BACKGROUND
investigations carried out in our laboratory over many years led to a general picture of the organization of the vertebrate genome and its evolution.
we recall here very briefly that the vertebrate genome is a mosaic of isochores, typically megabase-size dna segments that belong in a small number of families characterized by different gc levels, and that are tightly associated with basic genome properties such as gene density, gene expression, replication timing and recombination .
remarkably, the isochore family patterns  of mammals and birds were found to be strikingly different from those of amphibians and fishes.
most of our previous results were obtained through a compositional approach that mainly involved  dna fractionation using ultracentrifugation in density gradients in the presence of sequence-specific ligands;  cytogenetic analyses; and  gene and genome sequences, when this became possible.
the recent availability of a number of fully sequenced vertebrate genomes allowed us to quantify very precisely our previous results.
this approach was started by scanning gc levels  <cit>  in the first fully sequenced vertebrate genome, the human genome .
the number-average size of compositionally fairly homogeneous regions, the isochores, was found to be  <dig>  mb , the weight-average  <dig>  mb .
when isochores were pooled in bins of 1% gc, their distribution confirmed that they belonged in the five families that had been previously described   <cit> .
the euchromatic regions of human chromosomes were completely covered by  <dig> isochores that formed the ultimate chromosomal bands  <cit> .
in contrast, the genomes of the cold-blooded vertebrates  explored so far at the sequence level expectedly showed a much lower compositional heterogeneity and were characterized by less complex isochore patterns  <cit> .
we used here, as in previous papers  <cit> , the old-fashioned distinction between cold- and warm-blooded vertebrates in order to stress the link that we proposed to exist between genome structure  and body temperature , a point clearly made in our previous work .
in the present work we analyzed at the sequence level the genomes of eutherians not yet explored by us, namely chimpanzee , mouse  and dog , a marsupial, the opossum  and a monotreme, the platypus .
some comparative data from a reptile  and an amphibian  are also presented, even if these genome sequences are only available as scaffolds.
since we had previously investigated the fully sequenced human, fish and chicken genomes  <cit> , we could approach here the general problem of the organization and evolution of vertebrate genomes at the sequence level.
RESULTS
the isochore families: the patterns
when isochores from vertebrate genomes are pooled in bins of 1% gc  <cit> , or  <dig> % gc as in the present work, and plotted against their gc levels, isochore families appear, as expected from previous investigations.
a comparison of the isochore patterns of vertebrate genomes involves the assessment of the relative amounts of the isochore families and of their average gc levels.
these are reported below.
as expected, two primates  and a carnivore  showed a large similarity in the relative amounts of the isochore families, whereas in mouse l <dig> isochores were poorly represented and h <dig> isochores were essentially absent .
these values concern only a small part of h <dig>
they were not included in calculating the standard deviation in a.
values in parenthesis are likely to be at least in part artefactually large.
in opossum, l <dig> isochores were much more represented than in eutherians and gc-rich isochores h <dig> and h <dig> were very scarce .
this pattern might be due to interspersed repeats that represent about 50% of this genome.
this possibility was disproven, however, by two findings:  repeats were distributed over all isochore families with only a slightly higher concentration in gc-poor families; and  the base composition of repeats was quite close to those of "unique" contiguous sequences .
in contrast to the genomes of eutherians and chicken , which showed an average gc level of about 41%, and to the gc-poorer genome of opossum , the platypus genome, which has a size of approximately  <dig>  gb  showed a high gc level of  <dig> %.
this genome essentially consisted of l <dig> and h <dig> isochores with a small amount of h <dig> isochores , a result due in part to the missing assembly of gc-rich microchromosomes.
indeed, when the gc profile of the unassembled sequences was superimposed on the isochore profile of the platypus genome , it became clear that the unassembled parts essentially corresponded to gc-rich chromosomal regions .
in the chicken genome , which has a size about 1/ <dig> of the human genome, all isochore families were very slightly shifted toward gc-rich values compared to the human distribution.
moreover, l <dig> isochores were underrepresented in the genome and a gc-richest h <dig> isochore family was present, even if in very small amounts in the currently available assembly.
this data still lacks some microchromosomes, all of which are known to be very gc-rich  <cit> .
the isochore families of the fully sequenced fish genomes  were already described  <cit> .
in each of the four genome sequences only two isochore families were present , or predominant .
unfortunately, only scaffolds were available for the genomes of a reptile  and of an amphibian .
when- <dig> kb segments of these scaffolds were binned and compared with similar histograms for the human and medaka  genomes, the compositional heterogeneities of the anolis and xenopus genomes were found to be much lower than that of the human genome and rather close to that of the genome of medaka, the compositionally closest fish genome .
the isochore families: the gc levels and the dinucleotide frequencies
in spite of the different relative amounts of isochore families found within and among vertebrate classes , the average gc levels of isochores belonging to the different families were remarkably conserved .
because of their possible functional relevance in connection with chromatin structure  <cit> , dinucleotide frequencies were also assessed as observed/expected ratios in different isochore families.
these ratios were extremely close between human, mouse, opossum and platypus in each of the isochore families , whereas the ratios of anolis and xenopus  diverged slightly from the ratios seen in mammals.
in the human/fish comparison , practically no differences were found in aa, tt, at and ta, but cpg was higher in fish dna than in human, as expected from the lower body temperature of fishes  <cit> .
the isochore sizes
the average size of isochores in the different families showed a remarkable conservation in all vertebrates, from fish to human, again in spite of the differences in the relative amounts of isochore families .
this stability of isochore size within isochore families was accompanied, however, by systematic differences between isochore families, in particular  a larger size  and a larger spread of the gc-poorest isochore families ;  a smaller size  and a narrower size distribution of the gc-rich isochores; and  a regular decrease from l <dig> to h <dig> isochore families.
additional files  <dig>   <dig>   <dig>   <dig> and  <dig>  present the coordinates, sizes, gc levels and differences in gc between isochores for the genomes of chimpanzee, dog, mouse, opossum and platypus.
additional files  <dig>   <dig>   <dig>   <dig> and  <dig> display the corresponding gc profiles.
gene densities
the gene densities of all isochore families  showed an increase with increasing gc in both warm- and cold-blooded vertebrates, as expected from previous results .
in the case of xenopus, genes were localized on the scaffolds and gene densities were shown to follow the general trend of all vertebrates .
the only exception to this general rule was that of zebrafish, in which case the most represented l <dig> family had a slightly higher gene density compared to l <dig> isochores.
the reasons for such a situation are under current investigation.
gene density was not calculated in the case of anolis because the coordinates of genes on the scaffolds are not yet available.
discussion
the two modes of genome evolution: the transitional mode
it should be recalled that a transitional  mode in genome evolution was originally indicated by the gaussian analysis of buoyant density profiles of dna  from cold- and warm-blooded vertebrates  <cit> .
in this mode, large changes occurr in isochore patterns.
more specifically, gc-rich isochores were found to be absent or scarce in the carp and xenopus genome, respectively, compared to the genomes of human, mouse and chicken  <cit> , and similar differences were seen in orthologous genes  <cit> .
interestingly, the transitional mode of the genome evolution could also be observed at the cytogenetic level because the compositional heterogeneity of genomes is reflected in the chromosomal banding patterns .
these findings indicate the existence of correlations linking compositional heterogeneity, chromatin structure and banding patterns.
the transitional mode of evolution could now be checked on several vertebrate genomes at the sequence level with a much higher degree of precision.
the compositional differences of the genomes of human and xenopus were originally attributed to the different body temperature of warm- and cold-blooded vertebrates  <cit> .
this "thermodynamic stability hypothesis" accounted for the higher gc level of dna and rna.
moreover, it was noted that gc-rich codons preferentially encode aminoacids that confer thermal stability to the corresponding proteins.
this latter point was recently confirmed by showing  <cit>  that, out of  <dig>  human genes, those located in gc-rich isochores have an increased level of gc-rich codons leading to higher levels of stabilizing aminoacids  and lower levels of destabilizing aminoacids .
expectedly, the opposite was found in genes located in gc-poor isochores.
the thermodynamic stability hypothesis is now supported by several new findings  the isochore patterns of anolis, xenopus and fishes  below) lack the gc-richest isochores present in the human pattern;  the predominant gc-poor isochores of opossum might be related, at least in part, to the lower body temperature  of this marsupial;  the small shift to the gc-rich side of the isochore distribution of chicken and the presence of a small gc-richest h <dig> isochore family might be related to the higher body temperature   <cit>  of birds compared to mammals;  the shift to the gc-rich side of the tetraodon genome, a fish living in tropical freshwater contrasts with the isochore pattern of fugu, a fish  living at a lower temperature in the pacific ocean  <cit>  ;  the isochore patterns of reptiles, a class of vertebrates known to be characterized by different body temperatures and different thermal regulations cover a broad spectrum; indeed, genomes may either be even more compositionally homogeneous than the xenopus genome , or show the presence of gc-rich isochores ; the latter point was recently confirmed by comparing gc <dig>  of orthologous genes from alligator mississippiensis, human and chicken  <cit> ;  both mammals and birds, two classes of vertebrates derived at different times from different ancestral reptiles , showed the formation of the same families of gc-rich isochores , a clear indication of a convergent compositional evolution; likewise, a convergent evolution may be the explanation for the similarity of gc <dig> values of orthologous genes from alligator and chicken; indeed, there is no compelling reason to consider common descent from archosaurs as the explanation  <cit> , given the large phylogenetic distance  <cit> , the complex endo-ectothermic evolution of crocodiles  <cit> , and the contrasting data on the cold- or warm-bloodedness of the immediate ancestors of birds, dinosaurs;  the excess of at → gc over gc → at changes observed in the genes of gillichthys seta, a fish living at 40°c, compared to the orthologous genes of gillicthys mirabilis, a congeneric fish living at 20°c  <cit> ; interestingly, the former one was characterized by positive selection on some genes and by an expansion of a gc-rich minisatellites in gene-rich regions.
the explanation why only the gene-rich regions of the genome and not the whole genomes underwent a gc increase was provided by the finding that those regions have an open chromatin structure , whereas the gene-poor regions could be stabilized by their own compact chromatin.
this point is supported by the finding that, when the body temperature change is very rapid, as in the case of the divergence of g. seta from g. mirabilis , the gene-rich regions of the genome are stabilized by the regional expansion of a very gc-rich minisatellite .
since the thermal stability hypothesis is based on general physical-chemical properties, it would be expected to be valid very widely.
this is, indeed, the case as shown by the correlation of gc levels of paired sequences  of ribosomal 18s rnas with body temperature for vertebrates ranging from mammals to polar fishes  and by the correlation of gc levels and optimal growth temperatures of prokaryotes  <cit> .
while body temperature seems to be a major determinant of the compositional properties of genome, other factors may also play a role.
this is clearly indicated by the different isochore patterns of fishes.
in this case not only temperature, but other environmental factors such as salinity, oxygen level, ph etc.
are possibly involved.
the compositional differences found between eutherians and monotremes that have different body temperatures  require further investigations to be understood.
we know, however, that cpg and  <dig> mc values of monotremes are intermediate between the low values of eutherians and the high values of fishes  , as expected from their body temperature.
it should be stressed that, while the original observations pointed to a shifting mode of genome evolution in the case of the compositional transition between cold- and warm-blooded vertebrates, which is now confirmed on a sequence basis, the present results indicate the existence of a shifting mode even within cold-  and warm-blooded vertebrates .
the two modes of genome of genome evolution: the conservative mode
the other, conservative, mode, in which the isochore patterns are maintained over evolutionary time, was found in eutherian genomes that displayed the "general compositional pattern" .
some differences in the relative amounts of isochore families were observed, but they were within narrow limits and appeared to be essentially due to differences in the relative amounts of interspersed sequences, as well as to insertions/deletions.
moreover, when isochores from mhc loci of human and mouse  <cit> , or from synthenic chromosome regions of human and dog were examined , a high degree of conservation was found.
incidentally, the narrower isochore pattern of mouse was interpreted as due to an increased mutation rate  <cit>  and a poor repair mechanism  <cit> , two phenomena leading to some decrease of compositional heterogeneity.
the conservative mode was found in the present work to be further characterized by two remarkable properties that concerned the conservation in each isochore family of all vertebrate genomes investigated of  the average isochore size ; and  the gc levels and dinucleotide frequencies.
the conservation of the average isochore size may be correlated with the isochore role in chromosome organization.
indeed, it should be recalled here that the number of isochores estimated by us for the human genome, ~ <dig>  is in agreement with the maximum number,  <dig>  of the highest resolution bands as assessed by yunis et al.
<cit>  and that the boundaries of isochores coincide with those of chromosomal bands as obtained at the resolution of  <dig> bands .
moreover, isochores have been observed to coincide with replication units  <cit> .
as far as the larger size of the gc-poorest isochore families of vertebrates is concerned, this may be due to the preferred insertion in these families of interspersed repeated sequences, as well as to sequence expansion phenomena  <cit> .
unfortunately, the presence of gaps  or their surprising absence  may also contribute a possibly important artefactual component to the large size of gc-poor isochores  <cit> .
this implies that more complete sequence data will be needed in order to obtain reliable assessments of the gc-poorest isochore size of medaka, stickleback .
the conservation of gc level and dinucleotide frequencies of isochore families can be understood by recalling that these frequencies were consistently different in the different isochore families from the human genome  <cit> .
such differences are likely to influence protein/dna interactions and, therefore, chromatin structure, possibly through nucleosome positioning  <cit> .
in turn, the existence of five isochore families suggested that a discrete number of chromatin structures are present in eutherian mammals.
the different dnase accessibility of chromatin corresponding to isochores from different families  <cit>  may be viewed as an indication along this line.
the conservative mode of evolution was originally explained by "negative selection acting at a regional  level to eliminate any strong deviation from the presumably functionally optimal composition of isochores"  <cit> .
a number of findings, accumulated during the past twenty years  <cit>  and those presented in this paper, support this hypothesis.
an alternative proposal for the formation and maintenance of isochores was that "biased gene conversion  is probably the most likely cause of isochores"  <cit> .
this proposal has found a large number of supporters  do not show the formation of gc-rich isochores and just show a conservative mode of evolution.
the third problem is the lack of evidence, or even of models and hypotheses, concerning the expansion process from the rare, small-size bgc events   <cit>  to megabase regions.
in other words, if isochores were originating from bgc events, one should not expect the conservation of gc levels, sizes and  of the relative amounts of isochore families, nor the very high similarity of gc and gc <dig> levels in orthologous genes from eutherians and birds.
instead, one should see differences in compositional patterns, and such differences should concern individual classes, orders and families of vertebrates.
CONCLUSIONS
the present results reinforce our previous conclusions  concerning the mosaic organization of isochores in vertebrate genomes, the differences between the isochore patterns of warm- and cold-blooded vertebrates, the distribution of genes and the two modes  of genome evolution.
expectedly, the sequence level of the present data provide much more detailed pictures.
in particular, they lead for the first time to the discovery that gc levels, dinucleotide frequencies  and isochore sizes from corresponding isochore families are conserved in all vertebrate genomes.
these novel findings are not compatible with bgc as an explanation for the origin and conservation of isochores.
this leaves the original proposal of natural selection  <cit>  as the most plausible explanation for the origin and the maintenance of isochores, that represent, indeed, "a fundamental level of genome organization"  <cit> .

 investigations carried out in our laboratory over many years led to a general picture of the organization of the vertebrate genome and its evolution. we recall here very briefly that the vertebrate genome is a mosaic of isochores, typically megabase-size dna segments that belong in a small number of families characterized by different gc levels, and that are tightly associated with basic genome properties such as gene density, gene expression, replication timing and recombination. remarkably, the isochore family patterns of mammals and birds were found to be strikingly different from those of amphibians and fishes. most of our previous results were obtained through a compositional approach that mainly involved dna fractionation using ultracentrifugation in density gradients in the presence of sequence-specific ligands; cytogenetic analyses; and gene and genome sequences, when this became possible. the recent availability of a number of fully sequenced vertebrate genomes allowed us to quantify very precisely our previous results. this approach was started by scanning gc levels in the first fully sequenced vertebrate genome, the human genome. the number-average size of compositionally fairly homogeneous regions, the isochores, was found to be mb, the weight-average mb. when isochores were pooled in bins of 1% gc, their distribution confirmed that they belonged in the five families that had been previously described. the euchromatic regions of human chromosomes were completely covered by isochores that formed the ultimate chromosomal bands. in contrast, the genomes of the cold-blooded vertebrates explored so far at the sequence level expectedly showed a much lower compositional heterogeneity and were characterized by less complex isochore patterns. we used here, as in previous papers, the old-fashioned distinction between cold- and warm-blooded vertebrates in order to stress the link that we proposed to exist between genome structure and body temperature, a point clearly made in our previous work. in the present work we analyzed at the sequence level the genomes of eutherians not yet explored by us, namely chimpanzee, mouse and dog, a marsupial, the opossum and a monotreme, the platypus. some comparative data from a reptile and an amphibian are also presented, even if these genome sequences are only available as scaffolds. since we had previously investigated the fully sequenced human, fish and chicken genomes, we could approach here the general problem of the organization and evolution of vertebrate genomes at the sequence level.  the isochore families: the patterns when isochores from vertebrate genomes are pooled in bins of 1% gc, or % gc as in the present work, and plotted against their gc levels, isochore families appear, as expected from previous investigations. a comparison of the isochore patterns of vertebrate genomes involves the assessment of the relative amounts of the isochore families and of their average gc levels. these are reported below. as expected, two primates and a carnivore showed a large similarity in the relative amounts of the isochore families, whereas in mouse l isochores were poorly represented and h isochores were essentially absent. these values concern only a small part of h they were not included in calculating the standard deviation in a. values in parenthesis are likely to be at least in part artefactually large. in opossum, l isochores were much more represented than in eutherians and gc-rich isochores h and h were very scarce. this pattern might be due to interspersed repeats that represent about 50% of this genome. this possibility was disproven, however, by two findings: repeats were distributed over all isochore families with only a slightly higher concentration in gc-poor families; and the base composition of repeats was quite close to those of "unique" contiguous sequences. in contrast to the genomes of eutherians and chicken, which showed an average gc level of about 41%, and to the gc-poorer genome of opossum, the platypus genome, which has a size of approximately gb showed a high gc level of %. this genome essentially consisted of l and h isochores with a small amount of h isochores, a result due in part to the missing assembly of gc-rich microchromosomes. indeed, when the gc profile of the unassembled sequences was superimposed on the isochore profile of the platypus genome, it became clear that the unassembled parts essentially corresponded to gc-rich chromosomal regions. in the chicken genome, which has a size about 1/ of the human genome, all isochore families were very slightly shifted toward gc-rich values compared to the human distribution. moreover, l isochores were underrepresented in the genome and a gc-richest h isochore family was present, even if in very small amounts in the currently available assembly. this data still lacks some microchromosomes, all of which are known to be very gc-rich. the isochore families of the fully sequenced fish genomes were already described. in each of the four genome sequences only two isochore families were present, or predominant. unfortunately, only scaffolds were available for the genomes of a reptile and of an amphibian. when- kb segments of these scaffolds were binned and compared with similar histograms for the human and medaka genomes, the compositional heterogeneities of the anolis and xenopus genomes were found to be much lower than that of the human genome and rather close to that of the genome of medaka, the compositionally closest fish genome. the isochore families: the gc levels and the dinucleotide frequencies in spite of the different relative amounts of isochore families found within and among vertebrate classes, the average gc levels of isochores belonging to the different families were remarkably conserved. because of their possible functional relevance in connection with chromatin structure, dinucleotide frequencies were also assessed as observed/expected ratios in different isochore families. these ratios were extremely close between human, mouse, opossum and platypus in each of the isochore families, whereas the ratios of anolis and xenopus diverged slightly from the ratios seen in mammals. in the human/fish comparison, practically no differences were found in aa, tt, at and ta, but cpg was higher in fish dna than in human, as expected from the lower body temperature of fishes. the isochore sizes the average size of isochores in the different families showed a remarkable conservation in all vertebrates, from fish to human, again in spite of the differences in the relative amounts of isochore families. this stability of isochore size within isochore families was accompanied, however, by systematic differences between isochore families, in particular a larger size and a larger spread of the gc-poorest isochore families; a smaller size and a narrower size distribution of the gc-rich isochores; and a regular decrease from l to h isochore families. additional files and present the coordinates, sizes, gc levels and differences in gc between isochores for the genomes of chimpanzee, dog, mouse, opossum and platypus. additional files and display the corresponding gc profiles. gene densities the gene densities of all isochore families showed an increase with increasing gc in both warm- and cold-blooded vertebrates, as expected from previous results. in the case of xenopus, genes were localized on the scaffolds and gene densities were shown to follow the general trend of all vertebrates. the only exception to this general rule was that of zebrafish, in which case the most represented l family had a slightly higher gene density compared to l isochores. the reasons for such a situation are under current investigation. gene density was not calculated in the case of anolis because the coordinates of genes on the scaffolds are not yet available. discussion the two modes of genome evolution: the transitional mode it should be recalled that a transitional mode in genome evolution was originally indicated by the gaussian analysis of buoyant density profiles of dna from cold- and warm-blooded vertebrates. in this mode, large changes occurr in isochore patterns. more specifically, gc-rich isochores were found to be absent or scarce in the carp and xenopus genome, respectively, compared to the genomes of human, mouse and chicken, and similar differences were seen in orthologous genes. interestingly, the transitional mode of the genome evolution could also be observed at the cytogenetic level because the compositional heterogeneity of genomes is reflected in the chromosomal banding patterns. these findings indicate the existence of correlations linking compositional heterogeneity, chromatin structure and banding patterns. the transitional mode of evolution could now be checked on several vertebrate genomes at the sequence level with a much higher degree of precision. the compositional differences of the genomes of human and xenopus were originally attributed to the different body temperature of warm- and cold-blooded vertebrates. this "thermodynamic stability hypothesis" accounted for the higher gc level of dna and rna. moreover, it was noted that gc-rich codons preferentially encode aminoacids that confer thermal stability to the corresponding proteins. this latter point was recently confirmed by showing that, out of human genes, those located in gc-rich isochores have an increased level of gc-rich codons leading to higher levels of stabilizing aminoacids and lower levels of destabilizing aminoacids. expectedly, the opposite was found in genes located in gc-poor isochores. the thermodynamic stability hypothesis is now supported by several new findings the isochore patterns of anolis, xenopus and fishes below) lack the gc-richest isochores present in the human pattern; the predominant gc-poor isochores of opossum might be related, at least in part, to the lower body temperature of this marsupial; the small shift to the gc-rich side of the isochore distribution of chicken and the presence of a small gc-richest h isochore family might be related to the higher body temperature of birds compared to mammals; the shift to the gc-rich side of the tetraodon genome, a fish living in tropical freshwater contrasts with the isochore pattern of fugu, a fish living at a lower temperature in the pacific ocean; the isochore patterns of reptiles, a class of vertebrates known to be characterized by different body temperatures and different thermal regulations cover a broad spectrum; indeed, genomes may either be even more compositionally homogeneous than the xenopus genome, or show the presence of gc-rich isochores; the latter point was recently confirmed by comparing gc of orthologous genes from alligator mississippiensis, human and chicken; both mammals and birds, two classes of vertebrates derived at different times from different ancestral reptiles, showed the formation of the same families of gc-rich isochores, a clear indication of a convergent compositional evolution; likewise, a convergent evolution may be the explanation for the similarity of gc values of orthologous genes from alligator and chicken; indeed, there is no compelling reason to consider common descent from archosaurs as the explanation, given the large phylogenetic distance, the complex endo-ectothermic evolution of crocodiles, and the contrasting data on the cold- or warm-bloodedness of the immediate ancestors of birds, dinosaurs; the excess of at → gc over gc → at changes observed in the genes of gillichthys seta, a fish living at 40°c, compared to the orthologous genes of gillicthys mirabilis, a congeneric fish living at 20°c; interestingly, the former one was characterized by positive selection on some genes and by an expansion of a gc-rich minisatellites in gene-rich regions. the explanation why only the gene-rich regions of the genome and not the whole genomes underwent a gc increase was provided by the finding that those regions have an open chromatin structure, whereas the gene-poor regions could be stabilized by their own compact chromatin. this point is supported by the finding that, when the body temperature change is very rapid, as in the case of the divergence of g. seta from g. mirabilis, the gene-rich regions of the genome are stabilized by the regional expansion of a very gc-rich minisatellite. since the thermal stability hypothesis is based on general physical-chemical properties, it would be expected to be valid very widely. this is, indeed, the case as shown by the correlation of gc levels of paired sequences of ribosomal 18s rnas with body temperature for vertebrates ranging from mammals to polar fishes and by the correlation of gc levels and optimal growth temperatures of prokaryotes. while body temperature seems to be a major determinant of the compositional properties of genome, other factors may also play a role. this is clearly indicated by the different isochore patterns of fishes. in this case not only temperature, but other environmental factors such as salinity, oxygen level, ph etc. are possibly involved. the compositional differences found between eutherians and monotremes that have different body temperatures require further investigations to be understood. we know, however, that cpg and mc values of monotremes are intermediate between the low values of eutherians and the high values of fishes, as expected from their body temperature. it should be stressed that, while the original observations pointed to a shifting mode of genome evolution in the case of the compositional transition between cold- and warm-blooded vertebrates, which is now confirmed on a sequence basis, the present results indicate the existence of a shifting mode even within cold- and warm-blooded vertebrates. the two modes of genome of genome evolution: the conservative mode the other, conservative, mode, in which the isochore patterns are maintained over evolutionary time, was found in eutherian genomes that displayed the "general compositional pattern". some differences in the relative amounts of isochore families were observed, but they were within narrow limits and appeared to be essentially due to differences in the relative amounts of interspersed sequences, as well as to insertions/deletions. moreover, when isochores from mhc loci of human and mouse, or from synthenic chromosome regions of human and dog were examined, a high degree of conservation was found. incidentally, the narrower isochore pattern of mouse was interpreted as due to an increased mutation rate and a poor repair mechanism, two phenomena leading to some decrease of compositional heterogeneity. the conservative mode was found in the present work to be further characterized by two remarkable properties that concerned the conservation in each isochore family of all vertebrate genomes investigated of the average isochore size; and the gc levels and dinucleotide frequencies. the conservation of the average isochore size may be correlated with the isochore role in chromosome organization. indeed, it should be recalled here that the number of isochores estimated by us for the human genome, ~ is in agreement with the maximum number, of the highest resolution bands as assessed by yunis et al. and that the boundaries of isochores coincide with those of chromosomal bands as obtained at the resolution of bands. moreover, isochores have been observed to coincide with replication units. as far as the larger size of the gc-poorest isochore families of vertebrates is concerned, this may be due to the preferred insertion in these families of interspersed repeated sequences, as well as to sequence expansion phenomena. unfortunately, the presence of gaps or their surprising absence may also contribute a possibly important artefactual component to the large size of gc-poor isochores. this implies that more complete sequence data will be needed in order to obtain reliable assessments of the gc-poorest isochore size of medaka, stickleback. the conservation of gc level and dinucleotide frequencies of isochore families can be understood by recalling that these frequencies were consistently different in the different isochore families from the human genome. such differences are likely to influence protein/dna interactions and, therefore, chromatin structure, possibly through nucleosome positioning. in turn, the existence of five isochore families suggested that a discrete number of chromatin structures are present in eutherian mammals. the different dnase accessibility of chromatin corresponding to isochores from different families may be viewed as an indication along this line. the conservative mode of evolution was originally explained by "negative selection acting at a regional level to eliminate any strong deviation from the presumably functionally optimal composition of isochores". a number of findings, accumulated during the past twenty years and those presented in this paper, support this hypothesis. an alternative proposal for the formation and maintenance of isochores was that "biased gene conversion is probably the most likely cause of isochores". this proposal has found a large number of supporters do not show the formation of gc-rich isochores and just show a conservative mode of evolution. the third problem is the lack of evidence, or even of models and hypotheses, concerning the expansion process from the rare, small-size bgc events to megabase regions. in other words, if isochores were originating from bgc events, one should not expect the conservation of gc levels, sizes and of the relative amounts of isochore families, nor the very high similarity of gc and gc levels in orthologous genes from eutherians and birds. instead, one should see differences in compositional patterns, and such differences should concern individual classes, orders and families of vertebrates.  the present results reinforce our previous conclusions concerning the mosaic organization of isochores in vertebrate genomes, the differences between the isochore patterns of warm- and cold-blooded vertebrates, the distribution of genes and the two modes of genome evolution. expectedly, the sequence level of the present data provide much more detailed pictures. in particular, they lead for the first time to the discovery that gc levels, dinucleotide frequencies and isochore sizes from corresponding isochore families are conserved in all vertebrate genomes. these novel findings are not compatible with bgc as an explanation for the origin and conservation of isochores. this leaves the original proposal of natural selection as the most plausible explanation for the origin and the maintenance of isochores, that represent, indeed, "a fundamental level of genome organization". 
the present availability of a number of fully sequenced genomes ranging from fishes to mammals allowed us to carry out investigations that more precisely quantified our previous conclusions; showed that the different isochore families of vertebrate genomes are largely conserved in gc levels and dinucleotide frequencies, as well as in isochore size; and isochore family patterns can be either conserved or change within both warm- and cold-blooded vertebrates. on the basis of the results presented, we propose that the large conservation of gc levels and dinucleotide frequencies may reflect the conservation of chromatin structures; the conservation of isochore size may be linked to the role played by isochores in chromosome structure and replication; the formation, the maintainance and the changes of isochore patterns are due to natural selection. previous work from our laboratory showed that vertebrate genomes are mosaics of isochores, typically megabase-size dna segments that are fairly homogeneous in base composition; isochores belong to a small number of families characterized by different gc levels; isochore family patterns are different in fishes/amphibians and mammals/birds, the latter showing gc-rich isochore families that are absent or very scarce in the former; there are two modes of genome evolution, a conservative one in which isochore patterns basically do not change, and a transitional one, in which they do change; and isochores are tightly linked to a number of basic biological properties, such as gene density, gene expression, replication timing and recombination.
 previous work from our laboratory showed that vertebrate genomes are mosaics of isochores, typically megabase-size dna segments that are fairly homogeneous in base composition; isochores belong to a small number of families characterized by different gc levels; isochore family patterns are different in fishes/amphibians and mammals/birds, the latter showing gc-rich isochore families that are absent or very scarce in the former; there are two modes of genome evolution, a conservative one in which isochore patterns basically do not change, and a transitional one, in which they do change; and isochores are tightly linked to a number of basic biological properties, such as gene density, gene expression, replication timing and recombination.  the present availability of a number of fully sequenced genomes ranging from fishes to mammals allowed us to carry out investigations that more precisely quantified our previous conclusions; showed that the different isochore families of vertebrate genomes are largely conserved in gc levels and dinucleotide frequencies, as well as in isochore size; and isochore family patterns can be either conserved or change within both warm- and cold-blooded vertebrates.  on the basis of the results presented, we propose that the large conservation of gc levels and dinucleotide frequencies may reflect the conservation of chromatin structures; the conservation of isochore size may be linked to the role played by isochores in chromosome structure and replication; the formation, the maintainance and the changes of isochore patterns are due to natural selection. 