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.     

Sequence analysis and compositional properties of untranslated regions of human mRNAs

A detailed computer analysis of the untranslated regions, 5′-UTR and 3′- UTR, of human mRNA sequences is reported. The compositional properties of these regions, compared with those of the corresponding coding regions, indicate that 5′-UTR and 3′-UTR are less affected by the isochore compartmentalization than the corresponding third codon positions of mRNAs. The presence of higher functional constraints in 5′-UTR is also reported. Dinucleotide analysis shows a depletion of CpG and TpA in both sequences. A search for significant sequence motifs using the WORDUP algorithm reveals the patterns already known to have a functional role in the mRNA UTR, and several other motifs whose functional roles remain to be demonstrated. This type of analysis may be particularly useful for guiding site-directed mutagenesis experiments. In addition, it can be used for assessing the nature of anonymous sequences now produced in large amounts in megabase sequencing projects.     

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     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.     

The genome: an isochore ensemble and its evolution

The genomes of eukaryotes are mosaics of isochores. These are long DNA stretches that are fairly homogeneous in base composition and that belong to a small number of families characterized by different ratios of GC to AT and different short-sequence patterns (i.e., different DNA structures that interact with different proteins). This genome organization led to two discoveries: (1) the genomic code, which refers to two correlations, that of the composition of coding and contiguous noncoding sequences, and that of coding sequences and the structural properties of the encoded proteins; and (2) the genome phenotypes, which correspond to the patterns of isochore families in the genomes. These patterns indicate that genome evolution may proceed either according to a conservative mode or to a transitional (isochore shifting) mode, apparently depending upon whether the environment is constant or shifting. According to the neoselectionist theory, natural selection is responsible for both modes.     

Isochore pattern and gene distribution in the chicken genome

We report here investigations on the isochore pattern and the distribution of genes in the chromosomes of chicken. In spite of large differences in genome size and karyotype, the compositional properties and the gene distribution of the chicken genome are very similar to those recently published for the human genome, which is a good representative of most mammalian genomes. In fact, this similarity, which extends to the relative amounts and, also, to a large extent at least, to the average base composition of isochore families, is most interesting in view of the very large distance of mammals and birds for a common ancestor, which goes back to 310–340 million years ago. This raises important questions about genome evolution in vertebrates.     

Compositional mapping of the human dystrophin-encoding gene.

The genomes of warm-blooded vertebrates are mosaics of long DNA segments (> 300 kb, on the average), the isochores, homogeneous in GC levels, which belong to a small number of compositional families. In the present work, the human dystrophin-encoding gene, spanning more than 2.3 Mb in Giemsa band Xp21 (on the short arm of the X chromosome), was analyzed in its isochore organization by hybridizing cDNA probes, corresponding to eight contiguous segments of the coding sequence, on compositional fractions from human DNA. Five DNA regions of uniform (+/- 0.5%) GC content, separated by compositional discontinuities of about 2% GC, were found, so providing the first high-resolution compositional map obtained for a human genome locus and the first direct estimate of isochore size (360 kb to more than 770 kb, in the locus under consideration). One of the isochores contains 71% and another one 21% of deletion breakpoints found in patients suffering from Duchenne's and Becker's muscular dystrophies.     

Isochore patterns and gene distributions in fish genomes

The compositional approach developed in our laboratory many years ago revealed a large-scale compositional heterogeneity in vertebrate genomes, in which GC-rich and GC-poor regions, the isochores, were found to be characterized by high and low gene densities, respectively. Here we mapped isochores on fish chromosomes and assessed gene densities in isochore families. Because of the availability of sequence data, we have concentrated our investigations on four species, zebrafish (Brachydanio rerio), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis), which belong to four distant orders and cover almost the entire GC range of fish genomes. These investigations produced isochore maps that were drastically different not only from those of mammals (in that only two major isochore families were essentially present in each genome vs five in the human genome) but also from each other (in that different isochore families were represented in different genomes). Gene density distributions for these fish genomes were also obtained and shown to follow the expected increase with increasing isochore GC. Finally, we discovered a remarkable conservation of the average size of the isochores (which match replicon clusters in the case of human chromosomes) and of the average GC levels of isochore families in both fish and human genomes. Moreover, in each genome the GC-poorest isochore families comprised a group of "long isochores" (2-20 Mb in size), which were the lowest in GC and varied in size distribution and relative amount from one genome to the other.     

Compositional compartmentalization and gene composition in the genome of vertebrates

Summary The compositional distribution of coding sequences from five vertebrates (Xenopus, chicken, mouse, rat, and human) is shifted toward higher GC values compared to that of the DNA molecules (in the 35–85-kb size range) isolated from the corresponding genomes. This shift is due to the lower GC levels of intergenic sequences compared to coding sequences. In the cold-blooded vertebrate, the two distributions are similar in that GC-poor genes and GC-poor DNA molecules are largely predominant. In contrast, in the warm-blooded vertebrates, GC-rich genes are largely predominant over GC-poor genes, whereas GC-poor DNA molecules are largely predominant over GC-rich DNA molecules. As a consequence, the genomes of warm-blooded vertebrates show a compositional gradient of gene concentration. The compositional distributions of coding sequences (as well as of DNA molecules) showed remarkable differences between chicken and mammals, and between mouse (or rat) and human. Differences were also detected in the compositional distribution of housekeeping and tissue-specific genes, the former being more abundant among GC-rich genes.     

Purification of triosephosphate isomerase and isolation of its gene from the mosquito Culex tarsalis

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5′-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5′ flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.     

Compositional transitions in the nuclear genomes of cold-blooded vertebrates

Summary The compositional properties of DNAs from 122 species of fishes and from 18 other coldblooded vertebrates (amphibians and reptiles) were compared with those from 10 warm-blooded vertebrates (mammals and birds) and found to be substantially different. Indeed, DNAs from cold-blooded vertebrates are characterized by much lower intermolecular compositional heterogeneities and CsCl band asymmetries, by a much wider spectrum of modal buoyant densities in CsCl, by generally lower amounts of satellites, as well as by the fact that in no case do buoyant densities reach the high values found in the GC-richest components of DNAs from warm-blooded vertebrates.In the case of fish genomes, which were more extensively studied, different orders were generally characterized by modal buoyant densities that were different in average values as well as in their ranges. In contrast, different families within any given order were more often characterized by narrow ranges of modal buoyant densities, and no difference in modal buoyant density was found within any single genus (except for the genusAphyosemion, which should be split into several genera).The compositional differences that were found among species belonging to different orders and to different families within the same order are indicative of compositional transitions, which were shown to be essentially due to directional base substitutions. These transitions were found to be independent of geological time. Moreover, the rates of directional base substitutions were found to be very variable and to reach, in some cases, extremely high values, that were even higher than those of silent substitutions in primates. The taxonomic and evolutionary implications of these findings are discussed.     

Analysis of the phylogenetic distribution of isochores in vertebrates and a test of the thermal stability hypothesis

Warm-blooded vertebrates show large-scale variation in G + C content along their chromosomes, a pattern which appears to be largely absent from cold-blooded vertebrates. However, compositional variation in poikilotherms has generally been studied by ultracentrifugation rather than sequence analysis. In this paper, we investigate the compositional properties of coding sequences from a broad range of vertebrate poikilotherms using DNA sequence analysis. We find that on average poikilotherms have lower third-codon position GC contents (GC3) than homeotherms but that some poikilotherms have higher mean GC3 values. We find that most poikilotherms have lower variation in GC3 than homeotherms but that there is a correlation between GC12 and GC3 for some species, indicating that there is systematic variation in base composition across their genomes. We also demonstrate that the GC3 of genes in the zebrafish, Danio rerio, is correlated with that in humans, suggesting that vertebrates share a basic isochore structure. However, we find no correlation between either the mean GC3 or the standard deviation in GC3 and body temperature.     

Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions

Vertebrate genomes are mosaics of isochores. On the assumption that marked differences exist in the isochore structure between warm-blooded and cold-blooded animals, variations among vertebrates were previously attributed to adaptation to homeothermy. However, based on the data of coding regions from representatives of extant vertebrates, including a turtle, a crocodile (Archosauromorpha) and a few kinds of snakes (Lepidosauromorpha), it was recently hypothesized that the common ancestors of mammals, birds and extant reptiles already had the "warm-blooded" isochore structure. To test this hypothesis, the nucleotide sequences of alpha-globin genes including non-coding regions (introns) from two snakes, N. kaouthia and E. climacophora, were determined (accession number: AB104824, AB104825). The correlation between the GC contents in the introns and exons of alpha-globin genes from snakes and those from other vertebrates supports the above hypothesis. Similar analysis using data for exons and introns of other genes obtained from the GenBank (Release 131) also support the above hypothesis.     

The isochore organization and the compositional distribution of homologous coding sequences in the nuclear genome of plants.

The isochore structure of the nuclear genome of angiosperms described by Salinas et al. (1) was confirmed by using a different experimental approach, namely by showing that the levels of coding sequences from both dicots and Gramineae are linearly correlated with GC levels of the corresponding flanking sequences. The compositional distribution of homologous coding sequences from several orders of dicots and from Gramineae were also studied and shown to mimick the compositional distributions previously seen (1) for coding sequences in general, most coding sequences from Gramineae being much higher than those of the dicots explored. These differences were even stronger for third codon positions and led to striking codon usages for many coding sequences especially in the case of Gramineae.     

Compositional patterns in vertebrate genomes: Conservation and change in evolution

Summary The evolution of vertebrate genomes can be investigated by analyzing their regional compositional patterns, namely the compositional distributions of large DNA fragments (in the 30–100-kb size range), of coding sequences, and of their different codon positions. This approach has shown the existence of two evolutionary modes. In the conservative mode, compositional patterns are maintained over long times (many million years), in spite of the accumulation of enormous numbers of base substitutions. In the transitional, or shifting, mode, compositional patterns change into new ones over much shorter times.The conservation of compositional patterns, which has been investigated in mammalian genomes, appears to be due in part to some measure of compositional conservation in the base substitution process, and in part to negative selection acting at regional (isochore) levels in the genome and eliminating deviations from a narrow range of values, presumably corresponding to optimal functional properties. On the other hand, shifts of compositional patterns, such as those that occurred between cold-blooded and warm-blooded vertebrates, appear to be due essentially to both negative and positive selection again operating at the isochore level, largely under the influence of changes in environmental conditions, and possibly taking advantage of mutational biases in the replication/repair enzymes and/or in the enzyme make-up of nucleotide precursor pools. Other events (like translocations and changes in chromosomal structure) also play a role in the transitional mode of genome evolution.The present findings (1) indicate that isochores, which correspond to the DNA segments of individual or contiguous chromatin domains, represent selection units in the vertebrate genome; and (2) shed new light on the selectionist-neutralist controversy.This work was presented at the EMBO Workshop on Evolution (Cambridge, UK, 4–6 July 1988) and at the 16th International Congress of Genetics (Toronto, Canada, 20–27 August 1988)     

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.     

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     

Compositional patterns in reptilian genomes

Sauropsids form a complex group of vertebrates including squamates (lizards and snakes), turtles, crocodiles, sphenodon and birds (which are often considered as a separate class). Although avian genomes have been relatively well studied, the genomes of the other groups have remained only sparsely characterized. Moreover, the nuclear sequences available in databanks are still very limited. In the present study, we have analysed the compositional patterns, i.e. the GC (molar fraction of guanine and cytosine in DNA) distributions, of 31 reptilian (particularly snake) genomes by analytical ultracentrifugation of DNAs in CsCl gradients. The profiles were characterized by their modal buoyant density rho(o), mean buoyant density < rho>, asymmetry < rho>- rho(o), and heterogeneity H. The modal buoyant density distribution of reptilian DNAs clearly distinguishes two groups. The snakes fall in the same range of modal densities as most mammals, whereas crocodiles, turtles and lizards show higher values (>1.700 g/cm(3)). As far as the more important compositional properties of asymmetry and heterogeneity are concerned, previous studies showed that amphibians and fishes share relatively low values, whereas birds and mammals are characterized by highly heterogeneous and asymmetric patterns (with the exception of Muridae, which have a lower heterogeneity). The present results show that the snake genomes cover a broad range of asymmetry and heterogeneity values, whereas the genomes of crocodiles and turtles cover a narrow range that is intermediate between those of fishes/amphibians and those of mammals/birds.     

Localization of DNA Sequences Tightly Associated with the Synaptonemal Complex in Compositional Fractions of the Golden Hamster Genome*

Synaptonemal complex (SC) isolated from spermatocyte nuclei after their exhaustive hydrolysis by DNase II contains DNA sequences tightly associated with it (SCAR DNA). Here, the compositional properties of a cloned family of golden hamster SCAR DNA were studied. For this purpose, 27 SCAR DNA clones were hybridized with compositionally fractionated golden hamster genomic DNA. The sequences of the SCAR DNA family were mainly localized in the GC-poor isochore families L1 and L2, which accounted for 63% of hybridization signals. The remaining 37% of signals pertained to the GC-rich isochore families H1 and H2. Thus, SCAR DNA proved to be distributed throughout the genome, irrespective of differences in density and sequence type between isochore families. Moreover, the SCAR DNA sequences containing the regions of homology with LINE/SINE repeats were found in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that the SC and SCAR DNA participate in chromatin reorganization during meiosis prophase I, which should result in the attachment of chromatin loops to the lateral elements of SC throughout its length.