Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.     

A compositional map of the cen-q21 region of human chromosome 21

A compositional map of the centromere and of the subcentromeric region of the long arm of human chromosome 21 was established by determining the GC levels (GC is the molar fraction of guanine+cytosine in DNA) of 11 YACs (yeast artificial chromosomes) covering this 13–14 Mb region which extends from the α-satellite sequences of the C(entromeric) band qll.1, through R(everse) band q11.2, to the proximal part of G(iemsa) band q21. The entire region is made up of GC-poor, or L, isochores with only one GC-rich H1 isochore, at least 2 Mb in size, located in band q21. The almost identical GC levels of the centromeric α-satellite repeats (38.5%), of R band q11.2 (39%), and of G bands (38–40%) provide a direct demonstration that base composition cannot be the only cause of the cytogenetic differences between C, G, and the majority of R bands, namely the H3^- R bands (which do not contain the GC-richest H3 isochores). The results obtained also show that isochores may be as long as 6 Mb, at least in the GC-poor regions of the genome, and support previous observations suggesting that YACs from isochore borders are unstable and/or difficult to clone. Genes and CpG islands are very rare in the GC-poor region investigated, as expected from the fact that their concentration is proportional to the GC levels of the isochores in which they are contained.     

Isochore Structures in the Genome of the Plant <Emphasis Type="BoldItalic">Arabidopsis thaliana</Emphasis>

Arabidopsis thaliana is an important model system for the study of plant biology. We have analyzed the complete genome sequences of Arabidopsis by using a newly developed windowless method for the GC content computation, the cumulative GC profile. It is shown that the Arabidopsis genome is organized into a mosaic structure of isochores. All the centromeric regions are located in GC-rich isochores, called centromere-isochores, which are characterized by a high GC content but low gene and T-DNA insertion densities. This characteristic distinguishes centromere-isochores from the other class of GC-rich isochores, called GC-isochores, which have high gene and T-DNA insertion densities. Consequently, 15 isochores have been identified, i.e., 7 AT-isochores, 3 GC-isochores, and 5 centromere-isochores. The genes in centromere-isochores, which have the highest GC content, have much shorter intron lengths and lower intron numbers, compared to those of the other two types. There is also considerable difference in the numbers and lengths of transposable elements (TEs) between AT and GC-isochores, i.e., the TE number (length) of AT-isochores is 6.3 (7.3) times that of GC-isochores. It is generally believed that TEs are accumulated in the regions surrounding the centromeres. However, within these TE-rich regions, there are regions of extremely low TE numbers (TE deserts), which correspond to the positions of centromere-isochores. In addition, a heterochromatic knob is located at the boundary of an AT-isochore. Furthermore, we show that the differences in GC content among isochores are mainly due to the GC content variation of introns, the third codon positions and intergenic regions.[Reviewing Editor: Martin Kreitman]     

A compositional map of human chromosome 21.

GC-poor and GC-rich isochores, the long (greater than 300 kb) compositionally homogeneous DNA segments that form the genome of warm-blooded vertebrates, are located in G- and R-bands respectively of metaphase chromosomes. The precise correspondence between GC-rich isochores and R-band structure is still, however, an open problem, because GC-rich isochores are compositionally heterogeneous and only represent one-third of the genome, with the GC-richest family (which is by far the highest in gene concentration) corresponding to less than 5% of the genome. In order to clarify this issue and, more generally, to correlate DNA composition and chromosomal structure in an unequivocal way, we have developed a new approach, compositional mapping. This consists of assessing the base composition over 0.2-0.3 Mb (megabase) regions surrounding landmarks that were previously localized on the physical map. Compositional mapping was applied here to the long arm of human chromosome 21, using 53 probes that had already been used in physical mapping. The results obtained provide a direct demonstration that the DNA stretches of G-bands essentially correspond to GC-poor isochores, and that R-band DNA is characterized by a compositional heterogeneity that is much more striking than expected, in that it comprises isochores covering the full spectrum of GC levels. GC-poor isochores of R-bands may, however, correspond to 'thin' G-bands, as visualized at high resolution, leaving GC-rich and very GC-rich isochores as the real components of (high-resolution) R-band DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rate, not the spectrum, of base pair substitutions changes at a GC-content transition in the human NF1 gene region: implications for the evolution of the mammalian genome structure

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.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

How not to search for isochores: a reply to Cohen et Al

In a recent paper in these pages, Cohen et al. search for isochores in the human genome, based on a system of attributes that they assign to isochores. The putative isochores that they find and choose for presentation are almost all below 45% GC and cover only about 41% of the genome. Closer inspection reveals that the authors' methodology systematically loses GC-rich isochores because it does not anticipate the considerable fluctuations and corresponding long-range correlations that characterize mammalian DNA and that are highest in GC-rich DNA. Thus, they over-fragment GC-rich isochores (and also many GC-poor isochores) beyond recognition.     

Isochores and replication time zones: a perfect match

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.     

Vanishing GC-rich isochores in mammalian genomes

To understand the origin and evolution of isochores-the peculiar spatial distribution of GC content within mammalian genomes-we analyzed the synonymous substitution pattern in coding sequences from closely related species in different mammalian orders. In primate and cetartiodactyls, GC-rich genes are undergoing a large excess of GC --> AT substitutions over AT --> GC substitutions: GC-rich isochores are slowly disappearing from the genome of these two mammalian orders. In rodents, our analyses suggest both a decrease in GC content of GC-rich isochores and an increase in GC-poor isochores, but more data will be necessary to assess the significance of this pattern. These observations question the conclusions of previous works that assumed that base composition was at equilibrium. Analysis of allele frequency in human polymorphism data, however, confirmed that in the GC-rich parts of the genome, GC alleles have a higher probability of fixation than AT alleles. This fixation bias appears not strong enough to overcome the large excess of GC --> AT mutations. Thus, whatever the evolutionary force (neutral or selective) at the origin of GC-rich isochores, this force is no longer effective in mammals. We propose a model based on the biased gene conversion hypothesis that accounts for the origin of GC-rich isochores in the ancestral amniote genome and for their decline in present-day mammals.     

Directional fixation of mutations in vertebrate evolution

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

The Decline of Isochores in Mammals: An Assessment of the GC ContentVariation Along the Mammalian Phylogeny

Whether isochores, the large-scale variation of the GC content in mammalian genomes, are being maintained has recently been questioned. It has been suggested that GC-rich isochores originated in the ancestral amniote genome but that whatever force gave rise to them is no longer effective and that isochores are now disappearing from mammalian genomes. Here we investigated the evolution of the GC content of 41 coding genes in 6 to 66 species of mammals by estimating the ancestral GC content using a method which allows for different rates of substitution between sites. We found a highly significant decrease in the GC content during early mammalian evolution, as well as a weaker but still significant decrease in the GC content of GC-rich genes later in at least three groups of mammals: primates, rodents, and carnivores. These results are of interest because they confirm the recently suggested disappearance of GC-rich isochores in some mammalian genomes, and more importantly, they suggest that this disappearance started very early in mammalian evolution.This article contains online supplementary material.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Statistical analysis of vertebrate sequences reveals that long genes are scarce in GC-rich isochores

We compared the exon/intron organization of vertebrate genes belonging to different isochore classes, as predicted by their GC content at third codon position. Two main features have emerged from the analysis of sequences published in GenBank: (1) genes coding for long proteins (i.e., 500 aa) are almost two times more frequent in GC-poor than in GC-rich isochores; (2) intervening sequences (=sum of introns) are on average three times longer in GC-poor than in GC-rich isochores. These patterns are observed among human, mouse, rat, cow, and even chicken genes and are therefore likely to be common to all warm-blooded vertebrates. Analysis of Xenopus sequences suggests that the same patterns exist in cold-blooded vertebrates. It could be argued that such results do not reflect the reality because sequence databases are not representative of entire genomes. However, analysis of biases in GenBank revealed that the observed discrepancies between GC-rich and GC-poor isochores are not artifactual, and are probably largely underestimated. We investigated the distribution of microsatellites and interspersed repeats in introns of human and mouse genes from different isochores. This analysis confirmed previous studies showing that Ll repeats are almost absent from GC-rich isochores. Microsatellites and SINES (Alu, B1, B2) are found at roughly equal frequencies in introns from all isochore classes. Globally, the presence of repeated sequences does not account for the increased intron length in GC-poor isochores. The relationships between gene structure and global genome organization and evolution are discussed.     

Telomeres in Warm-Blooded Vertebrates Are Composed of GC-Rich Isochores

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.     

Isochore specificity of AUG initiator context of human genes

The efficiency of AUG start codon recognition in translation initiation is modulated by its sequence context. Here we investigated a non-redundant set of 5914 human genes and show that this context is different in genes located in different isochores. In particular, of the two main consensus start sequences, RCCaugR is five-fold more represented than AARaugR in genes from the GC-rich H3 isochores compared to genes from the GC-poor L isochores. Furthermore, genes located in GC-rich isochores have shorter 5' UTRs and stronger avoidance of upstream AUG than genes located in GC-poor isochores. This suggests that genes requiring highly efficient translation are located in GC-rich isochores and genes requiring fine modulation of expression are located in GC-poor isochores. This is in agreement with independent data from the literature concerning the location of housekeeping and tissue-specific genes, respectively.     

Patterns of Vertebrate Isochore Evolution Revealed by Comparison of Expressed Mammalian,Avian, and Crocodilian Genes

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. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Nicolas Galtier     

Autosomal localization of interstitial telomeric sites (ITS) in brook trout, Salvelinus fontinalis (Pisces, Salmonidae)

Cytogenetic analysis of brook trout performed with molecular and conventional methods led to identification of interstitial telomeric sites on one or two subtelocentric chromosomes within the same pair. Morphology and specific patterns of these chromosomes using fluorochromes associated with A/T- or G/C-rich DNA proved that these chromosomes are not sex related. The chromomycin-positive region was located on the short arms of the ITS bearing chromosome pair and flanked by telomeric sequences, suggesting that this part of the chromosome had been translocated from another one. Our observations confirm that GC-rich regions are highly mobile genetic structures, and led to ITS formation on brook trout chromosomes.