Gene distribution and isochore organization in the nuclear genome of plants.

The genomic distribution of 23 nuclear genes from three dicotyledons (pea, sunflower, tobacco) and five monocotyledons of the Gramineae family (barley, maize, rice, oat, wheat) was studied by localizing these genes in DNA fractions obtained by preparative centrifugation in Cs2SO4/BAMD density gradients. Each one of these genes (and of many other related genes and pseudogenes) was found to be located in DNA fragments (50-100 Kb in size) that were less than 1-2% GC apart from each other. This definitively demonstrates the existence of isochores in plant genomes, namely of compositionally homogeneous DNA regions at least 100-200 Kb in size. Moreover, the GC levels of the 23 coding sequences studied, of their first, second and third codon positions, and of the corresponding introns were found to be linearly correlated with the GC levels of the isochores harboring those genes. Compositional correlations displayed increasing slopes when going from second to first to third codon position with obvious effects on codon usage. Coding sequences for seed storage proteins and phytochrome of Gramineae deviate from the compositional correlations just described. Finally, CpG doublets of coding sequences were characterized by a shortage that decreased and vanished with increasing GC levels of the sequences. A number of these findings bear a striking similarity with results previously obtained for vertebrate genes.     

The compositional distribution of coding sequences and DNA molecules in humans and murids

Summary The compositional distributions of coding sequences and DNA molecules (in the 50-100-kb range) are remarkably narrower in murids (rat and mouse) compared to humans (as well as to all other mammals explored so far). In murids, both distributions begin at higher and end at lower GC values. A comparison of homologous coding sequences from murids and humans revealed that their different compositional distributions are due to differences in GC levels in all three codon positions, particularly of genes located at both ends of the distribution. In turn, these differences are responsible for differences in both codon usage and amino acids. When GC levels at first+second codon positions and third codon positions, respectively, of murid genes are plotted against corresponding GC levels of homologous human genes, linear relationships (with very high correlation coefficients and slopes of about 0.78 and 0.60, respectively) are found. This indicates a conservation of the order of GC levels in homologous genes from humans and murids. (The same comparison for mouse and rat genes indicates a conservation of GC levels of homologous genes.) A similar linear relationship was observed when plotting GC levels of corresponding DNA fractions (as obtained by density gradient centrifugation in the presence of a sequence-specific ligand) from mouse and human. These findings indicate that orderly compositional changes affecting not only coding sequences but also noncoding sequences took place since the divergence of murids. Such directional fixations of mutations point to the existence of selective pressures affecting the genome as a whole.     

The distribution of 5-methylcytosine in the nuclear genome of plants.

We have determined the 5-methylcytosine (5mC) content in high molecular weight DNA, from two dicot (tobacco and pea) and two monocot (wheat and maize) plant species, fractionated according to base composition. The results show that the proportion of 5mC in the genomic fractions increases linearly with their guanine + cytosine (G + C) content while the proportion of non-methylated cytosine remains almost constant. This can be interpreted as a consequence of a difference in mutation pressure related to spontaneous deamination of 5mC to thymine between the different compartments of plant genomes.     

The compositional organization and the expression of the Arabidopsis genome

The base composition patterns of genes, coding sequences and gene expression levels were analyzed in the available long sequences (contigs) of Arabidopsis. Chromosome 5 was analyzed in detail and all chromosomes for which sequence data are now available show essentially the same large-scale compositional properties. Guanine+cytosine levels of genes and of their coding regions, as well as gene densities and expression levels, all show a marked tendency to be higher in the distal regions of Arabidopsis chromosomes.     

真核生物基因组中的非编码序列

真核生物基因组绝大部分是非编码序列;绝大部分非编码序列以高度重复序列的形式存在,如卫星、小卫星、微卫星、长散布元件、短散布元件等;内含子、３’不译区作为结构基因的一部分被一同转录;ＲＮＡ基因转录具有明确功能的ＲＮＡ分子;顺式作用元件是目前已知的具有重要调控功能的非编码序列;非编码序列的存在与真核生物基因表达调控密切相关;目前非编码序列的研究已引起广泛的科学关注,利用数理方法研究其遗传信息的储存方式     

Correlations between coding and contiguous non-coding sequences in isochore families from vertebrate genomes

Many years ago compositional correlations were found to hold between coding and contiguous non-coding sequences. These correlations were essentially studied in whole genomes of mammals, which are characterized by strong compositional heterogeneities. Here we investigated whether these correlations also hold within the much more homogeneous isochore families. This point was checked not only in the case of mammals, but also in that of phylogenetically distant vertebrates, which are characterized by very different compositional patterns. Indeed, these are remarkably different in cold- and warm-blooded vertebrates. Fish genomes, for instance, are much more homogeneous than those of mammals and birds. The compositional correlations between coding sequences and the corresponding introns, or their 5′ and 3′ flanking regions, were studied in the isochore families of the fully sequenced genomes from four fishes (Brachydanio rerio, Oryzias latipes, Gasterosteus aculeatus and Tetraodon nigroviridis), human and chicken.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

基因组中开阅读框架长度的分布模型与基因组进化

分析了5种真核、15种细菌和10种古菌基因组中开阅读框架(open reading flame,ORF)的数目随长度的分布,发现不同生物的分布相似且有明显的规律性。用各种分布模型进行拟合比较,结果显示每种生物的这类分布均符合Г（α,β）分布,由此提出生物基因组中ORF的数目随长度的分布是Г（α,β）分布的假设。分析各生物基因组的拟合参数,发现α和β值与基因组进化存在明显的相关性;讨论了α和β值的生物进化意义,并给出了真核生物偏好使用长基因的结论;依照Г（α,β）分布估计了酵母基因组中ORF数目的上限为5870个。该方法对于研究生物基因组进化以及评估理论预测基因的可靠性具有建设性意义。     

Compositional compartmentalization and compositional patterns in the nuclear genomes of plants.

We report here results which indicate (i) that the nuclear genomes of angiosperms is characterized by a compositional compartmentalization and an isochore structure; and (ii) that the nuclear genomes of some Gramineae exhibit strikingly different compositional patterns compared to those of many dicots. Indeed, the compositional distribution of nuclear DNA molecules (in the 50-100 Kb size range) from three dicots (pea, sunflower and tobacco) and three monocots (maize, rice and wheat) were found to be centered around lower (41%) and higher (45% for rice, 48% for maize and wheat) GC levels, respectively (and to trail towards even higher GC values in maize and wheat). Experiments on gene localization in density gradient fractions showed a remarkable compositional homogeneity in vast (greater than 100-200 Kb) regions surrounding the genes. On the other hand, the compositional distribution of coding sequences (GenBank and literature data) from dicots (several orders) was found to be narrow, symmetrical and centered around 46% GC, that from monocots (essentially barley, maize and wheat) to be broad, asymmetrical and characterized by an upward trend towards high GC values, with the majority of sequences between 60 and 70% GC. Introns exhibited a similar compositional distribution, but lower GC levels, compared to exons from the same genes.     

Proto-oncogene homologous sequences in the sea urchin genome

Using probes specific for several oncogenes/proto-oncogenes we have performed gel blot hybridization analyses of genomic DNA isolated from the sea urchinStrongylocentrotus droebachiensis. Probes prepared from v-erbB, v-myc, c-myb and v-fps were found to hybridize with discrete fragments of HindIII digested genomic DNA. In contrast, probes prepared from v-abl, v-fos, v-sis, v-src, and v-mos either hybridized with multiple fragments, indicating non-specific binding, or failed to hybridize at all above background levels. These results clearly demonstrate the presence of proto-oncogene homologous sequences in the sea urchin genome.     

Diversity in isochore structure among cold-blooded vertebrates based on GC content of coding and non-coding sequences

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.     

Human cytomegalovirus genome: partial denaturation map and organization of genome sequences.

Contour-length measurements of both nondenatured and partially denatured DNA from purified extracellular human cytomegalovirus indicate that more than one size class of viral DNA is encapsidated. In addition to a size class averaging about 100 x 10(6) daltons, a much less abundant class of larger viral DNA molecules, 150 x 10(6) to 155 x 10(6) daltons, was extracted from purified extracellular virus. As predicted by melting-curve analysis, partial denaturation of human cytomegalovirus DNA generates denaturation maps showing distinctive adenine plus thymidine (A+T)-rich and guanine plus cytosine (G+C)-rich localizations. Alignment of partial denaturation maps of both 100 x 10(6)- and 150 x 10(6)- to 155 x 10(6)-dalton molecules from maximum overlap of common A+T- and G+C-rich zones clearly shows six unique zones contained in a length equal to the longest class, 150 x 10(6) to 155 x 10(6) daltons. However, various alignments of the smaller class of the molecules within the confines of the approximately 100 x 10(6)-dalton-length equivalent are nondistinctive. Of the six unique A+T- and G+C-rich zones, five are linked in a specific sequence and maintain the same relative orientation; these features indicate the absence of major inversions within these zones. The sixth unique zone may occur at either end of this five-zone series, but it was never found at both ends of the same molecule. Additionally, this terminal zone appears to undergo complete inversions at least at one end of the alignment, and perhaps at both. These data indicate that 150 x 10(6)- to 155 x 10(6)-dalton molecules comprise human cytomegalovirus-specific genetic information.     

Number and organization of actin-related sequences in the mouse genome

Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.     

The genome: an isochore ensemble and its evolution

The genomes of eukaryotes are mosaics of isochores. These are long DNA stretches that are fairly homogeneous in base composition and that belong to a small number of families characterized by different ratios of GC to AT and different short-sequence patterns (i.e., different DNA structures that interact with different proteins). This genome organization led to two discoveries: (1) the genomic code, which refers to two correlations, that of the composition of coding and contiguous noncoding sequences, and that of coding sequences and the structural properties of the encoded proteins; and (2) the genome phenotypes, which correspond to the patterns of isochore families in the genomes. These patterns indicate that genome evolution may proceed either according to a conservative mode or to a transitional (isochore shifting) mode, apparently depending upon whether the environment is constant or shifting. According to the neoselectionist theory, natural selection is responsible for both modes.     

The evolution of nuclear genome structure in seed plants

Plant nuclear genomes exhibit extensive structural variation in size, chromosome number, number and arrangement of genes, and number of genome copies per nucleus. This variation is the outcome of a set of highly active processes, including gene duplication and deletion, chromosomal duplication followed by gene loss, amplification of retrotransposons separating genes, and genome rearrangement, the latter often following hybridization and/or polyploidy. While these changes occur continuously, it is not surprising that some of them should be fixed evolutionarily and come to mark major clades. Large-scale duplications pre-date the radiation of Brassicaceae and Poaceae and correlate with the origin of many smaller clades as well. Nuclear genomes are largely colinear among closely related species, but more rearrangements are observed with increasing phylogenetic distance; however, the correlation between amount of rearrangement and time since divergence is not perfect. By changing patterns of gene expression and triggering genome rearrangements, novel combinations of genomes (hybrids) may be a driving force in evolution.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates.

Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.     

Detection of homologous oncogene sequences in the genome of Plasmodium falciparum

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.     

Biological implications of isochore boundaries in the human genome

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.