (as it preasumably is in parasitic land plants). A phylogenetic tree based on rbcL nucleotide sequences nested two Chlamydomonas species as a "pyrenoid-regained" clade within a monophyletic Chloromonas "pyrenoid-lost" clade. Character-state optimization with this tree suggested that the loss and the regain of pyrenoids were accompanied by eight synapomorphic amino acid replacements in the Rubisco large subunit, four of which are positioned in the region involved in its dimerization. However, both the atpB and the psaB sequence data gave robust support for a rather different set of phylogenetic relationships in which neither the "pyrenoid-lost" nor the "pyrenoid-regained" clade was resolved. The appearance of such clades in the rbcL-based tree may be an artifact of convergent evolutionary changes that have occurred in a region of the large subunit that determines whether Rubisco molecules will aggregate to form a visible pyrenoid.     

Rapid fixation of a distinctive sequence motif in the 3' noncoding region of the clade of West Nile virus invading North America

Phylogenetic analysis of complete genomes of West Nile virus (WNV) by a variety of methods supported the hypothesis that North American isolates of WNV constitute a monophyletic group, together with an isolate from Israel and one from Hungary. We used ancestral sequence reconstruction in order to obtain evidence for evolutionary changes that might be correlated with increased virulence in this clade (designated the N.A. clade). There was one amino acid change (I-->T at residue 356 of the NS3 protein) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed. There were four changes in the upstream portion of the 3' noncoding region (the AT-enriched region) that occurred in the ancestor of the N.A. clade and remained conserved in all N.A. clade genomes analyzed, changes predicted to alter RNA secondary structure. The AT-enriched region showed a higher rate of substitution in the branch ancestral to the N.A. clade, relative to polymorphism, than did the remainder of the noncoding regions, synonymous sites in coding regions, or nonsynonymous sites in coding regions. The high rate of occurrence of fixed nucleotide substitutions in this region suggests that positive Darwinian selection may have acted on this portion of the 3'NCR and that these fixed changes, possibly in concert with the amino acid change in NS3, may underlie phenotypic effects associated with increased virulence in North American WNV.     

Partial replacement of dietary protein nitrogen with dispensable amino acids in diets of Nile tilapia,Oreochromis niloticus

Eighteen groups of 40 juvenile tilapia (Oreochromis niloticus) were fed five experimental diets in which crude protein (30% dry matter) was replaced at a level of 25% by dispensable amino acids (DAA) in the free form, given singly (alanine, glutamic acid or glycine) or as a mixture and at a level of 50% by DAA mixture. At the 25% substitution level, irrespective of the nature of the DAA, growth was slightly reduced (10%), while glycaemia, nitrogen retention and excretion were unaffected. A 50% substitution with the DAA mixture resulted in a marked reduction in growth (50%), a modified pattern of glycaemia and a lower nitrogen retention associated with a higher nitrogen excretion. Results suggest that DAA should not account for more than 60% of the total amino acid supply in tilapia diets.     

A survey of nuclear ribosomal internal transcribed spacer substitution rates across angiosperms: an approximate molecular clock with life history effects

Background  

A full understanding of the patterns and processes of biological diversification requires the dating of evolutionary events, yet the fossil record is inadequate for most lineages under study. Alternatively, a molecular clock approach, in which DNA or amino acid substitution rates are calibrated with fossils or geological/climatic events, can provide indirect estimates of clade ages and diversification rates. The utility of this approach depends on the rate constancy of molecular evolution at a genetic locus across time and across lineages. Although the nuclear ribosomal internal transcribed spacer region (nrITS) is increasingly being used to infer clade ages in plants, little is known about the sources or magnitude of variation in its substitution rate. Here, we systematically review the literature to assess substitution rate variation in nrITS among angiosperms, and we evaluate possible correlates of the variation.     

氨基酸的置换数与生物大分子进化改变量的测度

以蛋白质分子的氨基酸置换数或核酸分子的核苷酸置换数为衡量尺度 ,说明生物大分子随时间的改变 (即分子进化速率 )保持相对恒定     

Models of amino acid substitution and applications to mitochondrial protein evolution

Models of amino acid substitution were developed and compared using maximum likelihood. Two kinds of models are considered. "Empirical" models do not explicitly consider factors that shape protein evolution, but attempt to summarize the substitution pattern from large quantities of real data. "Mechanistic" models are formulated at the codon level and separate mutational biases at the nucleotide level from selective constraints at the amino acid level. They account for features of sequence evolution, such as transition-transversion bias and base or codon frequency biases, and make use of physicochemical distances between amino acids to specify nonsynonymous substitution rates. A general approach is presented that transforms a Markov model of codon substitution into a model of amino acid replacement. Protein sequences from the entire mitochondrial genomes of 20 mammalian species were analyzed using different models. The mechanistic models were found to fit the data better than empirical models derived from large databases. Both the mutational distance between amino acids (determined by the genetic code and mutational biases such as the transition-transversion bias) and the physicochemical distance are found to have strong effects on amino acid substitution rates. A significant proportion of amino acid substitutions appeared to have involved more than one codon position, indicating that nucleotide substitutions at neighboring sites may be correlated. Rates of amino acid substitution were found to be highly variable among sites.      

Amino acid composition and the evolutionary rates of protein-coding genes

Summary Based on the rates of amino acid substitution for 60 mammalian genes of 50 codons or more, it is shown that the rate of amino acid substitution of a protein is correlated with its amino acid composition. In particular, the content of glycine residues is negatively correlated with the rate of amino acid substitution, and this content alone explains about 38% of the total variation in amino acid substitution rates among different protein families. The propensity of a polypeptide to evolve fast or slowly may be predicted from an index or indices of protein mutability directly derivable from the amino acid composition. The propensity of an amino acid to remain conserved during evolutionary times depends not so much on its being features prominently in active sites, but on its stability index, defined as the mean chemical distance [R. Grantham (1974) Science 185862–864] between the amino acid and its mutational derivatives produced by single-nucleotide substitutions. Functional constraints related to active and binding sites of proteins play only a minor role in determining the overall rate of amino acid substitution. The importance of amino acid composition in determining rates of substitution is illustrated with examples involving cytochrome c, cytochrome b₅,ras-related genes, the calmodulin protein family, and fibrinopeptides.     

Natural variation, functional divergence, and local adaptation of nucleotide binding site sequences in Rhododendron (Ericaceae)

To further understand natural variation and local adaptation in the evolution of plant defense, we analyzed polymorphism data of nucleotide-binding site (NBS) sequences of Rhododendron at both the species and population levels. Multiple duplication events were found in NBS sequence evolution in Rhododendron genomes, which resulted in six clades: A–F. Our results of several NBS clade pair comparisons showed significant evolutionary rate changes based on differences in substitution rates between NBS-encoding protein clades (type I functional divergence). Pairwise comparisons of NBS clades further revealed that many amino acids displayed radical biochemical property changes causing a shift in amino acid preferences between NBS-encoding protein clades (type II functional divergence). Such divergent evolution of NBSs is likely a consequence of positive selection related to differentiation of recognition signals in response to different pathogens. Primers specific to clades B and C, which differed in the number of radical amino acid changes causing type II functional divergence and levels of nucleotide diversities, were further used to amplify population clades B and C NBS sequences of Rhododendron formosanum populations. Higher levels of net nucleotide divergences (measured by D _a) between R. formosanum populations were found based on NBS sequences of population clade B compared to population clade C, suggesting local adaptation of population clade B NBS sequences. Local adaptation can be further inferred for R. formosanum population clade B NBS sequences because of significant Φ _ST based on variation in nonsynonymous substitutions. Furthermore, local adaptation was also suggested by no significant correlation of population pairwise F _ST between population clades B and C in R. formosanum.     

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8⁺ T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8⁺ T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8⁺ T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8⁺ T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.     

Inferring natural selection operating on conservative and radical substitution at single amino acid sites

Natural selection operating on amino acid substitution at single amino acid sites can be detected by comparing the rates of synonymous (r(S)) and nonsynonymous (r(N)) nucleotide substitution at single codon sites. Amino acid substitutions can be classified as conservative or radical according to whether they retain the properties of the substituted amino acid. Here methods for comparing the rates of conservative (r(C)) and radical (r(R)) nonsynonymous substitution with r(S) at single codon sites were developed to detect natural selection operating on these substitutions at single amino acid sites. A method for comparing r(C) and r(R) at single codon sites was also developed to detect biases toward these substitutions at single amino acid sites. Charge was used as the property of the amino acids. In a computer simulation, false-positive rates of these methods were always < 5%, unless termination sites were included in the computation of the numbers of sites and estimates of transition/transversion rate ratio were highly biased. The frequency of detection of natural selection operating on conservative substitution was almost independent of the presence of natural selection operating on radical substitution, and vice versa. Natural selection operating specifically on conservative and radical substitution was detected more efficiently by comparing r(S) with r(C) and r(S) with r(R) than by comparing r(S) with r(N). These methods also appeared to be robust against the occurrence of recombination during evolution. In an analysis of class I human leukocyte antigen, negative selection operating on conservative substitution, but not positive selection operating on radical substitution, was observed at some of the codon sites with r(R) > r(C), suggesting that r(R) > r(C) may not necessarily be an indicator of positive selection operating on radical substitution.     

Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus

The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).     

Partitioning the variation in mammalian substitution rates

We have used analysis of variance to partition the variation in synonymous and amino acid substitution rates between three effects (gene, lineage, and a gene-by-lineage interaction) in mammalian nuclear and mitochondrial genes. We find that gene effects are stronger for amino acid substitution rates than for synonymous substitution rates and that lineage effects are stronger for synonymous substitution rates than for amino acid substitution rates. Gene-by-lineage interactions, equivalent to overdispersion corrected for lineage effects, are found in amino acid substitutions but not in synonymous substitutions. The variance in the ratio of amino acid and synonymous substitution rates is dominated by gene effects, but there is also a significant gene-by-lineage interaction.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.