Nucleotide triplet based molecular phylogeny of class I and class II aminoacyl t-RNA synthetase in three domain of life process: bacteria, archaea, and eukarya.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. These important enzymes have been the subject of intense scientific inquiry for nearly half a century, but their complete evolutionary history has yet to emerge. Amino acids sequence based phylogeny has some limitation due to very low sequence similarity amongst the different tRNA synthetases and structure based phylogeny has also its limitation. In our study, tRNA nucleotide sequences of E. coli K12 (Bacteria), Saccharomyces cerevisiae (Eukarya), Thermococcus kodakaraensis KOD1, and Archaeoglobus fulgidus DSM 4304 (Archaea) were used for phylogenetic analysis. Our results complement the observation with the earlier studies based on multiple sequence alignment and structural alignment. We observed that relationship between archaeal tRNA synthetases are different that of bacteria and eucarya. Violation of Class rule of LysRS is observed here also. The uniqueness of this method is that it does not employ sequence alignment of complete nucleotide sequence of the corresponding gene.

Gene expression level shapes the amino acid usages in Prochlorococcus marinus MED4.

Prochlorococcus species are the first example of free-living bacteria with reduced genome. Codon and amino acid usages bias of Prochlorococcus marinus MED4 was investigated using all protein coding genes having length greater than or equal to 100 amino acids. Correspondence analysis on relative synonymous codon usage (RSCU) values shows that there is no such influence of translational selection in shaping the codon usage variation among the genes in this organism. However, amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analysis of the homologous genes of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 shows that amino acids conservation in highly expressed genes is significantly higher than lowly expressed genes. Based on our results we concluded that conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism.

Factors influencing synonymous codon and amino acid usage biases in Mimivirus.

Synonymous codon and amino acid usage biases have been investigated in 903 Mimivirus protein-coding genes in order to understand the architecture and evolution of Mimivirus genome. As expected for an AT-rich genome, third codon positions of the synonymous codons of Mimivirus carry mostly A or T bases. It was found that codon usage bias in Mimivirus genes is dictated both by mutational pressure and translational selection. Evidences show that four factors such as mean molecular weight (MMW), hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in Mimivirus proteins. Based on our observation, we suggest that genes involved in translation, DNA repair, protein folding, etc., have been laterally transferred to Mimivirus a long ago from living organism and with time these genes acquire the codon usage pattern of other Mimivirus genes under selection pressure.

Studies on codon usage in Thermoplasma acidophilum and its possible implications on the occurrences of lateral gene transfer.

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

Factors influencing the synonymous codon and amino acid usage bias in AT-rich Pseudomonas aeruginosa phage PhiKZ.

To reveal how the AT-rich genome of bacteriophage PhiKZ has been shaped in order to carry out its growth in the GC-rich host Pseudomonas aeruginosa, synonymous codon and amino acid usage bias of PhiKZ was investigated and the data were compared with that of P. aeruginosa. It was found that synonymous codon and amino acid usage of PhiKZ was distinct from that of P. aeruginosa. In contrast to P. aeruginosa, the third codon position of the synonymous codons of PhiKZ carries mostly A or T base; codon usage bias in PhiKZ is dictated mainly by mutational bias and, to a lesser extent, by translational selection. A cluster analysis of the relative synonymous codon usage values of 16 myoviruses including PhiKZ shows that PhiKZ is evolutionary much closer to Escherichia coli phage T4. Further analysis reveals that the three factors of mean molecular weight, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in PhiKZ proteins, whereas amino acid usage of P. aeruginosa proteins is mainly governed by grand average of hydropathicity, aromaticity and cysteine content. Based on these observations, we suggest that codons of the phage-like PhiKZ have evolved to preferentially incorporate the smaller amino acid residues into their proteins during translation, thereby economizing the cost of its development in GC-rich P. aeruginosa.

Synonymous codon usage bias in 16 Staphylococcus aureus phages: implication in phage therapy.

To reveal the factors influencing architecture of protein-coding genes in staphylococcal phages, relative synonymous codon usage variation has been investigated in 920 protein-coding genes of 16 staphylococcal phages. As expected for AT rich genomes, there are predominantly A and T ending codons in all 16 phages. Both Nc plot and correspondence analysis on relative synonymous codon usage indicates that mutation bias influences codon usage variation in the 16 phages. Correspondence analysis also suggests that translational selection and gene length also influence the codon usage variation in the phages to some extent and codon usage in staphylococcal phages is phage-specific but not S. aureus-specific. Further analysis indicates that among 16 staphylococcal phages, 44AHJD, P68 and K may be extremely virulent in nature as most of their genes have high translation efficiency. If this is true, then above three phages may be useful for curing staphylococcal infections.

Role of mutational bias and natural selection on genome-wide nucleotide bias in prokaryotic organisms.

Correlations between genomic GC contents and amino acid frequencies were studied in the homologous sequences of 12 eubacterial genomes. Results show that amino acids encoded by GC-rich codons increases significantly with genomic GC contents, whereas opposite trend was observed in case of amino acids encoded by GC-poor codons. Further studies show all the amino acids do not change in the predicted direction according to their genomic GC pressure, suggesting that protein evolution is not entirely dictated by their nucleotide frequencies. Amino acid substitution matrix calculated among hydrophobic, amphipathic and hydrophilic amino acid groups' shows that amphipathic and hydrophilic amino acids are more frequently substituted by hydrophobic amino acids than from hydrophobic to hydrophilic or amphipathic amino acids. This indicates that nucleotide bias induces a directional changes in proteome composition in such a way that underwent strong changes in hydropathy values. In fact, significant increases in hydrophobicity values have also been observed with the increase of genomic GC contents. Correlations between GC contents and amino acid compositions in three different predicted protein secondary structures show that hydropathy values increases significantly with GC contents in aperiodic and helix structures whereas strand structure remains insensitive with the genomic GC levels. The relative importance of mutation and selection on the evolution of proteins have been discussed on the basis of these results.

Comparative analysis of the base composition and codon usages in fourteen mycobacteriophage genomes.

To study the possible codon usage and base composition variation in the bacteriophages, fourteen mycobacteriophages were used as a model system here and both the parameters in all these phages and their plating bacteria, M. smegmatis had been determined and compared. As all the organisms are GC-rich, the GC contents at third codon positions were found in fact higher than the second codon positions as well as the first + second codon positions in all the organisms indicating that directional mutational pressure is strongly operative at the synonymous third codon positions. Nc plot indicates that codon usage variation in all these organisms are governed by the forces other than compositional constraints. Correspondence analysis suggests that: (i) there are codon usage variation among the genes and genomes of the fourteen mycobacteriophages and M. smegmatis, i.e., codon usage patterns in the mycobacteriophages is phage-specific but not the M. smegmatis-specific; (ii) synonymous codon usage patterns of Barnyard, Che8, Che9d, and Omega are more similar than the rest mycobacteriophages and M. smegmatis; (iii) codon usage bias in the mycobacteriophages are mainly determined by mutational pressure; and (iv) the genes of comparatively GC rich genomes are more biased than the GC poor genomes. Translational selection in determining the codon usage variation in highly expressed genes can be invoked from the predominant occurrences of C ending codons in the highly expressed genes. Cluster analysis based on codon usage data also shows that there are two distinct branches for the fourteen mycobacteriophages and there is codon usage variation even among the phages of each branch.

Towards a resolution on the inherent methodological weakness of the "effective number of codons used by a gene".

Recently Anders Fuglsang provided a modified way for calculating N(c) when biased discrepancy is present in a gene [Biochem. Biophys. Res. Commun. 317 (2004) 957]. Instead of taking the average codon homozygosity for each synonymous family type (as proposed by Wright) [Gene 87 (1990) 23] Fuglsang considered codon homozygosity of each amino acid individually. Marsashi and Najafabadi [Biochem. Biophys. Res. Commun. 324 (2004) 1] in their recent article demonstrated that the readjustment for overestimation at the level of individual amino acids results in loss of considerable amount of information. Immediately after the publication of Marsashi and Najafabadi, Fuglsang proposed that codon homozygosities can be calculated based on the classical population genetics [Biochem. Biophys. Res. Commun. 327 (2005) 1]. Though Fuglsang's approach is a novel one, it fails when any of the amino acids are absent in a gene. However, the inherent cause of overestimation at the level of individual amino acids is still obscured in the literature. Here in this communication we have presented a general condition where effective number of codons is overestimated using Wright's formula and also we propose a new way to calculate N(c), which is independent of amino acid composition.

Investigation on the causes of codon and amino acid usages variation between thermophilic Aquifex aeolicus and mesophilic Bacillus subtilis.

Base composition, codon usages and amino acid usages have been analyzed by taking 529 orthologous sequences of Aquifex aeolicus and Bacillus subtilis, having different optimal growth temperatures. These two bacteria do not have significant difference in overall GC composition, but GC(1+2) and GC3 levels were found to vary significantly. Significant increments in purine content and GC3 composition have been observed in the coding sequences of Aquifex aeolicus than its Bacillus subtilis counterparts. Correspondence analyses on codon and amino acid usages reveal that variation in base composition actually influences their codon and amino acid usages. Two selection pressures acting on the nucleotide level (GC3 and purine enrichment), causes variation in the amino acid usage differently in different protein secondary structures. Our results suggest that adaptation of amino acid usages in coil structure of Aquifex aeolicus proteins is under the control of both purine increment and GC3 composition, whereas the adaptation of the amino acids in the helical region of thermophilic bacteria is strongly influenced by the purine content. Evolutionary perspectives concerning the temperature adaptation of DNA and protein molecules of these two bacteria have been discussed on the basis of these results.

Evolutionary forces in shaping the codon and amino acid usages in Blochmannia floridanus.

Endosymbiotic relationship has great effect on ecological system. Codon and amino acid usages bias of endosymbiotic bacteria Blochmannia floridanus (whose host is an ant Camponotus floridanus) was investigated using experimentally known genes of this organism. Correspondence Analysis on RSCU values show that there exists only one single explanatory major axis that is linked to the strand specific mutational biases. Majority of the genes have a tendency to concentrate on the leading strand, which may be related to the adaptive property related to the replication mechanisms. Amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analyses of the orthologous genes of Escherichia coli and Blochmannia floridanus show that highly expressed genes are significantly more conserved than lowly expressed genes. Based on our results we concluded that strand specific mutational bias is strongly operational in selecting the codon usage in this organism. Replicational-transcriptional selection can be invoked from the presence of majority of highly expressed genes in the leading strand. Conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism. Hydrophobicity of the genes is the second major source in differentiating the genes according to their amino acid usages in this organism.

Synonymous codon usage in Lactococcus lactis: mutational bias versus translational selection.

In this study codon usage bias of all experimentally known genes of Lactococcus lactis has been analyzed. Since Lactococcus lactis is an AT rich organism, it is expected to occur A and/or T at the third position of codons and detailed analysis of overall codon usage data indicates that A and/or T ending codons are predominant in this organism. However, multivariate statistical analyses based both on codon count and on relative synonymous codon usage (RSCU) detect a large number of genes, which are supposed to be highly expressed are clustered at one end of the first major axis, while majority of the putatively lowly expressed genes are clustered at the other end of the first major axis. It was observed that in the highly expressed genes C and T ending codons are significantly higher than the lowly expressed genes and also it was observed that C ending codons are predominant in the duets of highly expressed genes, whereas the T endings codons are abundant in the quartets. Abundance of C and T ending codons in the highly expressed genes suggest that, besides, compositional biases, translational selection are also operating in shaping the codon usage variation among the genes in this organism as observed in other compositionally skewed organisms. The second major axis generated by correspondence analysis on simple codon counts differentiates the genes into two distinct groups according to their hydrophobicity values, but the same analysis computed with relative synonymous codon usage values could not discriminate the genes according to the hydropathy values. This suggests that amino acid composition exerts constraints on codon usage in this organism. On the other hand the second major axis produced by correspondence analysis on RSCU values differentiates the genes into two groups according to the synonymous codon usage for cysteine residues (rarest amino acids in this organism), which is nothing but a artifactual effect induced by the RSCU values. Other factors such as length of the genes and the positions of the genes in the leading and lagging strand of replication have practically no influence in the codon usage variation among the genes in this organism.

Reinvestigation on the causes of genomic GC variation between the orthologous genes of Mycobacterium tuberculosis and Mycobacterium leprae.

Genomic GC (overall G+C content of the coding sequences) variations were reinvestigated between the orthologous genes of Mycobacterium tuberculosis and Mycobacterium leprae species. It was observed that overall genomic GC variation between the species mainly originates from the combined effects GC(1) and GC(2) variations. But codons having identical amino acids with different codons (IA) (between the orthologous codon pairs) are responsible for the genomic GC(3) variation between the organisms, whereas orthologous codons having different amino acids (DA) between the two organisms are responsible for the variation of GC(1) levels. Further analyses indicate that duets and quartets are going in the same direction with same magnitude in changing the GC(3) levels for IA category, whereas GC(1) levels of duets of DA category decreases significantly from the overall GC(1) levels but GC(1) levels of quartets increases significantly from the overall GC(1) levels. GC(3) levels of informational genes for the IA category decrease more rapidly than the other functional categories of genes. The biological implications of these results have been discussed in this paper.

Compositional correlation and codon usage studies in Buchnera aphidicola.

Compositional distributions in three different codon positions as well as codon usage biases of all available DNA sequences of Buchnera aphidicola genome have been analyzed. It was observed that GC levels among the three codon positions is I>II>III as observed in other extremely high AT rich organisms. B. aphidicola being an AT rich organism is expected to have A and/or T at the third positions of codons. Overall codon usage analyses indicate that A and/or T ending codons are predominant in this organism and some particular amino acids are abundant in the coding region of genes. However, multivariate statistical analysis indicates two major trends in the codon usage variation among the genes; one being strongly correlated with the GC contents at the third synonymous positions of codons, and the other being associated with the expression level of genes. Moreover, codon usage biases of the highly expressed genes are almost identical with the overall codon usage biases of all the genes of this organism. These observations suggest that mutational bias is the main factor in determining the codon usage variation among the genes in B. aphidicola.

Gene expressivity is the main factor in dictating the codon usage variation among the genes in Pseudomonas aeruginosa.

Codon usage biases of all DNA sequences (length greater than or equal to 300 bp) from the complete genome of Pseudomonas aeruginosa have been analyzed. As P. aeruginosa is a GC-rich organism, G and/or C are expected to predominate in their codons. Overall codon usage data analysis indicates that indeed codons ending in G and/or C are predominant in this organism. But multivariate statistical analysis indicates that there is a single major trend in the codon usage variation among the genes in this organism, which has a strong negative correlation with the expressivities of the genes. The majority of the lowly expressed genes are scattered towards the positive end of the major axis whereas the highly expressed genes are clustered towards the negative end. This is the first report where the prokaryotic organism having highly skewed base composition is dictated mainly by translational selection, though some other factors such as the lengths of the genes as well as the hydrophobicity of genes also influence the codon usage variation among the genes in this organism in a minor way.

Studies on codon usage in Entamoeba histolytica.

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.

Studies on the relationships between the synonymous codon usage and protein secondary structural units.

The relationship between the synonymous codon usage and protein secondary structural elements (alpha helices and beta sheets) were reinvestigated by taking structural information of proteins from Protein Data Bank (PDB) and their corresponding mRNA sequences from GenBank for four different organisms E. coli, B. subtilis, S. cerevisiae, and Homo sapiens. It was observed that synonymous codon families have non-random codon usage, but there does not exist any species invariant universal correlation between the synonymous codon usage and protein secondary structural elements. The secondary structural units of proteins can be distinguished from the occurrences of bases at the second codon position.

Distribution of HIV-1 subtypes in female sex workers of Calcutta, India.

BACKGROUND & OBJECTIVES: Different routes for the transmission of HIV-1 in India have been reported and the majority of infections occurred through heterosexual route of transmission. In order to understand the dynamics of HIV-1 transmission, a systematic study was undertaken to determine the viral subtypes circulating among the female sex workers in Calcutta, India. METHODS: Peptide enzyme immunoassay (PEIA), heteroduplex mobility assay (HMA) and DNA sequence analysis were used to ascertain the HIV-1 subtypes. RESULTS: V3 serotyping of 52 HIV-1 seropositive samples identified 33 (60%) to be subtype C. A DNA fragment within C2-V3/C2-V5 regions of HIV-1 gp120 was amplified directly from the lymphocyte DNA to avoid any bias in selecting viral variants and used in HMA. Of the 40 samples analyzed, 38 (95%) belonged to subtype C and 2 were found to be non-typable. Further analysis of these 38 samples revealed that 26 (68%) had maximum homology to the C3-Indian reference strain (IND868), 11 (29%) were most homologous to C2-Zambian strain (ZM18) and 1 (3%) showed close resemblance to C1-Malawi strain (MA959). Nucleotide sequence of 11 subsamples encompassing about 325 base pairs was aligned for the Indian and other geographically distinct isolates. On distance and parsimony trees, most of the samples (8/11) clustered together as subtype C. INTERPRETATION & CONCLUSIONS: Subtype C was the major circulating HIV-1 strain in this geographical region, although variation within this subtype was also noticed. DNA sequence analysis was found to be the best method in determining the nature of the HIV-1 subtype followed by HMA and peptide enzyme immunoassay. These findings may have important implications for the design of effective vaccines in India and emphasizes the need for constant monitoring of the HIV-1 subtypes in different parts of India.

Compositional correlation studies among the three different codon positions in 12 bacterial genomes.

Compositional distributions in the three codon positions of the coding sequences of 12 fully sequenced prokaryotic genomes, which are publicly available, were investigated. A universal compositional correlation was observed in most of the genomes under investigation irrespective of their overall genomic GC contents. In all the genomes, the GC contents at the first codon positions are always greater than the overall GC contents of the genomes whereas the reverse is true in the case of second codon positions. GC contents at the third codon positions are higher than the overall genomic GC contents in high GC containing genomes, and the opposite situation was found in case of low GC genomes except for Helicobacter pylori. In high-GC rich genomes, the GC contents at the first + second codon positions are less than the GC contents at the third codon positions, and they are low in low-GC genomes except for Helicobacter pylori. The distributions of four bases at the three different positions were also investigated for all 12 organisms. It was observed that in high-GC genomes G is the most dominant base and in low-GC genomes A is the most dominant base in the first codon positions. But purine bases, i.e., (A + G), predominantly occur in the first codon position. In the second codon position, A is the most dominant base in most of the organisms and G is the least dominant base in all the organisms. There is no unique regular pattern of individual bases at the third codon positions; however, there are significant differences in the occurrences of (G + C) contents in the third codon positions among the different organisms. Calculations of dinucleotide frequencies in 12 different organisms indicate that in GC-rich genomes GG, GC, CC, and CG dinucleotides are the most dominant whereas the reverse is true in case of low-GC genomes. Biological implications of these results are discussed in this paper.

Similarities inferred from the studies of long range correlations among mitochondrial DNA sequences.

Existence of long range correlations within the DNA sequences of living organism has immense importance in understanding the language of DNA sequences. Recently it has been reported that long range correlations occur in DNA sequences. Some investigators claimed that these type of correlations occur only on intron containing DNA sequences. Some observers, however, have the opinion that long range correlations do not distinguish between the intron containing DNA sequences and intronless DNA sequences. The biological origin of long range correlations in the DNA sequences is not clearly known. In this paper we have demonstrated that long range correlations also occur on intronless mitochondrial DNA sequences, indicating that these special type of correlations are not the unique features for intron containing DNA sequences. We have also demonstrated that long range correlations simply originate in the region around which there is a large variation of pyrimidine and purine ratios. The similarities among the mitochondrial DNA sequences can be inferred by computing the fractal exponents in the region where there is a large variation of pyrimidine and purine ratio, as well as in the region where the ratio of pyrimidine and purine fluctuates in a nearly constant manner. In other words the similarities among the mitochondrial DNA sequences cannot be inferred by calculating the fractral exponents for the whole sequence.