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.     

Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores

We compared the exon/intron organization of vertebrate genes belonging to different isochore classes, as predicted by their GC content at third codon position. Two main features have emerged from the analysis of sequences published in GenBank: (1) genes coding for long proteins (i.e., 500 aa) are almost two times more frequent in GC-poor than in GC-rich isochores; (2) intervening sequences (=sum of introns) are on average three times longer in GC-poor than in GC-rich isochores. These patterns are observed among human, mouse, rat, cow, and even chicken genes and are therefore likely to be common to all warm-blooded vertebrates. Analysis of Xenopus sequences suggests that the same patterns exist in cold-blooded vertebrates. It could be argued that such results do not reflect the reality because sequence databases are not representative of entire genomes. However, analysis of biases in GenBank revealed that the observed discrepancies between GC-rich and GC-poor isochores are not artifactual, and are probably largely underestimated. We investigated the distribution of microsatellites and interspersed repeats in introns of human and mouse genes from different isochores. This analysis confirmed previous studies showing that Ll repeats are almost absent from GC-rich isochores. Microsatellites and SINES (Alu, B1, B2) are found at roughly equal frequencies in introns from all isochore classes. Globally, the presence of repeated sequences does not account for the increased intron length in GC-poor isochores. The relationships between gene structure and global genome organization and evolution are discussed.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Telomeres in Warm-Blooded Vertebrates Are Composed of GC-Rich Isochores

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.     

Bendable genes of warm-blooded vertebrates.

It is shown that in the genomes of warm-blooded vertebrates the elevation of genic GC-content is associated with an increase in the bendability of the DNA helix, which is both absolute and relative as compared with random sequences. This trend takes place both in exons and introns, being more pronounced in the latter. At the same time, the free energy of melting (delta G) of exons and introns increases only absolutely with elevation of GC-content, whereas it decreases as compared with random sequences (again, this trend is stronger in the introns). In genes of cold-blooded animals, plants, and unicellular organisms, these regularities are weaker and often not consistent. Generally, there is a negative correlation between bendability and melting energy at any fixed GC-content value. This effect is stronger in the introns. These findings suggest that GC-enrichment of genes in the homeotherm vertebrates can be caused by selection for increased bendability of DNA.     

Correlations between coding and contiguous non-coding sequences in isochore families from vertebrate genomes

Many years ago compositional correlations were found to hold between coding and contiguous non-coding sequences. These correlations were essentially studied in whole genomes of mammals, which are characterized by strong compositional heterogeneities. Here we investigated whether these correlations also hold within the much more homogeneous isochore families. This point was checked not only in the case of mammals, but also in that of phylogenetically distant vertebrates, which are characterized by very different compositional patterns. Indeed, these are remarkably different in cold- and warm-blooded vertebrates. Fish genomes, for instance, are much more homogeneous than those of mammals and birds. The compositional correlations between coding sequences and the corresponding introns, or their 5′ and 3′ flanking regions, were studied in the isochore families of the fully sequenced genomes from four fishes (Brachydanio rerio, Oryzias latipes, Gasterosteus aculeatus and Tetraodon nigroviridis), human and chicken.     

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.     

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.     

Diversity in isochore structure among cold-blooded vertebrates based on GC content of coding and non-coding sequences

To review the general consideration about the different compositional structure of warm and cold-blooded vertebrates genomes, we used of the increasing number of genetic sequences, including coding (exons) and non-coding (introns) regions, that have been deposited on the databases throughout last years. The nucleotide distributions of the third codon positions (GC3) have been analyzed in 1510 coding sequences (CDS) of fish, 1414 CDS of amphibians and 320 CDS of reptiles. Also, the relationship between GC content of 74, 56 and 25 CDS of fish, amphibians and reptiles, respectively and that of their corresponding introns (GCI) have been considerated. In accordance with recent data, sequence analysis showed the presence of very GC3-rich CDS in these poikilotherm vertebrates. However, very high diversity in compositional patterns among different orders of fish, amphibians and reptiles was found. Significant positive correlations between GC3 and GCI was also confirmed for the genes analyzed. Nevertheless, introns resulted to be poorer in GC than their corresponding CDS, this difference being larger than in human genome. Because the limited number of available sequences including exons and introns we must be cautious about the results derived from them. However, the indicious of higher GC richness of coding sequences than of their corresponding introns could aid to understand the discrepancy of sequence analysis with the ultracentrifugation studies in cold-blooded vertebrates that did not predict the existence of GC-rich isochores.     

Evolution of CpG islands within the myc gene family

CpG islands are discrete regions of DNA with significantly greater frequencies of CpG doublets than bulk genomic DNA. They are most frequently associated with the 5'-ends of housekeeping genes and are involved in the regulation of their expression. In this study, the structure and evolution of CpG islands within genes of the myc family were evaluated with the protein-coding sequences of animals and their transducing viruses. These evaluations relied on a gene tree for the entire myc family to test the origins of CpG islands within their two protein-coding exons. Overall, CG-very rich and CG-rich islands are associated with exon 2 of the different myc genes of warm-blooded vertebrates and with exon 3 of the N-myc and s-myc sequences of mammals, but not birds. These overall distributions of well-developed islands can be related to the major transitions of the CG-rich genomes of warm-blooded vertebrates from the CG-poor ones of other animals. In turn, the greater variability of well-developed islands within exon 3 of the N-myc gene and among the different retrogenes of the myc family can be attributed to their reduced functional constraints, as evidenced by their limited and very restricted patterns of expression, respectively.     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

Isochores and the evolutionary genomics of vertebrates

The nuclear genomes of vertebrates are mosaics of isochores, very long stretches (>300kb) of DNA that are homogeneous in base composition and are compositionally correlated with the coding sequences that they embed. Isochores can be partitioned in a small number of families that cover a range of GC levels (GC is the molar ratio of guanine+cytosine in DNA), which is narrow in cold-blooded vertebrates, but broad in warm-blooded vertebrates. This difference is essentially due to the fact that the GC-richest 10-15% of the genomes of the ancestors of mammals and birds underwent two independent compositional transitions characterized by strong increases in GC levels. The similarity of isochore patterns across mammalian orders, on the one hand, and across avian orders, on the other, indicates that these higher GC levels were then maintained, at least since the appearance of ancestors of warm-blooded vertebrates. After a brief review of our current knowledge on the organization of the vertebrate genome, evidence will be presented here in favor of the idea that the generation and maintenance of the GC-richest isochores in the genomes of warm-blooded vertebrates were due to natural selection.     

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.     

Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.