The effect of expression levels on codon usage in Plasmodium falciparum.

The usage of alternative synonymous codons in the completely sequenced, extremely A+T-rich parasite Plasmodium falciparum was studied. Confirming previous studies obtained with less than 3% of the total genes recently described, we found that A- and U-ending triplets predominate but translational selection increases the frequency of a subset of codons in highly expressed genes. However, some new results come from the analysis of the complete sequence. First, there is more variation in GC3 than previously described; second, the effect of natural selection acting at the level of translation has been analysed with real expression data at 4 different stages and third, we found that highly expressed proteins increment the frequency of energetically less expensive amino acids. The implications of these results are discussed.

Translational selection on codon usage in Xenopus laevis.

A correspondence analysis of codon usage in Xenopus laevis revealed that the first axis is strongly correlated with the base composition at third codon positions. The second axis discriminates between putatively highly expressed genes and the other coding sequences, with expression levels being confirmed by the analysis of Expressed sequence tag frequencies. The comparison of codon usage of the sequences displaying the extreme values on the second axis indicates that several codons are statistically more frequent among the highly expressed (mainly housekeeping) genes. Translational selection appears, therefore, to influence synonymous codon usage in Xenopus.

Evidence for translational selection in codon usage in Echinococcus spp.

We analysed the intragenomic variation in codon usage in Echinococcus spp. by correspondence analysis. This approach detected a trend among genes which was correlated with expression levels. Among the (presumed) highly expressed sequences we found an increased usage of a subset of codons, almost all of them G- or C- ending. Since an increase in these bases at the synonymous sites is against the mutational bias (these genomes are slightly A+-T- rich), we conclude that codon usage in Echinococcus is the result of an equilibrium between compositional pressure and selection, the latter acting at the level of translation, mainly on highly expressed genes. This is the first report where translational selection for codon usage is detected among Platyhelminthes.

Translational selection shapes codon usage in the GC-rich genome of Chlamydomonas reinhardtii.

In unicellular species codon usage is determined by mutational biases and natural selection. Among prokaryotes, the influence of these factors is different if the genome is skewed towards AT or GC, since in AT-rich organisms translational selection is absent. On the other hand, in AT-rich unicellular eukaryotes the two factors are present. In order to understand if GC-rich genomes display a similar behavior, the case of Chlamydomonas reinhardtii was studied. Since we found that translational selection strongly influences codon usage in this species, we conclude that there is not a common pattern among unicellular organisms.

Codon usage in Chlamydia trachomatis is the result of strand-specific mutational biases and a complex pattern of selective forces.

The patterns of synonymous codon choices of the completely sequenced genome of the bacterium Chlamydia trachomatis were analysed. We found that the most important source of variation among the genes results from whether the sequence is located on the leading or lagging strand of replication, resulting in an over representation of G or C, respectively. This can be explained by different mutational biases associated to the different enzymes that replicate each strand. Next we found that most highly expressed sequences are located on the leading strand of replication. From this result, replicational-transcriptional selection can be invoked. Then, when the genes located on the leading strand are studied separately, the correspondence analysis detects a principal trend which discriminates between lowly and highly expressed sequences, the latter displaying a different codon usage pattern than the former, suggesting selection for translation, which is reinforced by the fact that Ks values between orthologous sequences from C. trachomatis and Chlamydia pneumoniae are much smaller in highly expressed genes. Finally, synonymous codon choices appear to be influenced by the hydropathy of each encoded protein and by the degree of amino acid conservation. Therefore, synonymous codon usage in C.trachomatis seems to be the result of a very complex balance among different factors, which rises the problem of whether the forces driving codon usage patterns among microorganisms are rather more complex than generally accepted.

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica.

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.

Trends of amino acid usage in the proteins from the unicellular parasite Giardia lamblia.

Correspondence analysis of amino acid frequencies was applied to 75 complete coding sequences from the unicellular parasite Giardia lamblia, and it was found that three major factors influence the variability of amino acidic composition of proteins. The first trend strongly correlated with (a) the cysteine content and (b) the mean weight of the amino acids used in each protein. The second trend correlated with the global levels of hydropathy and aromaticity of each protein. Both axes might be related with the defense of the parasite to oxygen free radicals. Finally, the third trend correlated with the expressivity of each gene, indicating that in G. lamblia highly expressed sequences display a tendency to preferentially use a subset of the total amino acids.

The correlation of protein hydropathy with the base composition of coding sequences.

The "universal correlation" (D'Onofrio, G., Bernardi, G., 1992. A universal compositional correlation among codon positions. Gene 110, 81-88.) that holds between and or ( values are the average values of the coding sequences of each genome analyzed) at both the inter- and intra-genomic level, was re-analyzed on a vastly larger dataset. The results showed a slight, but significant, difference in the vs. correlations exhibited by prokaryotes and eukaryotes. This finding prompted an analysis of the correlation between and the amino acid frequencies in the encoded proteins, which has shown that positive correlations exist between values of coding sequences and the hydropathy of the corresponding proteins. These correlations are due to the fact that hydrophobic and amphypathic amino acids increase, whereas hydrophilic amino acids decrease with increasing values. Hydropathy values of prokaryotic proteins are systematically higher than those of eukaryotes, but the slopes of the regression lines are identical. The lower hydrophobicity of eukaryotic proteins is due to differences in the amino acid composition. In particular, the twofold higher cysteine (and disulfide bond) level of eukaryotic proteins compared to prokaryotic proteins most probably compensates for their lower hydrophobicity. This supports the viewpoint that hydrophobicity plays a structural and functional role as far as protein stability is concerned.

Compositional correlations in the chicken genome.

This paper analyses the compositional correlations that hold in the chicken genome. Significant linear correlations were found among the regions studied-coding sequences (and their first, second, and third codon positions), flanking regions (5' and 3'), and introns-as is the case in the human genome. We found that these compositional correlations are not limited to global GC levels but even extend to individual bases. Furthermore, an analysis of 1037 coding sequences has confirmed a correlation among GC(3), GC(2), and GC(1). The implications of these results are discussed.

Evolutionary genomics of vertebrates and its implications.

The discovery that the vertebrate genomes of warm-blooded vertebrates are mosaics of isochores, long DNA segments homogeneous in base composition, yet belonging to families covering a broad spectrum of GC levels, has led to two major observations. The first is that gene density is strikingly non-uniform in the genome of all vertebrates, gene concentration increasing with increasing GC levels. (Although the genomes of cold-blooded vertebrates are characterized by smaller compositional heterogeneities than those of warm-blooded vertebrates and high GC levels are not attained, their gene distribution is basically similar to that of warm-blooded vertebrates.) The second observation is that the GC-richest and gene-richest isochores underwent a compositional transition (characterized by a strong increase in GC level) between cold- and warm-blooded vertebrates. Evidence to be discussed favors the idea that this compositional transition and the ensuing highly heterogeneous compositional pattern was due to, and was maintained by, natural selection.

Synonymous codon choices in the extremely GC-poor genome of Plasmodium falciparum: compositional constraints and translational selection.

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection.

Heterogeneity in codon usage in the flatworm Schistosoma mansoni.

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC3, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC3, 5', 3', and flanking (5' + 3' + introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded.

Compositional constraints in the extremely GC-poor genome of Plasmodium falciparum.

We have analyzed the compositional properties of coding (protein encoding) and non-coding sequences of Plasmodium falciparum, a unicellular parasite characterized by an extremely AT-rich genome. GC% levels, base and dinucleotide frequencies were studied. We found that among the various factors that contribute to the properties of the sequences analyzed, the most relevant are the compositional constraints which operate on the whole genome.

Cytogenetic and molecular analysis of the satellite DNA of the genus Ctenomys (Rodentia Octodontidae) from Uruguay.

The genus of subterranean rodents Ctenomys presents the widest range of variability in diploid number among mammals (from 2n = 10 to 2n = 70). In Uruguay, this variability is observed in karyotypes with 2n = 44, 50 or 58 and two geographically isolated populations with 70 chromosomes but different karyotypic structure. The last three populations were analyzed in the present study. They present a satellite DNA, which was isolated from genomic DNA after AluI digestion. In situ hybridization showed that this satellite DNA is located in the centromeric region of a few chromosomes, coincident with Hoechst 33258 staining and C-banding patterns. A similar satellite DNA was detected in Argentinian species of this genus. We established that, in spite of differences in number of positive heterochromatic blocks per karyotype, the C value is the same in the three populations studied. The nature and possible evolutionary path of this repeated DNA is discussed.

The analysis of protein coding genes suggests monophyly of Trypanosoma.

We analyze evolutionary relationships among members of the family Trypanosomatidae, with particular emphasis on whether protein coding genes support paraphyly of the genus Trypanosoma. Phylogenetic reconstruction based on three different protein coding genes (glyceraldehyde-3-phosphate dehydrogenase, trypanothione reductase, and alpha-tubulin) suggests that Trypanosoma is monophyletic. Moreover, pairwise comparisons of other protein coding genes show that the distances between Trypanosoma cruzi and T. brucei are significantly smaller than are the distances between each Trypanosoma species and Crithidia or Leishmania. These results contradict recent published phylogenies based on nuclear rRNA genes which suggested that T. cruzi is more closely related to Leishmania and Crithidia than to T. brucei.

Compositional correlations in the nuclear genes of the flatworm Schistosoma mansoni.

We have investigated the genome organization in the flatworm Schistosoma mansoni. First, we analyzed the compositional distributions of the three codon positions. Second, we investigated the correlations that exist between (1) the GC levels of exons against flanking regions, (2) the GC levels of third codon positions against flanking regions, (3) the dinucleotide frequencies of exons against flanking regions, and (4) the GC levels of 5' against 3' regions. The modality of the distribution of third codon positions, together with the significant correlations found, leads us to propose that the nuclear genome of this species is compositionally compartmentalized.

Compositional properties of nuclear genes from Plasmodium falciparum.

We have analyzed the compositional distributions of coding sequences and their different codon positions, as well as the codon usage of the nuclear genes of Plasmodium falciparum, a parasite characterized by an extremely GC-poor genome. As expected, coding sequences are AT-rich, codon usage is strongly biased towards A or T in third codon positions, and some particular amino acids (aa) are especially abundant in the encoded proteins. Remarkably, however, no difference was detected between housekeeping (HK) and antigen (Ag) genes, in spite of differences in expression level and evolutionary constraints. Moreover, all the features found in P. falciparum are very similar to those found in a bacterium characterized by a very GC-poor genome, Staphylococcus aureus. These findings stress the importance of compositional constraints in determining codon usage and aa utilisation.

The compositional compartments of the nuclear genomes of Trypanosoma brucei and T. cruzi.

Fractionation of DNA from Trypanosoma brucei and T. equiperdum by centrifugation in a Cs2SO4/BAMD density gradient indicated that these genomes are compositionally compartmentalized, a conclusion confirmed by the analysis of the compositional distribution of third codon positions from T. brucei and T. cruzi. In order to investigate whether this compartmentalization is accompanied by the often different properties of coding sequences, we have analyzed and compared the compositional compartments with respect to dinucleotide frequency and amino-acid usage of the encoded proteins of all gene sequences available in the GenBank database from T. brucei and T. cruzi. In all cases, the compartments displayed remarkable differences. These results are similar to findings obtained in highly compartmentalized genomes, like those of warm-blooded vertebrates.

Dinucleotide biases in the platyhelminth Schistosoma mansoni.

The analysis of dinucleotide biases in coding and flanking regions, introns, rDNA and repetitive sequences, in the flatworm Schistosoma mansoni is reported. Except for rDNA, all regions display CpG avoidance and TpG plus CpA excess, which might be evidence of the presence of 5mC. The distribution and hierarchies of dinucleotides differ from the data published for invertebrate and vertebrate coding sequences.