Compositional properties of coding sequences and mammalian phylogeny

The compositional distributions of large DNA fragments reflect those of the isochores that make up vertebrate genomes and can provide novel phylogenetic insights in the case of mammalian genomes (see Sabeur et al. 1993). This approach has been complemented here by an analysis of the compositional patterns of coding sequences and their codon positions (which also reflect the isochore pattern) and by a comparison of the base compositions of codon positions from homologous genes in a number of pairs of species. The results obtained using these two approaches support the existence of a general compositional pattern for mammalian genomes and of a distinct pattern for Myomorpha. The other two “special” patterns identified in a megachiropteran and in pangolin could not be tested here. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Compositional heterogeneity within and among isochores in mammalian genomes. I. CsCl and sequence analyses

GC level distributions of a species' nuclear genome, or of its compositional fractions, encode key information on structural and functional properties of the genome and on its evolution. They can be calculated either from absorbance profiles of the DNA in CsCl density gradients at sedimentation equilibrium, or by scanning long contigs of largely sequenced genomes. In the present study, we address the quantitative characterization of the compositional heterogeneity of genomes, as measured by the GC distributions of fixed-length fragments. Special attention is given to mammalian genomes, since their compartmentalization into isochores implies two levels of heterogeneity, intra-isochore (local) and inter-isochore (global). This partitioning is a natural one, since large-scale compositional properties vary much more among isochores than within them. Intra-isochore GC distributions become roughly Gaussian for long fragments, and their standard deviations decrease only slowly with increasing fragment length, unlike random sequences. This effect can be explained by 'long-range' correlations, often overlooked, that are present along isochores.     

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     

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.     

The pig genome: compositional analysis and identification of the gene-richest regions in chromosomes and nuclei

The isochore organization of the mammalian genome comprises a general pattern and some special patterns, the former being characterized by a wider compositional distribution of the DNA fragments. The large majority of the mammalian genomes belong to the former, and only some groups, such as the Myomorpha sub-order of Rodentia, belong to the latter. Here we describe the compositional organization of the pig (Sus scrofa) genome that belongs to the general mammalian pattern. We investigated (i) the compositional distribution of the genes by analysis of their GC3 levels (the GC levels at the third codon positions), and (ii) the correlation between the GC3 value of orthologous genes from pig and other vertebrates (human, calf, mouse, chicken, and Xenopus). As expected, the highest gene concentration corresponded to the H3 isochore family, and the highest GC3 correlations were observed in the pig/human and pig/calf comparisons. Then we identified, by in situ hybridization of the GC-richest H3 isochores, the pig chromosomal regions endowed by the highest gene-density that largely corresponded to the telomeric chromosomal bands. Moreover, we observed that these gene-rich bands are syntenic with the previously identified GC-richest/gene richest H3+ bands of the human chromosomes. At the cell nucleus level, we observed that the gene-dense region corresponded to the more internal compartment, as previously found in human and avian cell nuclei.     

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     

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.     

TUCAN/CARDINAL and DRAL participate in a common pathway for modulation of NF-kappaB activation

Prior to genome sequencing, information on base composition (GC level) and its variation in mammalian genomes could be obtained using density gradient ultracentrifugation. Analyses using this approach led to the conclusion that mammalian genomes are organized into mosaics of fairly homogeneous regions, called isochores. We present an initial compositional overview of the chromosomes of the recently available draft human genome sequence, in the form of color-coded moving window plots and corresponding GC level histograms. Results obtained from the draft human genome sequence agree well with those obtained or deduced earlier from CsCl experiments. The draft sequence now permits the visualization of the mosaic organization of the human genome at the DNA sequence level.     

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.     

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.     

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.     

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 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.     

Evidence of distinct gene functional patterns in GC-poor and GC-rich isochores in Bos taurus

Vertebrate genomes are mosaics of megabase-size DNA segments with a fairly homogeneous base composition, called isochores. They are divided into five families characterized by different guanine-cytosine (GC) levels and linked to several functional and structural properties. The increased availability of fully sequenced genomes allows the investigation of isochores in several species, assessing their level of conservation across vertebrate genomes. In this work, we characterized the isochores in Bos taurus using the ARS-UCD1.2 genome version. The comparison of our results with the well-studied human isochores and those of other mammals revealed a large conservation in isochore families, in number, average GC levels and gene density. Exceptions to the established increase in gene density with the increase in isochores (GC%) were observed for the following gene biotypes: tRNA, small nuclear RNA, small nucleolar RNA and pseudogenes that have their maximum number in H2 and H1 isochores. Subsequently, we assessed the ontology of all gene biotypes looking for functional classes that are statistically over- or under-represented in each isochore. Receptor activity and sensory perception pathways were significantly over-represented in L1 and L2 (GC-poor) isochores. This was also validated for the horse genome. Our analysis of housekeeping genes confirmed a preferential localization in GC-rich isochores, as reported in other species. Finally, we assessed the SNP distribution of a bovine high-density SNP chip across the isochores, finding a higher density in the GC-rich families, reflecting a potential bias in the chip, widely used for genetic selection and biodiversity studies.     

Compositional bimodality and evolution of retroviral genomes.

The compositional distributions of genomes, genes (and their third codon positions) and long terminal repeats from retroviruses of warm-blooded vertebrates are characterized by a striking bimodality which is accompanied by a remarkable compositional homogeneity within each retroviral genome. A first, major class of retroviral genomes is GC-rich, whereas a second, minor class is GC-poor. Representative expressed viral genomes from the two classes integrate in GC-rich and GC-poor isochores, respectively, of host genomes. The first class comprises all oncoviruses (except B-types and some D-types), the second, lentiviruses, spumaviruses, as well as B-type and some D-type oncoviruses (e.g., mouse mammary tumor virus and simian retroviruses type D, respectively). The compositional bimodal distribution of retroviral genomes and the accompanying compositional homogeneity within each retroviral genome appear to be the result of the compositional evolution of retroviral genomes in their integrated form.     

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.     

Comparative Compositional Mapping of Chicken and Quail Chromosomes

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA–DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.     

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 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)