Protein-coding Nucleotide Sequences Research Articles

A Novel Method to Detect Proteins Evolving at Correlated Rates: Identifying New Functional Relationships between Coevolving Proteins

Interacting proteins evolve at correlated rates, possibly as the result of evolutionary pressures shared by functional groups and/or coevolution between interacting proteins. This evolutionary signature can be exploited to learn more about protein networks and to infer functional relationships between proteins on a genome-wide scale. Multiple methods have been introduced that detect correlated evolution using amino acid distances. One assumption made by these methods is that the neutral rate of nucleotide substitution is uniform over time; however, this is unlikely and such rate heterogeneity would adversely affect amino acid distance methods. We explored alternative methods that detect correlated rates using protein-coding nucleotide sequences in order to better estimate the rate of nonsynonymous substitution at each branch (d(N)) normalized by the underlying synonymous substitution rate (d(S)). Our novel likelihood method, which was robust to realistic simulation parameters, was tested on Drosophila nuclear pore proteins, which form a complex with well-documented physical interactions. The method revealed significantly correlated evolution between nuclear pore proteins, where members of a stable subcomplex showed stronger correlations compared with those proteins that interact transiently. Furthermore, our likelihood approach was better able to detect correlated evolution among closely related species than previous methods. Hence, these sequence-based methods are a complementary approach for detecting correlated evolution and could be applied genome-wide to provide candidate protein-protein interactions and functional group assignments using just coding sequences.

Estimates of the Effect of Natural Selection on Protein-Coding Content

Analysis of natural selection is key to understanding many core biological processes, including the emergence of competition, cooperation, and complexity, and has important applications in the targeted development of vaccines. Selection is hard to observe directly but can be inferred from molecular sequence variation. For protein-coding nucleotide sequences, the ratio of nonsynonymous to synonymous substitutions (ω) distinguishes neutrally evolving sequences (ω = 1) from those subjected to purifying (ω < 1) or positive Darwinian (ω > 1) selection. We show that current models used to estimate ω are substantially biased by naturally occurring sequence compositions. We present a novel model that weights substitutions by conditional nucleotide frequencies and which escapes these artifacts. Applying it to the genomes of pathogens causing malaria, leprosy, tuberculosis, and Lyme disease gave significant discrepancies in estimates with ∼10–30% of genes affected. Our work has substantial implications for how vaccine targets are chosen and for studying the molecular basis of adaptive evolution.

Phylogenetic Window Analysis for Detecting Chronological Changes in Natural Selection

Natural selection operating at the amino acid sequence level can be detected by comparing the rates of syn- onymous (rS) and nonsynonymous (rN) substitutions for the protein-coding nucleotide sequence, where relationships rN > rS and rN < rS conventionally indicate positive and negative selection, respectively. The direction and magnitude of natural selection operating on a protein may change during evolution because the environmental conditions may vary along with time. Here a phylogenetic window analysis method is proposed for examining the chronological change in natural selec- tion and for detecting natural selection that has operated temporarily in the phylogenetic tree. The phylogenetic window was defined as an interval between two time points in the phylogenetic tree, which was constructed under the assumption of a molecular clock. The total numbers of synonymous and nonsynonymous changes that have occurred for all the parts of branches overlapping with the window were compared to detect natural selection. When this method was applied to the analysis of the intra-host evolution for hypervariable region 1 of hepatitis C virus (HCV), which was known as the major target of humoral immunity, it was found that the pattern of chronological change in natural selection was heterogeneous among patients. The rN/rS value was sometimes elevated temporarily, where positive selection was detected, suggesting that the antigenic evolution was punctuated during chronic infection of HCV.

Characteristics of Human and Mouse Orthologous Protein-Coding Nucleotide Sequences with Large G+C Content Variations

Characteristics of human and mouse orthologous gene sequences which have large G+C content variations were investigated in this study. The orthologous gene pairs were classified into two groups according to the deviation between human and mouse G+C content at the third codon position (GC3) and were subsequently analyzed. In one group, mouse genes had higher GC3 than the corresponding human genes and in another group, human genes had higher GC3 than mouse. Furthermore, the orthologous pairs were separated based on the deviation between human or mouse GC3 and the G+C content at the third codon position of identical codons (IC3), to examine the effect of increased or decreased G+C content in human or mouse sequences. The nucleotide substitution patterns between human and mouse sequences in the two groups were remarkably distinct, and consistent with the state of G+C-rich or G+C-poor sequences. The effect of increase or decrease of G+C content in human or mouse sequences was not clear in the nucleotide substitution patterns. The chromosomal locations of human and mouse orthologous gene pairs were different between the two groups. The genes located on an identical syntenic segment showed the trend of having similar G+C content. Moreover, the same gene order of some genes on different chromosomes of both species demonstrated the gene rearrangements between human and mouse. Our study indicated that the chromosomal locations and rearrangements are associated with the GC3 variation between human and mouse sequences.

Negative Correlation of G+C Content at Silent Substitution Sites Between Orthologous Human and Mouse Protein-Coding Sequences

We conducted a genome-wide analysis of variations in guanine plus cytosine (G+C) content at the third codon position at silent substitution sites of orthologous human and mouse protein-coding nucleotide sequences. Alignments of 3776 human protein-coding DNA sequences with mouse orthologs having >50 synonymous codons were analyzed, and nucleotide substitutions were counted by comparing sequences in the alignments extracted from gap-free regions. The G+C content at silent sites in these pairs of genes showed a strong negative correlation (r = -0.93). Some gene pairs showed significant differences in G+C content at the third codon position at silent substitution sites. For example, human thymine-DNA glycosylase was A+T-rich at the silent substitution sites, while the orthologous mouse sequence was G+C-rich at the corresponding sites. In contrast, human matrix metalloproteinase 23B was G+C-rich at silent substitution sites, while the mouse ortholog was A+T-rich. We discuss possible implications of this significant negative correlation of G+C content at silent sites.

Complete nucleotide sequence of Japanese flounder (Paralichthys olivaceus) mitochondrial genome: structural properties and cue for resolving teleostean relationships.

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.

Structure of the α 1 subunit of horse Na,K-ATPase gene

Genomic DNA for Na,K-ATPase α 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the α 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an M r of 112 264. The putative amino acid sequence of horse α 1 is 96–97% homologous to those of other mammalian species.

Stage-specific selection of alternative transcriptional initiation sites from the 5C actin gene of Drosophila melanogaster.

The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.