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.     

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.      

Two classes of genes in plants

Two classes of genes were identified in three Gramineae (maize, rice, barley) and six dicots (Arabidopsis, soybean, pea, tobacco, tomato, potato). One class, the GC-rich class, contained genes with no, or few, short introns. In contrast, the GC-poor class contained genes with numerous, long introns. The similarity of the properties of each class, as present in the genomes of maize and Arabidopsis, is particularly remarkable in view of the fact that these plants exhibit large differences in genome size, average intron size, and DNA base composition. The functional relevance of the two classes of genes is stressed by (1) the conservation in homologous genes from maize and Arabidopsis not only of the number of introns and of their positions, but also of the relative size of concatenated introns; and (2) the existence of two similar classes of genes in vertebrates; interestingly, the differences in intron sizes and numbers in genes from the GC-poor and GC-rich classes are much more striking in plants than in vertebrates.     

Mapping Insertions,Deletions and SNPs on Venter's Chromosomes

Background

The very recent availability of fully sequenced individual human genomes is a major revolution in biology which is certainly going to provide new insights into genetic diseases and genomic rearrangements.

Results

We mapped the insertions, deletions and SNPs (single nucleotide polymorphisms) that are present in Craig Venter''s genome, more precisely on chromosomes 17 to 22, and compared them with the human reference genome hg17. Our results show that insertions and deletions are almost absent in L1 and generally scarce in L2 isochore families (GC-poor L1+L2 isochores represent slightly over half of the human genome), whereas they increase in GC-rich isochores, largely paralleling the densities of genes, retroviral integrations and Alu sequences. The distributions of insertions/deletions are in striking contrast with those of SNPs which exhibit almost the same density across all isochore families with, however, a trend for lower concentrations in gene-rich regions.

Conclusions

Our study strongly suggests that the distribution of insertions/deletions is due to the structure of chromatin which is mostly open in gene-rich, GC-rich isochores, and largely closed in gene-poor, GC-poor isochores. The different distributions of insertions/deletions and SNPs are clearly related to the two different responsible mechanisms, namely recombination and point mutations.     

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.     

Presence of isochore structures in reptile genomes suggested by the relationship between GC contents of intron regions and those of coding regions

Vertebrate genomes are mosaics of isochores. On the assumption that marked differences exist in the isochore structure between warm-blooded and cold-blooded animals, variations among vertebrates were previously attributed to adaptation to homeothermy. However, based on the data of coding regions from representatives of extant vertebrates, including a turtle, a crocodile (Archosauromorpha) and a few kinds of snakes (Lepidosauromorpha), it was recently hypothesized that the common ancestors of mammals, birds and extant reptiles already had the "warm-blooded" isochore structure. To test this hypothesis, the nucleotide sequences of alpha-globin genes including non-coding regions (introns) from two snakes, N. kaouthia and E. climacophora, were determined (accession number: AB104824, AB104825). The correlation between the GC contents in the introns and exons of alpha-globin genes from snakes and those from other vertebrates supports the above hypothesis. Similar analysis using data for exons and introns of other genes obtained from the GenBank (Release 131) also support the above hypothesis.     

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 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.     

The compositional patterns of the avian genomes and their evolutionary implications

The compositional distributions of large (main-band) DNA fragments from eight birds belonging to eight different orders (including both paleognathous and neognathous species) are very broad and extremely close to each other. These findings, which are paralleled by the compositional similarity of homologous coding sequences and their codon positions, support the idea that birds are a monophyletic group.The compositional distribution of third-codon positions of genes from chicken, the only avian species for which a relatively large number of coding sequences is known, is very broad and bimodal, the minor GC-richer peak reaching 100% GC. The very high compositional heterogeneity of avian genomes is accompanied (as in the case of mammalian genomes) by a very high speciation rate compared to cold-blooded vertebrates which are characterized by genomes that are much less heterogeneous. The higher GC levels attained by avian compared to mammalian genomes might be correlated with the higher body temperature (41–43°C) of birds compared to mammals (37°C).A comparison of GC levels of coding sequences and codon positions from man and chicken revealed very close average GC levels and standard deviations. Homologous coding sequences and codon positions from man and chicken showed a surprisingly high degree of compositional similarity which was, however, higher for GC-poor than for GC-rich sequences. This indicates that GC-poor isochores of warm-blooded vertebrates reflect the composition of the isochores of the genome of the common reptilian ancestor of mammals and birds, which underwent only a small compositional change at the transition from cold- to warm-blooded vertebrates. In contrast, the GC-rich isochores of birds and mammals are the result of large compositional changes at the same evolutionary transition, where were in part different in the two classes of warm-blooded vertebrates.Correspondence to: G. Bernaadi     

Compositional bimodality and evolution of retroviral genomes.

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

Localization of DNA sequences tightly associated with synaptonemal complex in compositional fractions of golden hamster

The synaptonemal complex isolated from the spermatocyte nuclei by exhaustive hydrolysis of the latter by DNase II contains tightly associated DNA sequences (SCAR DNA). Here we studied the compositional properties of a cloned family of SCAR DNA of golden hamster, namely we performed the localization of 27 SCAR DNA clones on compositionally fractionated genomic DNA from golden hamster. We observed that sequences of the SCAR DNA family are mainly localized in the GC-poor isochore families L1 and L2, that showed 63% hybridization signals. This means that 37% of signals is referred to the GC-rich isochores, indicating the presence of SCAR DNA overall the genome, even if each isochore family presents differences in density and sequence type. Moreover, the SCAR DNA sequences containing regions of homology with LINE/SINE repeats were observed in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that SC and SCAR DNA participate in the chromatin organization during the meiosis prophase I, which should result in the attachment of chromatin loops to lateral elements of SC along the whole length of the latter.     

The distributions of "new" and "old" Alu sequences in the human genome: the solution of a "mystery"

The distribution in the human genome of the largest family of mobile elements, the Alu sequences, has been investigated for the past 30 years, and the vast majority of Alu sequences were shown to have the highest density in GC-rich isochores. Ten years ago, it was discovered, however, that the small "youngest" (most recently transposed) Alu families had a strikingly different distribution compared with the "old" families. This raised the question as to how this change took place in evolution. We solved what was considered to be a "mystery" by 1) revisiting our previous results on the integration and stability of retroviral sequences, and 2) assessing the densities of acceptor sites TTTT/AA in isochore families. We could conclude 1) that the open state of chromatin structure plays a crucial role in allowing not only the initial integration of retroviral sequences but also that of the youngest Alu sequences, and 2) that the distribution of old Alus can be explained as due to Alu sequences being unstable in the GC-poor isochores but stable in the compositionally matching GC-rich isochores, again in line with what happens in the case of retroviral sequences.     

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

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

Evolution of isochores in rodents

The most deviant isochore pattern within mammals was found in rat and mouse; most other mammals possess a different kind of isochore organization called the "general pattern." However, isochore patterns remain largely unknown in rodents other than mouse and rat. To investigate the taxonomic distribution of isochore patterns in rodents, we sequenced the nuclear gene LCAT (lecithin:cholesterol acyltransferase) from 17 rodents species (bringing the total of LCAT sequences in rodent to 19) and compared their GC contents at third codon positions and in introns. We also analyzed an extensive sequence database from rodents other than rat and mouse. All murid LCAT sequences are much poorer in GC than all nonrodent LCAT sequences, and the hamster sequence database shows exactly the same isochore pattern as rat and mouse. Thus, all murids share the same special isochore pattern--GC homogenization. LCAT sequences are GC-poor in hystricomorphs too, but the guinea pig sequence database indicates that large changes in GC content occur without an overall modification of the isochore pattern. This novel mode of isochore evolution is called GC reordering. LCAT sequences also show that the evolution of isochores in sciurids and glirids is nonconservative in comparison with that in nonrodents. Thus, at least two novel patterns of isochore evolution were found. No rodent investigated to date shared the general mammalian pattern.      

Deciphering heterogeneity in pig genome assembly Sscrofa9 by isochore and isochore-like region analyses

Background

The isochore, a large DNA sequence with relatively small GC variance, is one of the most important structures in eukaryotic genomes. Although the isochore has been widely studied in humans and other species, little is known about its distribution in pigs.

Principal Findings

In this paper, we construct a map of long homogeneous genome regions (LHGRs), i.e., isochores and isochore-like regions, in pigs to provide an intuitive version of GC heterogeneity in each chromosome. The LHGR pattern study not only quantifies heterogeneities, but also reveals some primary characteristics of the chromatin organization, including the followings: (1) the majority of LHGRs belong to GC-poor families and are in long length; (2) a high gene density tends to occur with the appearance of GC-rich LHGRs; and (3) the density of LINE repeats decreases with an increase in the GC content of LHGRs. Furthermore, a portion of LHGRs with particular GC ranges (50%–51% and 54%–55%) tend to have abnormally high gene densities, suggesting that biased gene conversion (BGC), as well as time- and energy-saving principles, could be of importance to the formation of genome organization.

Conclusion

This study significantly improves our knowledge of chromatin organization in the pig genome. Correlations between the different biological features (e.g., gene density and repeat density) and GC content of LHGRs provide a unique glimpse of in silico gene and repeats prediction.     

Compositional compartmentalization and gene composition in the genome of vertebrates

Summary The compositional distribution of coding sequences from five vertebrates (Xenopus, chicken, mouse, rat, and human) is shifted toward higher GC values compared to that of the DNA molecules (in the 35–85-kb size range) isolated from the corresponding genomes. This shift is due to the lower GC levels of intergenic sequences compared to coding sequences. In the cold-blooded vertebrate, the two distributions are similar in that GC-poor genes and GC-poor DNA molecules are largely predominant. In contrast, in the warm-blooded vertebrates, GC-rich genes are largely predominant over GC-poor genes, whereas GC-poor DNA molecules are largely predominant over GC-rich DNA molecules. As a consequence, the genomes of warm-blooded vertebrates show a compositional gradient of gene concentration. The compositional distributions of coding sequences (as well as of DNA molecules) showed remarkable differences between chicken and mammals, and between mouse (or rat) and human. Differences were also detected in the compositional distribution of housekeeping and tissue-specific genes, the former being more abundant among GC-rich genes.     

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.     

Isochore patterns and gene distributions in fish genomes

The compositional approach developed in our laboratory many years ago revealed a large-scale compositional heterogeneity in vertebrate genomes, in which GC-rich and GC-poor regions, the isochores, were found to be characterized by high and low gene densities, respectively. Here we mapped isochores on fish chromosomes and assessed gene densities in isochore families. Because of the availability of sequence data, we have concentrated our investigations on four species, zebrafish (Brachydanio rerio), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis), which belong to four distant orders and cover almost the entire GC range of fish genomes. These investigations produced isochore maps that were drastically different not only from those of mammals (in that only two major isochore families were essentially present in each genome vs five in the human genome) but also from each other (in that different isochore families were represented in different genomes). Gene density distributions for these fish genomes were also obtained and shown to follow the expected increase with increasing isochore GC. Finally, we discovered a remarkable conservation of the average size of the isochores (which match replicon clusters in the case of human chromosomes) and of the average GC levels of isochore families in both fish and human genomes. Moreover, in each genome the GC-poorest isochore families comprised a group of "long isochores" (2-20 Mb in size), which were the lowest in GC and varied in size distribution and relative amount from one genome to the other.