Compositional patterns in the nuclear genome of cold-blooded vertebrates

Summary DNA preparations obtained from 122 species of fishes, 5 species of amphibians, and 13 species of reptiles were investigated in their compositional properties by analytical equilibrium centrifugation in CsCl density gradients. These species represented 21 orders of Osteichthyes, 3 orders of Chondrichthyes, 2 orders of amphibians, and 3 orders of reptiles. Modal buoyant densities of fish DNAs ranged from 1.696 to 1.707 g/cm³, the vast majority of values falling, however, between 1.699 and 1.704 g/cm³, which is the range covered by the DNAs of amphibians and reptiles. In all cases, DNA bands in CsCl were only weakly asymmetrical and only very rarely were accompanied by separate satellite bands (mostly on the GC-rich side). Intermolecular compositional heterogeneities were low in the vast majority of cases, and, like CsCl band asymmetries, at least partially due to cryptic or poorly resolved satellites. The present findings indicate, therefore, that DNAs from cold-blooded vertebrates are characterized by a number of common properties, namely a very wide spectrum of modal buoyant densities, low intermolecular compositional heterogeneities, low CsCl band asymmetries, and, in most cases, small amounts of satellite DNAs. In the case of fish DNAs a negative correlation was found between the GC level and the haploid size (c value) of the genome. If polyploidization is neglected, this phenomenon appears to be mainly due to the fact that increases and decreases in GC are associated with contraction and expansion phenomena, respectively, of intergenic noncoding sequences, which are GC poor relative to coding sequences.     

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.     

Diversity and phylogenetic implications of CsCl profiles from rodent DNAs

Buoyant density profiles of high-molecular-weight DNAs sedimented in CsCl gradients, i.e., compositional distributions of 50- to 100-kb genomic fragments, have revealed a clear difference between the murids so far studied and most other mammals, including other rodents. Sequence analyses have revealed other, related, compositional differences between murids and nonmurids. In the present study, we obtained CsCl profiles of 17 rodent species representing 13 families. The modal buoyant densities obtained for rodents span the full range of values observed in other eutherians. More remarkably, the skewness (asymmetry, mean - modal buoyant density) of the rodent profiles extends to values well below those of other eutherians. Scatterplots of these and related CsCl profile parameters show groups of rodent families that agree largely with established rodent taxonomy, in particular with the monophyly of the Geomyoidea superfamily and the position of the Dipodidae family within the Myomorpha. In contrast, while confirming and extending previously reported differences between the profiles of Myomorpha and those of other rodents, the CsCl data question a traditional hypothesis positing Gliridae within Myomorpha, as does the recently sequenced mitochondrial genome of dormouse. Analysis of CsCl profiles is presented here as a rapid, robust method for exploring rodent and other vertebrate systematics.     

An ultracentrifugation analysis of two hundred fish genomes

The goal of this study was to provide a comprehensive view of the compositional characteristics of fish genomes. We therefore expanded the number of fish species that we had explored so far in their DNAs by analytical ultracentrifugation in CsCl density gradient from 122 to 201. This study included representatives from three out of nine orders of Elasmobranchs (sharks and rays), both orders of dipnoan lungfishes, and both orders of chondrosteans (sturgeons and bichirs). We also studied 19 out of 38 teleostean orders, which represent all but four (minor) superorders of the subdivision Teleostei, a group comprising about 23,600 species (96% of all extant fishes). This leaves for further studies two subclasses, Holocephali (chimaeras), and Coelacanthimorpha (gombessas). In spite of this substantial increase in the number of species and orders analysed, all average properties (the modal buoyant density, rho(0), the average buoyant density, , the CsCl profile asymmetry, A, and the compositional heterogeneity, H), and all their ranges were unchanged compared to a previous study [J. Mol. Evol. 31 (1990) 265]. This suggests that, in all likelihood, the properties reported in the present paper can be considered as generally valid for all fish genomes.     

Buoyant densities of DNA of mammals

One characteristic of DNA, CsCl buoyant density peak values, was determined for DNA samples isolated from 93 species belonging to 11 orders of mammals. The CsCl buoyant density values varied over a very narrow range, 1.696–1.701 g/cm³. Satellite DNAs were found in a number of species. The function and origin of these satellite DNAs are not known.This work was supported by grant DRG-269 from the Damon Runyon Memorial Fund for Cancer Research, Inc., and GB-6657 from the National Science Foundation.     

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.     

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 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     

Directional fixation of mutations in vertebrate evolution

Summary We have made pairwise comparisons between the coding sequences of 21 genes from coldblooded vertebrates and 41 homologous sequences from warm-blooded vertebrates. In the case of 12 genes, GC levels were higher, especially in third codon positions, in warm-blooded vertebrates compared to cold-blooded vertebrates. Six genes showed no remarkable difference in GC level and three showed a lower level. In the first case, higher GC levels appear to be due to a directional fixation of mutations, presumably under the influence of body temperature (see Bernardi and Bernardi 1986b). These GC-richer genes of warm-blooded vertebrates were located, in all cases studied, in isochores higher in GC than those comprising the homologous genes of cold-blooded vertebrates. In the third case, increases appear to be due to a limited formation of GC-rich isochores which took place in some cold-blooded vertebrates after the divergence of warm-blooded vertebrates. The directional changes in the GC content of coding sequences and the evolutionary conservation of both increased and unchanged GC levels are in keeping with the existence of compositional constraints on the genome.     

Organization of nucleotide sequences in the chicken genome

The four major components of chicken DNA were prepared by density gradient centrifugation and characterized in several basic properties: relative amounts, dG + dC content, buoyant densities, compositional heterogeneity, and reassociation kinetics. While the relative amounts and the compositions of the major components of chicken DNA were similar to those found in mammalian genomes, their compositional heterogeneities were found to be narrower. The relative amounts of interspersed repeated and unique sequences were strikingly different in different components and also different from those found in the corresponding major components of mouse and human DNAs. If one takes into consideration that major DNA components (a) account for practically all of main-band DNA and (b) derive by preparative breakage from very long DNA segments of fairly homogeneous composition, the isochores, our findings indicate that the distribution of interspersed repeats is different in different chromosomal regions and is species-specific.     

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs₂SO₄/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes. A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively. The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm³ in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores. The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.     

Using analytical ultracentrifugation to study compositional variation in vertebrate genomes

Although much attention has recently been directed to analytical ultracentrifugation (AUC), the revival of interest has hardly addressed the applications of this technology in genome analysis, and the extent to which AUC studies can quickly and effectively complement modern sequence-based analyses of genomes, e.g. by anticipating, extending or checking results that can be obtained by cloning and sequencing. In particular, AUC yields a quick overview of the base compositional structure of a species' genome even if no DNA sequences are available and the species is unlikely to be sequenced in the near future. The link between AUC and DNA sequences dates back to 1959, when a precise linear relation was discovered between the GC (guanine+cytosine) level of DNA fragments and their buoyant density in CsCl as measured at sedimentation equilibrium. A 24-hour AUC run of a high molecular weight sample of a species' total DNA already yields the GC distribution of its genome. AUC methods based on this principle remain sensitive tools in the age of genomics, and can now be fine-tuned by comparing CsCl absorbance profiles with the corresponding sequence histograms. The CsCl profiles of vertebrates allow insight into structural and functional properties that correlate with base composition, and their changes during vertebrate evolution can be monitored by comparing CsCl profiles of different taxa. Such comparisons also allow consistency checks of phylogenetic hypotheses at different taxonomic levels. We here discuss some of the information that can be deduced from CsCl profiles, with emphasis on mammalian DNAs.     

Density gradient analysis of newly replicated DNA from synchronized mouse lymphoma cells

The replication of DNA in synchronous cultures of mouse lymphoma cells was investigated by use of CsCl density gradient centrifugation. We found that the buoyant density of newly replicated DNA depended upon the particular stage of S phase in which synthesis occurred. In early S phase, newly replicated DNA exhibited buoyant densities which were slightly higher, on the average, than that of pre-existing DNA. As S phase progressed, newly replicated DNA shifted to lower buoyant densities, until, near the end of S phase, densities less than pre-existing DNA were observed. These observations are discussed in terms of their possible relevance to base compositional differences between nucleotide sequences made in early as opposed to middle or late S phase.     

Different hydrophobicities of orthologous proteins from Xenopus and human

A compositional transition was previously detected by comparing orthologous coding sequences from cold- and warm-blooded vertebrates (see Bernardi, G., Hughes, S., Mouchiroud, D., 1997. The major compositional transitions in the vertebrate genome. J. Mol. Evol. 44, S44-S51 for a review). The transition is characterized by higher GC levels (GC is the molar ratio of guanine+cytosine in DNA) and, especially, by higher GC3 levels (GC3 is the GC level of third codon positions) in coding sequences from warm-blooded vertebrates. This transition essentially affects GC-rich genes, although the nucleotide substitution rate is of the same order of magnitude in both GC-poor and GC-rich genes. In order to understand the evolutionary basis of the changes, we have compared the hydrophobicity of orthologous proteins from Xenopus and human. Although the differences are small in proteins encoded by coding sequences ranging from 0 to 65% in GC3, they are large in the proteins encoded by sequences characterized by GC3 values higher than 65%. The latter proteins are more hydrophobic in human than in Xenopus.     

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 major shifts of human duplicated genes

Since many gene duplications in the human genome are ancient duplications going back to the origin of vertebrates, the question may be asked about the fate of such duplicated genes at the compositional genome transitions that occurred between cold- and warm-blooded vertebrates. Indeed, at that transition, about half of the (GC-poor) genes of cold-blooded vertebrates (the genes of the gene-dense "ancestral genome core") underwent a GC enrichment to become the genes of the "genome core" of warm-blooded vertebrates. Since the compositional distribution of the human duplicated genes investigated (1111 pairs) mimics the general distribution of human genes (about 50% GC(3)-poor and 50% GC(3)-rich genes, the border being at 60% GC(3)), we considered two possibilities, namely that the compositional transition affected either (i) about half of the copies on a random basis, or (ii) preferentially only one copy of the duplicated genes. The two possibilities could be distinguished if each copy is put into one of two subsets according to its GC(3) level. Indeed, in the first case, the two distributions would be similar, whereas in the second case, the two distributions would be different, one copy having maintained the ancestral GC-poor composition, and one copy having undergone the compositional change. Using this approach, we could show that, by far and large, one copy of the duplicated genes preferentially underwent the GC enrichment. This result implies that this copy, which had possibly acquired a different function and/or regulation, was preferentially translocated into the gene-dense compartment of the genome, the "ancestral genome core", namely the "gene space" which underwent the compositional transition at the emergence of warm-blooded vertebrates.     

The fractionation of calf thymus DNA by multiple formation of sedimentable complexes with homologous lysine-rich histone KAP. Derivative melting curves and ultracentrifugation in CsCl concentration gradients

The process of fractionation of total calf thymus DNA using a step precipitation of DNA by means of increasing concentrations of the homologous histone KAP was investigated. In addition to the known fractions three so far undescribed ones/in thymus/,characterized by buoyant densities in CsCl equal 1.692, 1.706 and 1.728 g/ccm, were identified. Considerable amounts of preparations seriously enriched in individual satellite fractions were obtained. The ability of GC-rich satellite DNAs to form more soluble complexes with histone KAP is suggested as reason for the observed fractionation.     

Recombination in yeast mitochondrial DNA

When haploid yeast strains containing mitochondrial DNAs (mtDNAs) of different buoyant densities are mated, the resulting zygotes contain a mixed population of mitochondria and mitochondrial DNAs. During vegetative growth of diploid cells formed from such a cross between a petite strain with mtDNA of density 1.677 g cm^?3 and a respiratory competent strain with mtDNA of density 1.684 g cm^?3, mtDNAs with intermediate buoyant densities are obtained. Virtually all newly synthesized mtDNA in diploid ρ^? progeny has the intermediate buoyant density. Therefore, within 2 generations of growth of the diploid cells, the intermediate buoyant density species predominate. In crosses between a respiratory competent strain and other petite strains with different values of genetic suppressiveness, it was found that the amount of recombination yielding mtDNAs of intermediate buoyant densities roughly parallels the degree of suppressiveness. Individual clones of respiratory deficient cells from such crosses were also isolated to confirm that stable mtDNAs with intermediate buoyant densities were obtained. Thus, it is apparent that some form of recombination takes place within the mtDNAs of yeast cells that results in stable mtDNA species.     

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.