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The organization of the genome nucleotide (AT/GC) composition in vertebrates remains poorly understood despite the numerous genome assemblies available. Particularly, the origin of the AT/GC heterogeneity in amniotes, in comparison to the homogeneity in anamniotes, is controversial. Recently, several exceptions to this dichotomy were confirmed in an ancient fish lineage with mammalian AT/GC heterogeneity. Hence, our current knowledge necessitates a reevaluation considering this fact and utilizing newly available data and tools. We analyzed fish genomes in silico with as low user input as possible to compare previous approaches to assessing genome composition. Our results revealed a disparity between previously used plots of GC% and histograms representing the authentic distribution of GC% values in genomes. Previous plots heavily reduced the range of GC% values in fish to comply with the alleged AT/GC homogeneity and AT-richness of their genomes. We illustrate how the selected sequence size influences the clustering of GC% values. Previous approaches that disregarded chromosome and genome sizes, which are about three times smaller in fish than in mammals, distorted their results and contributed to the persisting confusion about fish genome composition. Chromosome size and their transposons may drive the AT/GC heterogeneity apparent on mammalian chromosomes, whereas far less in fishes.

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., (© 2020 Stichting International Foundation for Animal Genetics.)

The recent availability of a number of fully sequenced genomes (including marine organisms) allowed to map very precisely the isochores, based on DNA sequences, confirming the results obtained before genome sequencing by the ultracentrifugation in CsCl. In fact, the analytical profile of human DNA showed that the vertebrate genome is a mosaic of isochores, typically megabase-size DNA segments that belong to a small number of families characterized by different GC levels. In this review, we will concentrate on some general genome features regarding the compositional organization from different organisms and their evolution, ranging from vertebrates to invertebrates until unicellular organisms. Since isochores are tightly linked to biological properties such as gene density, replication timing, and recombination, the new level of detail provided by the isochore map helped the understanding of genome structure, function, and evolution. All the findings reported here confirm the idea that the isochores can be considered as a "fundamental level of genome structure and organization." We stress that we do not discuss in this review the origin of isochores, which is still a matter of controversy, but we focus on well established structural and physiological aspects.

Epilepsy is a common complex disorder most frequently associated with psychiatric and neurological diseases. Massive parallel sequencing of individual or cohort genomes and exomes led the identification of several disease associated genes. We review here the candidate genes in epilepsy genetics with focus on exome and gene panel data. Together with the examination of brain expressed genes and post synaptic proteome the results show that: (1) Non-metabolic epilepsies and autism candidate genes tend to be AT-rich and (2) large transcript size and local AT-richness are characteristic features of genes involved in developmental brain disorders and synaptic functions. These results point to the preferential location of core epilepsy and autism candidate genes in late replicating, GC-poor chromosomal regions (isochores). These results indicate that the genomic alterations leading to some brain disorders are confined to responsive chromatin areas harboring brain critical genes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10-11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes--i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms--nucleosome positioning motifs--are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.

Total GC-content in the platelet phosphofructokinase gene of Zebra Finch (Taeniopygia guttata) is low (37.53±0.51%), while there are short areas (about 300 nucleotides in length) with increased GC-content overlapping its exon 4 and exon 17. GC-content in third codon positions (3GC) of those two exons is equal to 88.42 and 80.00%, respectively, while overall 3GC of the coding region is equal to 49.9%. Similar distribution of GC-content has been found in platelet phosphofructokinase genes of other birds from Passeriformes order. According to the results of phylogenetic analysis, formation of those areas with high G+C started from 91.4 to 47.1millionyears ago, since there are no such peaks of GC-content in homologous genes of other birds and reptiles. There are clusters of transcription factor binding sites in those areas with higher GC-content, as well as microRNA precursors conserved in Zebra Finch and Flycatcher genes. According to our hypothesis those intragenic isochores (intrachores) may be consequences of autonomous microRNA precursor transcription at certain period(s) of embryogenesis and gametogenesis, when the platelet phosphofructokinase gene itself is not expressed. Transcription-associated mutational pressure existing during those periods may cause the increase in rates of AT to GC mutations in those genes which are transcribed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In meiotic prophase I attached to the lateral elements of the synaptonemal complex chromatin fibrils form loops. SCARs DNA (Synaptonemal Complex Associated Regions of DNA) is a family of genomic DNA sequences tightly associated with the synaptonemal complex and lying at the loop basements. Isochore compositional fractions of the human and chicken genomes were used as 32P-labeled probes for hybridization with the SCARs DNA isolated previously from the spermatocyte nucleus of the golden hamster Mesocricetus auratus. The localization of sequences, similar to golden hamster SCARs DNA in the human and chicken genome isochores was established. It was concluded that SCARs DNA localization into isochore com- partments of examined genomes is evolutionary conservative.

Background: The genomes of multicellular eukaryotes are compartmentalized in mosaics of isochores, large and fairly homogeneous stretches of DNA that belong to a small number of families characterized by different average GC levels, by different gene concentration (that increase with GC), different chromatin structures, different replication timing in the cell cycle, and other different properties. A question raised by these basic results concerns how far back in evolution the compartmentalized organization of the eukaryotic genomes arose., Results: In the present work we approached this problem by studying the compositional organization of the genomes from the unicellular eukaryotes for which full sequences are available, the sample used being representative. The average GC levels of the genomes from unicellular eukaryotes cover an extremely wide range (19%-60% GC) and the compositional patterns of individual genomes are extremely different but all genomes tested show a compositional compartmentalization., Conclusions: The average GC range of the genomes of unicellular eukaryotes is very broad (as broad as that of prokaryotes) and individual compositional patterns cover a very broad range from very narrow to very complex. Both features are not surprising for organisms that are very far from each other both in terms of phylogenetic distances and of environmental life conditions. Most importantly, all genomes tested, a representative sample of all supergroups of unicellular eukaryotes, are compositionally compartmentalized, a major difference with prokaryotes.

The genomes of many vertebrates show a characteristic heterogeneous distribution of GC content, the so-called GC isochore structure. The origin of isochores has been explained via the mechanism of GC-biased gene conversion (gBGC). However, although the isochore structure is declining in many mammalian genomes, the heterogeneity in GC content is being reinforced in the avian genome. Despite this discrepancy, which remains unexplained, examinations of individual substitution frequencies in mammals and birds are both consistent with the gBGC model of isochore evolution. On the other hand, a negative correlation between substitution and recombination rate found in the chicken genome is inconsistent with the gBGC model. It should therefore be important to consider along with gBGC other consequences of recombination on the origin and fate of mutations, as well as to account for relationships between recombination rate and other genomic features. We therefore developed an analytical model to describe the substitution patterns found in the chicken genome, and further investigated the relationships between substitution patterns and several genomic features in a rigorous statistical framework. Our analysis indicates that GC content itself, either directly or indirectly via interrelations to other genomic features, has an impact on the substitution pattern. Further, we suggest that this phenomenon is particularly visible in avian genomes due to their unusually low rate of chromosomal evolution. Because of this, interrelations between GC content and other genomic features are being reinforced, and are as such more pronounced in avian genomes as compared with other vertebrate genomes with a less stable karyotype.

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing., Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented., Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

Several studies on adult tissues agree on the presence of a positive effect of the genomic and genic base composition on mammalian gene expression. Recent literature supports the idea that during developmental processes GC-poor genomic regions are preferentially implicated. We investigate the relationship between the compositional properties of the isochores and of the genes with their respective expression activity during developmental processes. Using RNA-seq data from two distinct developmental stages of the mouse cortex, embryonic day 18 (E18) and postnatal day 7 (P7), we established for the first time a developmental-related transcriptome map of the mouse isochores. Additionally, for each stage we estimated the correlation between isochores' GC level and their expression activity, and the genes' expression patterns for each isochore family. Our analyses add evidence supporting the idea that during development GC-poor isochores are preferentially implicated, and confirm the positive effect of genes' GC level on their expression activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There are mathematical constants that describe universal relationship between variables, and physical/chemical constants that are invariant measurements of physical quantities. In a similar spirit, we have collected a set of parameters that characterize the human genome. Some parameters have a constant value for everybody's genome, others vary within a limited range. The following nine human genome parameters are discussed here, number of bases (genome size), number of chromosomes (karyotype), number of protein-coding gene loci, number of transcription factors, guanine-cytosine (GC) content, number of GC-rich gene-rich isochores, density of polymorphic sites, number of newly generated deleterious mutations in one generation, and number of meiotic crossovers. Comparative genomics and theoretical predictions of some parameters are discussed and reviewed. This collection only represents a beginning of compiling a more comprehensive list of human genome parameters, and knowing these parameter values is an important part in understanding human evolution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The availability of fully sequenced genomes and the implementation of transcriptome technologies have increased the studies investigating the expression profiles for a variety of tissues, conditions, and species. In this study, using RNA-seq data for three distinct tissues (brain, liver, and muscle), we investigate how base composition affects mammalian gene expression, an issue of prime practical and evolutionary interest., Results: We present the transcriptome map of the mouse isochores (DNA segments with a fairly homogeneous base composition) for the three different tissues and the effects of isochores' base composition on their expression activity. Our analyses also cover the relations between the genes' expression activity and their localization in the isochore families., Conclusions: This study is the first where next-generation sequencing data are used to associate the effects of both genomic and genic compositional properties to their corresponding expression activity. Our findings confirm previous results, and further support the existence of a relationship between isochores and gene expression. This relationship corroborates that isochores are primarily a product of evolutionary adaptation rather than a simple by-product of neutral evolutionary processes.

This report shows that isochore-like structures can be found not only in warm-blooded animals, some reptiles, fishes and yeast, but also in certain archaeal species. In perfectly shaped isochore-like structures (in "protoisochores") from Sulfolobus acidocaldarius and Thermofilum pendens genomes the difference in 3GC levels between genes from different "protoisochores" is about 30%. In these archaeal species GC-poor "protoisochores" are situated near the origin of replication, while GC-rich "protoisochores" are situated near the terminus of replication. There is a strong linear dependence between position of a gene and its 3GC level in S. acidocaldarius (an average difference in 3GC per 100,000 base pairs is equal to 3.6%). Detailed analyses of nucleotide usage biases in genes from leading and lagging strands led us to the suggestion that 3GC in genes situated near terminus of replication grows due to higher rates of thymine oxidation producing T to C transitions in lagging strands., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in analysis of GC content and rates of evolution.

It is well established that changes in the phenotype depend much more on changes in gene expression than on changes in protein-coding genes, and that cis-regulatory sequences and chromatin structure are two major factors influencing gene expression. Here, we investigated these factors at the genome-wide level by focusing on the trinucleotide patterns in the 0.1- to 25-kb regions flanking the human genes that are present in the GC-poorest L1 and GC-richest H3 isochore families, the other families exhibiting intermediate patterns. We could show 1) that the trinucleotide patterns of the 25-kb gene-flanking regions are representative of the very different patterns already reported for the whole isochores from the L1 and H3 families and, expectedly, identical in upstream and downstream locations; 2) that the patterns of the 0.1- to 0.5-kb regions in the L1 and H3 isochores are remarkably more divergent and more specific when compared with those of the 25-kb regions, as well as different in the upstream and downstream locations; and 3) that these patterns fade into the 25-kb patterns around 5kb in both upstream and downstream locations. The 25-kb findings indicate differences in nucleosome positioning and density in different isochore families, those of the 0.1- to 0.5-kb sequences indicate differences in the transcription factors that bind upstream and downstream of genes. These results indicate differences in the regulation of genes located in different isochore families, a point of functional and evolutionary relevance.

Isochores are large regions of relatively homogeneous nucleotide composition and are present in the genomes of all mammals and birds that have been sequenced to date. The newly sequenced genome of Anolis carolinensis provides the first opportunity to quantify isochore structure in a nonavian reptile. We find Anolis to have the most compositionally homogeneous genome of all amniotes sequenced thus far, a homogeneity exceeding that for the frog Xenopus. Based on a Bayesian algorithm, Anolis has smaller and less GC-rich isochores compared with human and chicken. Correlates generally associated with GC-rich isochores, including shorter introns and higher gene density, have all but disappeared from the Anolis genome. Using genic GC as a proxy for isochore structure so as to compare with other vertebrates, we found that GC content has substantially decreased in the lineage leading to Anolis since diverging from the common ancestor of Reptilia ∼275 Ma, perhaps reflecting weakened or reversed GC-biased gene conversion, a nonadaptive substitution process that is thought to be important in the maintenance and trajectory of isochore evolution. Our results demonstrate that GC composition in Anolis is not associated with important features of genome structure, including gene density and intron size, in contrast to patterns seen in mammal and bird genomes.

The origin, evolution, and functional relevance of genomic variations in GC content are a long-debated topic, especially in mammals. Most of the existing literature, however, has focused on a small number of model species and/or limited sequence data sets. We analyzed more than 1000 orthologous genes in 33 fully sequenced mammalian genomes, reconstructed their ancestral isochore organization in the maximum likelihood framework, and explored the evolution of third-codon position GC content in representatives of 16 orders and 27 families. We showed that the previously reported erosion of GC-rich isochores is not a general trend. Several species (e.g., shrew, microbat, tenrec, rabbit) have independently undergone a marked increase in GC content, with a widening gap between the GC-poorest and GC-richest classes of genes. The intensively studied apes and (especially) murids do not reflect the general placental pattern. We correlated GC-content evolution with species life-history traits and cytology. Significant effects of body mass and genome size were detected, with each being consistent with the GC-biased gene conversion model.

We carried out an in silico analysis of the complete genome sequences of 14 species, including eukaryotes, prokaryotes, and archaea, to investigate the proportion of amplified fragment length polymorphism bands that are homoplasious for the different species, as well as the distribution of fragment lengths. We investigated several possible reasons for the disagreement, previously observed in Arabidopsis thaliana, between the observed fragment length distribution and the null random sequence distribution, which occurs in the direction of a deficit of fragments of small length and an excess of those of large length with respect to the null distribution. We made the following findings: 1) The positive relationship previously found between the percentage of homoplasy and genome size is a direct consequence of the number of observed bands and the GC content. For the same number of observed bands, the percentage of homoplasy is independent of the genome size of the species. 2) The disagreement between the observed fragment length distribution and the null random sequence distribution observed in A. thaliana is a phenomenon that also occurs in other species. 3) This disagreement is due neither to the structure of the genomes in isochores nor the possible impact of indels in reducing the number of restriction sites, two hypotheses discussed in the literature. 4) Nor is the disagreement eliminated by using restriction enzymes with balanced motifs. 5) The discrepancy seems to be caused, rather, by the nonrandom distribution of restriction enzyme motifs.

DNA methylation is a major epigenetic modification of the genome that affects basic biological functions, such as gene expression and cell development. We used the human genome sequences and the DNA methylation data that are available in order to establish a map of the levels of GC and methylation in isochores. We also looked for the correlations that hold between GC levels and the distribution of the (1) dinucleotide CpG, (2) ratio 5mC/CpG, and (3) CpG islands. Our results show that methylation levels, CpG frequencies, and the density of CpG islands are positively correlated with the GC level of isochores. In contrast, the correlation between the 5mC/CpG ratio and GC is a negative one because the increase in methylation lags behind that of CpG, to reach a plateau in the GC-richest, gene-richest isochore families H2 and H3. In conclusion, there are more CpG targets that remain unmethylated in the GC-richest, gene-richest isochores in comparison with the other isochores. This conclusion supports the idea that the widespread methylation under consideration here has a general inhibitory effect on gene expression.

Isochore is the genome-wide mosaic structure in guanine-cytosine (GC) content. The origin of isochores is thought to have emerged in the ancestral amniote genome, and the GC-rich isochore is eroded in the mammalian lineages. However, there are many enigmas in the isochore evolution: 1) although all the mammalians, birds, and even reptiles, which are clearly polyphyletic, have isochore, opossum and platypus lack GC-rich and GC-poor isochore classes; 2) although the isochore is predicted to vanish according to a fairly robust theory, a completely opposite conclusion was led in some mammalian lineages; and 3) the major three hypotheses on the isochore evolution cannot explain observed evidences without flaws. So far compositional evolution has been studied under the assumption that per base pair rate of GC-->AT (u) and AT-->GC (v) mutations are temporally constant (the constant model). With this model alone, however, it is difficult to explain the isochore evolution. We propose a simple model for compositional evolution based on the temporal per base pair rate of mutations (the variable model). In this model, rates u and v vary depending on temporal GC contents. Mathematically, the variable model is an expansion of the constant model. By using high-density human single nucleotide polymorphism data, we compared the variable model with the constant model. Although the variable model gave consistent results with the constant model, it can potentially describe the complicated isochore evolution, which the constant model cannot explain. The versatile characteristics of the variable model may shed new light on the mysterious isochore evolution.

Background: Previous investigations from our laboratory were largely focused on the genome organization of vertebrates. We showed that these genomes are mosaics of isochores, megabase-size DNA sequences that are fairly homogeneous in base composition yet belong to a small number of families that cover a wide compositional spectrum. A question raised by these results concerned how far back in evolution an isochore organization of the eukaryotic genome arose., Results: The present investigation deals with the compositional patterns of the invertebrates for which full genome sequences, or at least scaffolds, are available. We found that (i) a mosaic of isochores is the long-range organization of all the genomes that we investigated; (ii) the isochore families from the invertebrate genomes matched the corresponding families of vertebrates in GC levels; (iii) the relative amounts of isochore families were remarkably different for different genomes, except for those from phylogenetically close species, such as the Drosophilids., Conclusion: This work demonstrates not only that an isochore organization is present in all metazoan genomes analyzed that included Nematodes, Arthropods among Protostomia, Echinoderms and Chordates among Deuterostomia, but also that the isochore families of invertebrates share GC levels with the corresponding families of vertebrates.

The isochore theory depicts the genomes of warm-blooded vertebrates as a mosaic of long genomic regions that are characterized by relatively homogeneous GC content. In the absence of genomic data, the GC content at third-codon positions of protein-coding genes (GC3) was commonly used as a proxy for the GC content of isochores. Oddly, in the postgenomic era, GC3 is still sometimes used as a proxy for the GC composition of isochores. Here, we use genic and genomic sequences from human, chimpanzee, cow, mouse, rat, chicken, and zebrafish to show that GC3 only explains a very small proportion of the variation in GC content of long genomic sequences flanking the genes (GCf), and what little correlation there is between GC3 and GCf was found to decay rapidly with distance from the gene. The coefficient of variation of GC3 was found to be much larger than that of GCf and, therefore, GC3 and GCf values are not comparable with each other. Comparisons of orthologous gene pairs from 1) human and chimpanzee and 2) mouse and rat show strong correlations between their GC3 values, but very weak correlations between their GCf values. We conclude that the GC content of third-codon position cannot be used as stand-in for isochoric composition.

Background: Previous work from our laboratory showed that (i) vertebrate genomes are mosaics of isochores, typically megabase-size DNA segments that are fairly homogeneous in base composition; (ii) isochores belong to a small number of families (five in the human genome) characterized by different GC levels; (iii) 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; (iv) there are two modes of genome evolution, a conservative one in which isochore patterns basically do not change (e.g., among mammalian orders), and a transitional one, in which they do change (e.g., between amphibians and mammals); and (v) isochores are tightly linked to a number of basic biological properties, such as gene density, gene expression, replication timing and recombination., Results: The present availability of a number of fully sequenced genomes ranging from fishes to mammals allowed us to carry out investigations that (i) more precisely quantified our previous conclusions; (ii) 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 (iii) isochore family patterns can be either conserved or change within both warm- and cold-blooded vertebrates., Conclusion: On the basis of the results presented, we propose that (i) the large conservation of GC levels and dinucleotide frequencies may reflect the conservation of chromatin structures; (ii) the conservation of isochore size may be linked to the role played by isochores in chromosome structure and replication; (iii) the formation, the maintainance and the changes of isochore patterns are due to natural selection.

Replication timing is frequently discussed superficially in terms of its relationship to transcriptional activity via chromatin structure. However, so little is known about what regulates where and when replication initiates that it has been impossible to identify mechanistic and causal relationships. Moreover, much of our knowledge base has been anecdotal, derived from analyses of a few genes in unrelated cell lines. Recent studies have revisited long-standing hypotheses using genome-wide approaches. In particular, the foundation of this field was recently shored up with incontrovertible evidence that cellular differentiation is accompanied by coordinated changes in replication timing and transcription. These changes accompany subnuclear repositioning, and take place at the level of megabase-sized domains that transcend localized changes in chromatin structure or transcription. Inferring from these results, we propose that there exists a key transition during the middle of S-phase and that changes in replication timing traversing this period are associated with subnuclear repositioning and changes in the activity of certain classes of promoters.

Vertebrate genomes are characterized with CpG deficiency, particularly for GC-poor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5' T to 5' A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5' Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected (obs/exp) values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes (except sea urchin) but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5' T and 5' A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

The human genome, a typical mammalian genome, is made up of long (approximately 1-Mb, on average) regions, the isochores, that are fairly homogeneous in base composition and belong in five families characterized by different GC levels. An analysis of di- and tri-nucleotide densities in the isochores from the five families has shown large differences. These different "short-sequence designs:" (i) account for the fractionation of human DNA (and vertebrate DNA in general) when using sequence-specific ligands in density gradients, (ii) are very similar in whole isochores and in the corresponding intergenic sequences and introns, (iii) are reflected in different codon usages, (iv) lead to amino acid differences that increase the thermal stability of the proteins encoded by genes located in increasingly GC-rich isochore families, and (v) correspond to different chromatin structures.

Background: To a greater or lesser extent, eukaryotic nuclear genomes contain fragments of their mitochondrial genome counterpart, deriving from the random insertion of damaged mtDNA fragments. NumtS (Nuclear mt Sequences) are not equally abundant in all species, and are redundant and polymorphic in terms of copy number. In population and clinical genetics, it is important to have a complete overview of NumtS quantity and location. Searching PubMed for NumtS or Mitochondrial pseudo-genes yields hundreds of papers reporting Human NumtS compilations produced by in silico or wet-lab approaches. A comparison of published compilations clearly shows significant discrepancies among data, due both to unwise application of Bioinformatics methods and to a not yet correctly assembled nuclear genome. To optimize quantification and location of NumtS, we produced a consensus compilation of Human NumtS by applying various bioinformatics approaches., Results: Location and quantification of NumtS may be achieved by applying database similarity searching methods: we have applied various methods such as Blastn, MegaBlast and BLAT, changing both parameters and database; the results were compared, further analysed and checked against the already published compilations, thus producing the Reference Human Numt Sequences (RHNumtS) compilation. The resulting NumtS total 190., Conclusion: The RHNumtS compilation represents a highly reliable reference basis, which may allow designing a lab protocol to test the actual existence of each NumtS. Here we report preliminary results based on PCR amplification and sequencing on 41 NumtS selected from RHNumtS among those with lower score. In parallel, we are currently designing the RHNumtS database structure for implementation in the HmtDB resource. In the future, the same database will host NumtS compilations from other organisms, but these will be generated only when the nuclear genome of a specific organism has reached a high-quality level of assembly.

Unraveling the evolutionary forces responsible for variations of neutral substitution patterns among taxa or along genomes is a major issue for detecting selection within sequences. Mammalian genomes show large-scale regional variations of GC-content (the isochores), but the substitution processes at the origin of this structure are poorly understood. We analyzed the pattern of neutral substitutions in 1 Gb of primate non-coding regions. We show that the GC-content toward which sequences are evolving is strongly negatively correlated to the distance to telomeres and positively correlated to the rate of crossovers (R2 = 47%). This demonstrates that recombination has a major impact on substitution patterns in human, driving the evolution of GC-content. The evolution of GC-content correlates much more strongly with male than with female crossover rate, which rules out selectionist models for the evolution of isochores. This effect of recombination is most probably a consequence of the neutral process of biased gene conversion (BGC) occurring within recombination hotspots. We show that the predictions of this model fit very well with the observed substitution patterns in the human genome. This model notably explains the positive correlation between substitution rate and recombination rate. Theoretical calculations indicate that variations in population size or density in recombination hotspots can have a very strong impact on the evolution of base composition. Furthermore, recombination hotspots can create strong substitution hotspots. This molecular drive affects both coding and non-coding regions. We therefore conclude that along with mutation, selection and drift, BGC is one of the major factors driving genome evolution. Our results also shed light on variations in the rate of crossover relative to non-crossover events, along chromosomes and according to sex, and also on the conservation of hotspot density between human and chimp., Competing Interests: The authors have declared that no competing interests exist.

Background: The phylogenetic distribution of large-scale genome structure (i.e. mosaic compositional patchiness) has been explored mainly by analytical ultracentrifugation of bulk DNA. However, with the availability of large, good-quality chromosome sequences, and the recently developed computational methods to directly analyze patchiness on the genome sequence, an evolutionary comparative analysis can be carried out at the sequence level., Results: The local variations in the scaling exponent of the Detrended Fluctuation Analysis are used here to analyze large-scale genome structure and directly uncover the characteristic scales present in genome sequences. Furthermore, through shuffling experiments of selected genome regions, computationally-identified, isochore-like regions were identified as the biological source for the uncovered large-scale genome structure. The phylogenetic distribution of short- and large-scale patchiness was determined in the best-sequenced genome assemblies from eleven eukaryotic genomes: mammals (Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, and Canis familiaris), birds (Gallus gallus), fishes (Danio rerio), invertebrates (Drosophila melanogaster and Caenorhabditis elegans), plants (Arabidopsis thaliana) and yeasts (Saccharomyces cerevisiae). We found large-scale patchiness of genome structure, associated with in silico determined, isochore-like regions, throughout this wide phylogenetic range., Conclusion: Large-scale genome structure is detected by directly analyzing DNA sequences in a wide range of eukaryotic chromosome sequences, from human to yeast. In all these genomes, large-scale patchiness can be associated with the isochore-like regions, as directly detected in silico at the sequence level.

Chromosome replication timing is biphasic (early-late) in the cell cycle of vertebrates and of most (possibly all) eukaryotes. In the present work we have compared the extended, detailed replication timing maps that are available, namely those of human chromosomes 6, 11q, and 21q, with chromosomal bands as visualized at low (400 bands), high (850 bands), and highest (3,200 isochores) resolution. We have observed that the replicons located in a given isochore practically always show either all early or all late replication timing and that early-replicating isochores are short and GC-rich and late-replicating isochores are long and GC-poor. In the vast majority of cases, replicons are clustered in isochores, which are themselves most often clustered in early- or late-replication timing zones and may often reach the size of high-resolution bands and, very rarely, even that of low-resolution bands. Finally, we show that our results should be representative for the whole human genome and thus help to predict replication timing zones in all chromosomes.

The distribution of guanine and cytosine nucleotides throughout a genome, or the GC content, is associated with numerous features in mammals; understanding the pattern and evolutionary history of GC content is crucial to our efforts to annotate the genome. The local GC content is decaying toward an equilibrium point, but the causes and rates of this decay, as well as the value of the equilibrium point, remain topics of debate. By comparing the results of 2 methods for estimating local substitution rates, we identify 620 Mb of the human genome in which the rates of the various types of nucleotide substitutions are the same on both strands. These strand-symmetric regions show an exponential decay of local GC content at a pace determined by local substitution rates. DNA segments subjected to higher rates experience disproportionately accelerated decay and are AT rich, whereas segments subjected to lower rates decay more slowly and are GC rich. Although we are unable to draw any conclusions about causal factors, the results support the hypothesis proposed by Khelifi A, Meunier J, Duret L, and Mouchiroud D (2006. GC content evolution of the human and mouse genomes: insights from the study of processed pseudogenes in regions of different recombination rates. J Mol Evol. 62:745-752.) that the isochore structure has been reshaped over time. If rate variation were a determining factor, then the current isochore structure of mammalian genomes could result from the local differences in substitution rates. We predict that under current conditions strand-symmetric portions of the human genome will stabilize at an average GC content of 30% (considerably less than the current 42%), thus confirming that the human genome has not yet reached equilibrium.

The human genome is composed of large sequence segments with fairly homogeneous GC content, namely isochores, which have been linked to many important functions; biological implications of most isochore boundaries, however, remain elusive, partly due to the difficulty in determining these boundaries at high resolution. Using the segmentation algorithm based on the quadratic divergence, we re-determined all 79 boundaries of previously identified human isochores at single-nucleotide resolution, and then compared the boundary coordinates with other genome features. We found that 55.7% of isochore boundaries coincide with termini of repeat elements; 45.6% of isochore boundaries coincide with termini of highly conserved sequences based on alignment of 17 vertebrate genomes, i.e., the highly conserved genome sequence switches to a less or non-conserved one at the isochore boundary; some isochore boundaries coincide with abrupt change of CpG island distribution (note that one boundary can associate with more than one genome feature). In addition, sequences around isochore boundaries are highly conserved. It seems reasonable to deduce that the boundaries of all the isochores studied here would be replication timing sites in the human genome. These results suggest possible key roles of the isochore boundaries and may further our understanding of the human genome organization.

Background: For eukaryotes, there is almost no strand bias with regard to base composition, with exceptions for origins of replication and transcription start sites and transcribed regions. This paper revisits the question for subsequences of DNA taken at random from the genome., Results: For a typical mammal, for example mouse or human, there is a small strand bias throughout the genomic DNA: there is a correlation between (G - C) and (A - T) on the same strand, (that is between the difference in the number of guanine and cytosine bases and the difference in the number of adenine and thymine bases). For small subsequences - up to 1 kb - this correlation is weak but positive; but for large windows - around 50 kb to 2 Mb - the correlation is strong and negative. This effect is largely independent of GC%. Transcribed and untranscribed regions give similar correlations both for small and large subsequences, but there is a difference in these regions for intermediate sized subsequences. An analysis of the human genome showed that position within the isochore structure did not affect these correlations. An analysis of available genomes of different species shows that this contrast between large and small windows is a general feature of mammals and birds. Further down the evolutionary tree, other organisms show a similar but smaller effect. Except for the nematode, all the animals analysed showed at least a small effect., Conclusion: The correlations on the large scale may be explained by DNA replication. Transcription may be a modifier of these effects but is not the fundamental cause. These results cast light on how DNA mutations affect the genome over evolutionary time. At least for vertebrates, there is a broad relationship between body temperature and the size of the correlation. The genome of mammals and birds has a structure marked by strand bias segments.

We show that although the currently available isochore mapping methods agree on the isochore classification of about two-thirds of the human DNA, they produce significantly different results with regard to the location of isochore boundaries and isochore length distribution. We present a new consensus isochore assignment method based on majority voting and provide IsoBase, a comprehensive on-line database of isochore maps for all completely sequenced vertebrate 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.

Vertebrate genomes are mosaics of isochores, defined as long (>100 kb) regions with relatively homogeneous within-region base composition. Birds and mammals have more GC-rich isochores than amphibians and fish, and the GC-rich isochores of birds and mammals have been suggested to be an adaptation to homeothermy. If this hypothesis is correct, all poikilothermic (cold-blooded) vertebrates, including the nonavian reptiles, are expected to lack a GC-rich isochore structure. Previous studies using various methods to examine isochore structure in crocodilians, turtles, and squamates have led to different conclusions. We collected more than 6000 expressed sequence tags (ESTs) from the American alligator to overcome sample size limitations suggested to be the fundamental problem in the previous reptilian studies. The alligator ESTs were assembled and aligned with their human, mouse, chicken, and western clawed frog orthologs, resulting in 366 alignments. Analyses of third-codon-position GC content provided conclusive evidence that the poikilothermic alligator has GC-rich isochores, like homeothermic birds and mammals. We placed these results in a theoretical framework able to unify available models of isochore evolution. The data collected for this study allowed us to reject the models that explain the evolution of GC content using changes in body temperature associated with the transition from poikilothermy to homeothermy. Falsification of these models places fundamental constraints upon the plausible pathways for the evolution of isochores.

Hidden Markov models (HMMs) are effective tools to detect series of statistically homogeneous structures, but they are not well suited to analyse complex structures. For example, the duration of stay in a state of a HMM must follow a geometric law. Numerous other methodological difficulties are encountered when using HMMs to segregate genes from transposons or retroviruses, or to determine the isochore classes of genes. The aim of this paper is to analyse these methodological difficulties, and to suggest new tools for the exploration of genome data. We show that HMMs can be used to analyse complex gene structures with bell-shaped length distribution by using convolution of geometric distributions. Thus, we have introduced macros-states to model the distributions of the lengths of the regions. Our study shows that simple HMM could be used to model the isochore organisation of the mouse genome. This potential use of markovian models to help in data exploration has been underestimated until now.

The isochore structure of a genome is observable by variation in the G+C (guanine and cytosine) content within and between the chromosomes. Describing the isochore structure of vertebrate genomes is a challenging task, and many computational methods have been developed and applied to it. Here we apply a well-known least-squares optimal segmentation algorithm to isochore discovery. The algorithm finds the best division of the sequence into k pieces, such that the segments are internally as homogeneous as possible. We show how this simple segmentation method can be applied to isochore discovery using as input the G+C content of sliding windows on the sequence. To evaluate the performance of this segmentation technique on isochore detection, we present results from segmenting previously studied isochore regions of the human genome. Detailed results on the MHC locus, on parts of chromosomes 21 and 22, and on a 100 Mb region from chromosome 1 are similar to previously suggested isochore structures. We also give results on segmenting all 22 autosomal human chromosomes. An advantage of this technique is that oversegmentation of G+C rich regions can generally be avoided. This is because the technique concentrates on greater global, instead of smaller local, differences in the sequence composition. The effect is further emphasized by a log-transformation of the data that lowers the high variance that is observed in G+C rich regions. We conclude that the least-squares optimal segmentation method is computationally efficient and yields results close to previous biologically motivated isochore structures.

The vertebrate genome is a mosaic of GC-poor and GC-rich isochores, megabase-sized DNA regions of fairly homogeneous base composition that differ in relative amount, gene density, gene expression, replication timing, and recombination frequency. At the emergence of warm-blooded vertebrates, the gene-rich, moderately GC-rich isochores of the cold-blooded ancestors underwent a GC increase. This increase was similar in mammals and birds and was maintained during the evolution of mammalian and avian orders. Neither the GC increase nor its conservation can be accounted for by the random fixation of neutral or nearly neutral single-nucleotide changes (i.e., the vast majority of nucleotide substitutions) or by a biased gene conversion process occurring at random genome locations. Both phenomena can be explained, however, by the neoselectionist theory of genome evolution that is presented here. This theory fully accepts Ohta's nearly neutral view of point mutations but proposes in addition (i) that the AT-biased mutational input present in vertebrates pushes some DNA regions below a certain GC threshold; (ii) that these lower GC levels cause regional changes in chromatin structure that lead to deleterious effects on replication and transcription; and (iii) that the carriers of these changes undergo negative (purifying) selection, the final result being a compositional conservation of the original isochore pattern in the surviving population. Negative selection may also largely explain the GC increase accompanying the emergence of warm-blooded vertebrates. In conclusion, the neoselectionist theory not only provides a solution to the neutralist/selectionist debate but also introduces an epigenomic component in genome evolution.

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

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties., (Copyright 2007 S. Karger AG, Basel.)

Several studies of nucleotide substitution patterns in mammalian species suggested that GC-rich isochores might be vanishing in mammalian genomes. However, the number of genes and the number of genomes included in these studies might not have given a reliable broad view of the trend in GC change in mammals. It is therefore worth exploiting this issue with a broader coverage of mammalian genomes using a reliable approach, the maximum likelihood approach. We have applied two maximum likelihood methods to infer the ancestral GC contents of 176 mammalian genes from representative eutherian species and at least one marsupial species. Except for a large GC decrease in marsupial genes, we found no general decreasing trend in GC content in GC-rich genes or in other genes among eutherian mammals; indeed, the GC content of GC-rich genes appears to have increased in recent times in some genomes, e.g., the rabbit. For the large GC decrease in marsupials, it could be mainly due to the great reduction in chromosome number, which could lead to a large reduction in recombination rate and thus also a large reduction in the rate of gene conversion. Since many eutherian mammals still maintain a fairly large number of chromosomes, it is unlikely that GC-rich isochores are vanishing in these mammals.

Mammalian genomes are organised into a mosaic of regions (in general more than 300 kb in length), with differing, relatively homogeneous G+C contents. The G+C content is the basic characteristic of isochores, but they have also been associated with many other biological properties. For instance, the genes are more compact and their density is highest in G+C rich isochores. Various ways of locating isochores in the human genome have been developed, but such methods use only the base composition of the DNA sequences. The present paper proposes a new method, based on a hidden Markov model, which takes into account several of the biological properties associated with the isochore structure of a genome. This method leads to good segmentation of the human genome into isochores, and also permits a new analysis of the known heterogeneity of G+C rich isochores: most (60%) of the G+C poor genes embedded in G+C rich isochores have UTR sequences characteristic of G+C rich genes. This genomic feature is discussed in the context of both evolution and genome function.