Succinate dehydrogenase localization in the mitochondria of the renal tubules of cold- and warm-blooded vertebrates

Using electron microscopic histochemical technique, studies have been made on the activity of succinic dehydrogenase in the kidneys of the cod Gadus morrhua and dog. It was shown that chelate granules indicating localization of the enzyme in the mitochondria of nephronal cells, concentrate mainly in two zones -- between the membranes and inside the cristae. This distribution of the enzyme implies the presence of two pools of succinic dehydrogenase in the mitochondria which are utilized at different stages of oxidative phosphorylation. Succinic dehydrogenase content of the cristae is lower in cod than in dog.     

The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.     

Comprehensive comparison between locations of orthologous genes on archaeal and bacterial genomes

MOTIVATION: Following an extensive search for orthologous genes between the complete genomes from archaea and bacteria, the spatial association of the orthologs has been investigated in terms of synteny, the conservation of the order of neighboring genes. However, the relationships between the relative locations of remote orthologs over entire genomes have not been shown. RESULTS: Comprehensive comparisons between the locations of orthologs on nineteen archaeal and bacterial genomes are presented by the location to location correspondence based on the gene-location distance. When the two genomes are rotated such that a pair of orthologs with the shortest distance is set in the same angle, a statistically significant number of orthologs maintain their relative locations between the genomes. Even by the short distances at the 5% significance level, the rotations are restricted within a narrow range, suggesting an intrinsic angle for realizing similar locations between the orthologs in each genome pair. Furthermore, the rotations in the restricted range agree with the replication origin and terminus sites for the analyzed genomes where such sites are known. The relationship between location-maintained orthologs and gene function is also discussed.     

Single-copy sequence homology among the GC-richest isochores of the genomes from warm-blooded vertebrates

We have hybridized a human DNA fraction corresponding to the GC-richest and gene-richest isochore family, H3, on compositional fractions of DNAs from 12 mammalian species and three avian species, representing eight and three orders, respectively. Under conditions in which repetitive sequences are competed out, the H3 isochore probe only or predominantly hybridized on the GC-richest fractions of main-band DNA from all the species investigated. These results indicate that single-copy sequences from the human H3 isochores share homology with sequences located in the compositionally corresponding compartments of the vertebrate genomes tested. These sequences are likely to be essentially formed by conserved coding sequences. The present results add to other lines of evidence indicating that isochore patterns are highly conserved in warm-blooded vertebrate genomes. Moreover, they refine recent reports (Sabeur et al., 1993; Kadi et al., 1993), and correct them in some details and also in demonstrating that the shrew genome does not exhibit the general mammalian pattern, but a special pattern.Correspondence to: G. Bernardi     

The length of chromatin loops in meiotic prophase I of warm-blooded vertebrates depends on the DNA compositional organization

In meiotic prophase I, chromatin fibrils attached to the lateral elements of the synaptonemal complexes (SC) form loops. SCAR DNA (synaptonemal complex associated regions of DNA) is a family of genomic DNA tightly associated with the SC and located at the chromatin loop basements. Using the hybridization technique, it was demonstrated that localization of SCAR DNA was evolutionarily conserved in the isochore compositional fractions of the three examined genomes of warm-blooded vertebrates—human, chicken, and golden hamster. The introduction of the concept of the comparative loops (CL) of DNA that form of chromatin attach to SC in the isochore compositional fractions provided the calculation of their length. An inverse proportional relationship between the length of CL DNA and the GC level in the isochore compartments of the studied warm-blooded vertebrate genomes was revealed. An exception was the GCpoorest L1 isochore family. For different compositional isochores of the human and chicken genomes, the number of genes in the CL DNA was evaluated. A model of the formation of GC-rich isochores in vertebrate genomes, according to which there was not only an increase in the GC level but also the elimination of functionally insignificant noncoding DNA regions, as well as joining of isochores decreasing in size, was suggested.     

The compositional transition of vertebrate genomes: an analysis of the secondary structure of the proteins encoded by human genes

Fluctuations and increments of both C(3) and G(3) levels along the human coding sequences were investigated comparing two sets of Xenopus/human orthologous genes. The first set of genes shows minor differences of the GC(3) levels, the second shows considerable increments of the GC(3) levels in the human genes. In both data sets, the fluctuations of C(3) and G(3) levels along the coding sequences correlated with the secondary structures of the encoded proteins. The human genes that underwent the compositional transition showed a different increment of the C(3) and G(3) levels within and among the structural units of the proteins. The relative synonymous codon usage (RSCU) of several amino acids were also affected during the compositional transition, showing that there exists a correlation between RSCU and protein secondary structures in human genes. The importance of natural selection for the formation of isochore organization of the human genome has been discussed on the basis of these results.     

CpG islands, genes and isochores in the genomes of vertebrates

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [A?ssani and Bernardi, Gene 106 (1991) 173-183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5'-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the 'extragenic' CpG islands located in the 5'-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including 'extragenic' CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including 'extragenic' CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.     

Successful lateral transfer requires codon usage compatibility between foreign genes and recipient genomes

We present evidence supporting the notion that codon usage (CU) compatibility between foreign genes and recipient genomes is an important prerequisite to assess the selective advantage of imported functions, and therefore to increase the fixation probability of horizontal gene transfer (HGT) events. This contrasts with the current tendency in research to predict recent HGTs in prokaryotes by assuming that acquired genes generally display poor CU. By looking at the CU level (poor, typical, or rich) exhibited by putative xenologs still resembling their original CU, we found that most alien genes predominantly present typical CU immediately upon introgression, thereby suggesting that the role of CU amelioration in HGT has been overemphasized. In our strategy, we first scanned a representative set of 103 complete prokaryotic genomes for all pairs of candidate xenologs (exported/imported genes) displaying similar CU. We applied additional filtering criteria, including phylogenetic validations, to enhance the reliability of our predictions. Our approach makes no assumptions about the CU of foreign genes being typical or atypical within the recipient genome, thus providing a novel unbiased framework to study the evolutionary dynamics of HGT.     

Correlations between the compositional properties of human genes,codon usage,and amino acid composition of proteins

Summary We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unity slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.     

Relationship between codon usage and sequence-dependent curvature of genomes

Static DNA curvature distributions of full-sequenced genomes and large DNA contigs from different organisms were calculated. Very distinctive differences among histogram profiles coming from archaebacteria, eubacteria, and eukaryotes were observed. Eubacterial profiles were, on average, more curved than were archaeal and eukaryotic profiles. A comparative analysis between real and randomized DNA sequences revealed that eubacterial genomes presented, overall, higher curvature values than random sequences. An opposite portrait was exhibited by archaeal and eukaryotic genomes. They displayed a lower frequency of curved regions than their corresponding randomized sequences. The contributions of coding and intergenic regions to the curvature profile were also analyzed. Intergenic regions, on average, were found to be more curved than the overall genomic sequences, especially in prokaryotic organisms. Nevertheless, because of their small size with respect to coding regions, the contribution of intergenic sequences to the overall curvature profile tended to be minor. A clear relationship between codon usage and DNA curvature was demonstrated, and a proposal of the possible coevolution of both systems is discussed. Finally, we present a procedure to quantify the deviation of a curvature profile from randomness through a formal statistical analysis.     

Clusters of orthologous genes for 41 archaeal genomes and implications for evolutionary genomics of archaea

Background  

An evolutionary classification of genes from sequenced genomes that distinguishes between orthologs and paralogs is indispensable for genome annotation and evolutionary reconstruction. Shortly after multiple genome sequences of bacteria, archaea, and unicellular eukaryotes became available, an attempt on such a classification was implemented in Clusters of Orthologous Groups of proteins (COGs). Rapid accumulation of genome sequences creates opportunities for refining COGs but also represents a challenge because of error amplification. One of the practical strategies involves construction of refined COGs for phylogenetically compact subsets of genomes.     

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     

Further evidence of microcolinearity between barley and rice genomes at two orthologous regions

Two genetic markers, BCD135 and RZ567 were used to select clones from genomic BAC libraries of barley and rice for sequencing and subsequent sequence comparisons. A set of two orthologous BACs each from barley and rice was selected by hybridization with BCD135 and RZ567 cDNA probes. A total of 556-kb stretch including two barley BACs (773K135 and 745C13) and two orthologous rice BACs (24K23 and 49D11) was completely sequenced. Comparative sequence analysis between orthologous BACs from the two species revealed presence of two conserved genes at BCD135 region and only one gene at the RZ567 regions. The two conserved genes were in the same order and orientation in both the species however, separated by significantly larger distance in barley. The larger distance between two barley genes was mainly due to presence of different retrotransposable elements and their derivatives (78.9% of the intergenic region) that expanded the barley BCD135 region at the rate of 9.1X. An additional gene of unknown function was also inserted along with several retrotransposable elements between two conserved genes at barley BCD135 region. More genome expansion rate (10X) around barley RZ567 locus was estimated by extremely high proportion (> 70%) of retrotransposons. Among different retrotransposons, the Sabrina elements rather than BARE were more prevalent in both the regions. Contrary to it, the BCD135 region of rice was composed of only 17.1% retrotransposable elements and no significant retrotransposons except 14 miniature inverted transposable elements (MITEs) were observed in its RZ567 region. The sequence comparison between orthologous regions of rice and barley genomes was useful for gene identification and determination of individual gene structure indicating the possibility of effective utilization of rice genome sequences in understanding the large genome of barley. (The sequence data described in this paper have been submitted to the GenBank data library under the accession no. AF474072 (773K14), AF474071 (745C13), AF480497 (24K23) and AF480496 (49D11)).     

CpG islands: features and distribution in the genomes of vertebrates

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.     

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.     

The compositional patterns of the avian genomes and their evolutionary implications

The compositional distributions of large (main-band) DNA fragments from eight birds belonging to eight different orders (including both paleognathous and neognathous species) are very broad and extremely close to each other. These findings, which are paralleled by the compositional similarity of homologous coding sequences and their codon positions, support the idea that birds are a monophyletic group.The compositional distribution of third-codon positions of genes from chicken, the only avian species for which a relatively large number of coding sequences is known, is very broad and bimodal, the minor GC-richer peak reaching 100% GC. The very high compositional heterogeneity of avian genomes is accompanied (as in the case of mammalian genomes) by a very high speciation rate compared to cold-blooded vertebrates which are characterized by genomes that are much less heterogeneous. The higher GC levels attained by avian compared to mammalian genomes might be correlated with the higher body temperature (41–43°C) of birds compared to mammals (37°C).A comparison of GC levels of coding sequences and codon positions from man and chicken revealed very close average GC levels and standard deviations. Homologous coding sequences and codon positions from man and chicken showed a surprisingly high degree of compositional similarity which was, however, higher for GC-poor than for GC-rich sequences. This indicates that GC-poor isochores of warm-blooded vertebrates reflect the composition of the isochores of the genome of the common reptilian ancestor of mammals and birds, which underwent only a small compositional change at the transition from cold- to warm-blooded vertebrates. In contrast, the GC-rich isochores of birds and mammals are the result of large compositional changes at the same evolutionary transition, where were in part different in the two classes of warm-blooded vertebrates.Correspondence to: G. Bernaadi