Comparative Compositional Mapping of Chicken and Quail Chromosomes

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

Comparative compositional mapping of chicken and quail chromosomes

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

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 chromosome maps of the human genome

The human genome is a mosaic of isochores, which are long DNA segments (300 kbp) relatively homogeneous in G+C. Human isochores were first identified by density-gradient ultracentrifugation of bulk DNA, and differ in important features, e.g. genes are found predominantly in the GC-richest isochores. Here, we use a reliable segmentation method to partition the longest contigs in the human genome draft sequence into long homogeneous genome regions (LHGRs), thereby revealing the isochore structure of the human genome. The advantages of the isochore maps presented here are: (1) sequence heterogeneities at different scales are shown in the same plot; (2) pair-wise compositional differences between adjacent regions are all statistically significant; (3) isochore boundaries are accurately defined to single base pair resolution; and (4) both gradual and abrupt isochore boundaries are simultaneously revealed. Taking advantage of the wide sample of genome sequence analyzed, we investigate the correspondence between LHGRs and true human isochores revealed through DNA centrifugation. LHGRs show many of the typical isochore features, mainly size distribution, G+C range, and proportions of the isochore classes. The relative density of genes, Alu and long interspersed nuclear element repeats and the different types of single nucleotide polymorphisms on LHGRs also coincide with expectations in true isochores. Potential applications of isochore maps range from the improvement of gene-finding algorithms to the prediction of linkage disequilibrium levels in association studies between marker genes and complex traits. The coordinates for the LHGRs identified in all the contigs longer than 2 Mb in the human genome sequence are available at the online resource on isochore mapping: http://bioinfo2.ugr.es/isochores.     

Distribution of DNA methylation,CpGs, and CpG islands in human isochores

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 structures in the mouse genome

The distribution of the G+C content in the mouse genome has been studied using a windowless technique. We have found that: (i). Abrupt variations of the G+C content from a GC-rich region to a GC-poor region, and vice versa, occur frequently at some sites along the sequence of the mouse genome. (ii). Long domains with relatively homogeneous G+C content (isochores) exist, which usually have sharp boundaries. Consequently, 28 isochores longer than 1 Mb have been identified in the mouse genome. A homogeneity index was used to quantify the variations of the G+C content within isochores. The precise boundaries, sizes, and G+C contents of these isochores have been determined. The windowless technique for the G+C content computation was also used to analyze the DNA sequence containing the mouse MHC region, which has a GC-poor isochore. This isochore is located at the central part of the sequence with boundaries at 468459 and 812716 bp, where the sequence is extended from the centromeric end to the telomeric end. In addition, the analysis of a segment of the rat genome shows that the rat genome also has clear isochore structures.     

The Biased Distribution of Alus in Human Isochores Might Be Driven by Recombination

Alu retrotransposons do not show a homogeneous distribution over the human genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores. However, since they preferentially insert into the L isochores, the question arises: What is the evolutionary mechanism that shifts the Alu density maximum from L to H isochores? To disclose the role played by each of the potential mechanisms involved in such biased distribution, we carried out a genome-wide analysis of the density of the Alus as a function of their evolutionary age, isochore membership, and intron vs. intergene location. Since Alus depend on the retrotransposase encoded by the LINE1 elements, we also studied the distribution of LINE1 to provide a complete evolutionary scenario. We consecutively check, and discard, the contributions of the Alu/LINE1 competition for retrotransposase, compositional matching pressure, and Alu overrepresentation in introns. In analyzing the role played by unequal recombination, we scan the genome for Alu trimers, a direct product of Alu–Alu recombination. Through computer simulations, we show that such trimers are much more frequent than expected, the observed/expected ratio being higher in L than in H isochores. This result, together with the known higher selective disadvantage of recombination products in H isochores, points to Alu–Alu recombination as the main agent provoking the density shift of Alus toward the GC-rich parts of the genome. Two independent pieces of evidence—the lower evolutionary divergence shown by recently inserted Alu subfamilies and the higher frequency of old stand-alone Alus in L isochores—support such a conclusion. Other evolutionary factors, such as population bottlenecks during primate speciation, may have accelerated the fast accumulation of Alus in GC-rich isochores.     

DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria

We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobre?a and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species.     

Isochore chromosome maps of eukaryotic genomes

Analytical DNA ultracentrifugation revealed that eukaryotic genomes are mosaics of isochores: long DNA segments (>300 kb on average) relatively homogeneous in G+C. Important genome features are dependent on this isochore structure, e.g. genes are found predominantly in the GC-richest isochore classes. However, no reliable method is available to rigorously partition the genome sequence into relatively homogeneous regions of different composition, thereby revealing the isochore structure of chromosomes at the sequence level. Homogeneous regions are currently ascertained by plain statistics on moving windows of arbitrary length, or simply by eye on G+C plots. On the contrary, the entropic segmentation method is able to divide a DNA sequence into relatively homogeneous, statistically significant domains. An early version of this algorithm only produced domains having an average length far below the typical isochore size. Here we show that an improved segmentation method, specifically intended to determine the most statistically significant partition of the sequence at each scale, is able to identify the boundaries between long homogeneous genome regions displaying the typical features of isochores. The algorithm precisely locates classes II and III of the human major histocompatibility complex region, two well-characterized isochores at the sequence level, the boundary between them being the first isochore boundary experimentally characterized at the sequence level. The analysis is then extended to a collection of human large contigs. The relatively homogeneous regions we find show many of the features (G+C range, relative proportion of isochore classes, size distribution, and relationship with gene density) of the isochores identified through DNA centrifugation. Isochore chromosome maps, with many potential applications in genomics, are then drawn for all the completely sequenced eukaryotic genomes available.     

An isochore map of the human genome based on the Z curve method

The distribution of the G+C content in the human genome has been studied by using a windowless technique derived from the Z curve method. The most important findings presented in this paper are twofold. First, abrupt variations of the G+C content along human chromosome sequences are the main variation patterns of G+C content. It is found that at some sites, the G+C content undergoes abrupt changes from a G+C-rich region to a G+C-poor region alternatively and vice versa. Second, it is shown that long domains with relatively homogeneous G+C content along each chromosome do exist. These domains are thought to be isochores, which usually have sharp boundaries. Consequently, 56 isochores longer than 3 Mb have been identified in chromosomes 1-22, X and Y. Boundaries, size and G+C content of each isochore identified are listed in detail. As an example to demonstrate the power of the method, the boundary between the Classes III and II isochores of the MHC sequence has been determined and found to be at 2,477,936, which is in good agreement with the experimental evidence. A homogeneity index is introduced to measure the homogeneity of G+C content in isochores. We emphasize that the homogeneity of G+C content is relative. The isochores in which the G+C content keeps absolutely constant do not exist. Isochore structures appear to be a basic organization of the human genome. Due to the relevance to many important biological functions, the clarification of isochore structures will provide much insight into the understanding of the human genome.     

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.     

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.     

The genome of Plasmodium cynomolgi is partitioned into separable domains which appear to differ in sequence stability.

The genome of Plasmodium cynomolgi is partitioned into at least 7 distinct genetic domains. Each domain is apparently uniform in DNA density and is separable from the others by CsCl density centrifugation in the presence of Hoechst dye. The protein-encoding genes that were tested are localized in the two heaviest density domains (isochores). The ribosomal genes are in two lighter isochores as well as in one of the isochores that contains protein encoding genes. Telomeric sequences are mainly, if not exclusively, in the lightest isochores, indicating that position with regard to chromosome ends may correlate with density. Blocks of a tandemly-repeating sequence which mark genetically hypervariable chromosome regions in malaria parasites are located in all isochores. However, the rate of change associated with the blocks of sequence is much slower in some isochores than in others. This indicates that the rate of genetic change in these parasites may differ with isochore and chromosomal position. These results may also have more general biological implications since they suggest that the genetic instability often noted for tandem repeat sequences in the eukaryotic genome may be limited to only a distinct subset of the genomic complement of such sequence blocks.     

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.     

Active and inactive genes localize preferentially in the periphery of chromosome territories

The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the non-expressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three- dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed.     

Isochore structures in the chicken genome

The availability of the complete chicken genome sequence provides an unprecedented opportunity to study the global genome organization at the sequence level. Delineating compositionally homogeneous G + C domains in DNA sequences can provide much insight into the understanding of the organization and biological functions of the chicken genome. A new segmentation algorithm, which is simple and fast, has been proposed to partition a given genome or DNA sequence into compositionally distinct domains. By applying the new segmentation algorithm to the draft chicken genome sequence, the mosaic organization of the chicken genome can be confirmed at the sequence level. It is shown herein that the chicken genome is also characterized by a mosaic structure of isochores, long DNA segments that are fairly homogeneous in the G + C content. Consequently, 25 isochores longer than 2 Mb (megabases) have been identified in the chicken genome. These isochores have a fairly homogeneous G + C content and often correspond to meaningful biological units. With the aid of the technique of cumulative GC profile, we proposed an intuitive picture to display the distribution of segmentation points. The relationships between G + C content and the distributions of genes (CpG islands, and other genomic elements) were analyzed in a perceivable manner. The cumulative GC profile, equipped with the new segmentation algorithm, would be an appropriate starting point for analyzing the isochore structures of higher eukaryotic genomes.     

Delineating relative homogeneous G+C domains in DNA sequences

The concept of homogeneity of G+C content is always relative and subjective. This point is emphasized and quantified in this paper using a simple example of one sequence segmented into two subsequences. Whether the sequence is homogeneous or not can be answered by whether the two-subsequence model describes the DNA sequence better than the one-sequence model. There are at least three equivalent ways of looking at the 1-to-2 segmentation: Jensen-Shannon divergence measure, log likelihood ratio test, and model selection using Bayesian information criterion. Once a criterion is chosen, a DNA sequence can be recursively segmented into multiple domains. We use one subjective criterion called segmentation strength based on the Bayesian information criterion. Whether or not a sequence is homogeneous and how many domains it has depend on this criterion. We compare six different genome sequences (yeast S. cerevisiae chromosome III and IV, bacterium M. pneumoniae, human major histocompatibility complex sequence, longest contigs in human chromosome 21 and 22) by recursive segmentations at different strength criteria. Results by recursive segmentation confirm that yeast chromosome IV is more homogeneous than yeast chromosome III, human chromosome 21 is more homogeneous than human chromosome 22, and bacterial genomes may not be homogeneous due to short segments with distinct base compositions. The recursive segmentation also provides a quantitative criterion for identifying isochores in human sequences. Some features of our recursive segmentation, such as the possibility of delineating domain borders accurately, are superior to those of the moving-window approach commonly used in such analyses.     

Development of T. aestivum L.eH. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum(2n=2x=14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring(CS)eH. californicum amphidiploid(2n=6x=56, AABBDDHH) was established. By genomic in situ hybridization(GISH) and multicolor fluorescent in situ hybridization(FISH) using repetitive DNA clones(pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheatealien chromosome lines, including four disomic addition lines(DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines(MtH7L,MtH1 S, MtH1 L, DtH6 S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line(DSH4) and one translocation line(TH7S/1BL), were identified from the progenies derived from the crosses of CSeH. californicum amphidiploid with common wheat varieties. A total of 482 EST(expressed sequence tag) or SSR(simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2,H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2Hc, 6Hc, 3Hcand 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hcand 4Hc, respectively, by referring to SSR markers located on rye chromosomes.