Single-copy sequence homology among the GC-richest isochores of the genomes from warm-blooded vertebrates

We have hybridized a human DNA fraction corresponding to the GC-richest and gene-richest isochore family, H3, on compositional fractions of DNAs from 12 mammalian species and three avian species, representing eight and three orders, respectively. Under conditions in which repetitive sequences are competed out, the H3 isochore probe only or predominantly hybridized on the GC-richest fractions of main-band DNA from all the species investigated. These results indicate that single-copy sequences from the human H3 isochores share homology with sequences located in the compositionally corresponding compartments of the vertebrate genomes tested. These sequences are likely to be essentially formed by conserved coding sequences. The present results add to other lines of evidence indicating that isochore patterns are highly conserved in warm-blooded vertebrate genomes. Moreover, they refine recent reports (Sabeur et al., 1993; Kadi et al., 1993), and correct them in some details and also in demonstrating that the shrew genome does not exhibit the general mammalian pattern, but a special pattern.Correspondence to: G. Bernardi     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Different hydrophobicities of orthologous proteins from Xenopus and human

A compositional transition was previously detected by comparing orthologous coding sequences from cold- and warm-blooded vertebrates (see Bernardi, G., Hughes, S., Mouchiroud, D., 1997. The major compositional transitions in the vertebrate genome. J. Mol. Evol. 44, S44-S51 for a review). The transition is characterized by higher GC levels (GC is the molar ratio of guanine+cytosine in DNA) and, especially, by higher GC3 levels (GC3 is the GC level of third codon positions) in coding sequences from warm-blooded vertebrates. This transition essentially affects GC-rich genes, although the nucleotide substitution rate is of the same order of magnitude in both GC-poor and GC-rich genes. In order to understand the evolutionary basis of the changes, we have compared the hydrophobicity of orthologous proteins from Xenopus and human. Although the differences are small in proteins encoded by coding sequences ranging from 0 to 65% in GC3, they are large in the proteins encoded by sequences characterized by GC3 values higher than 65%. The latter proteins are more hydrophobic in human than in Xenopus.     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.     

Replication timing of two human common fragile sites: FRA1H and FRA2G

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.     

Molecular cytogenetics of the equidae. II. purification and cytological localization of a (G + C)-rich satellite DNA from Equus hemionus onager and cross-species hybridization to E. asinus chromosomes

A (G + C)-rich satellite DNA component (p = 1.716 g/ml) has been fractionated from the total DNA of the Iranian subspecies of the Asiatic wild ass, Equus hemionus onager, by successive dactinomycin-CsCl and netropsin sulfate-CsCl isopycnic gradients. Complementary 3H-RNA (cRNA) transcribed from the satellite DNA hybridized predominantly to the centromeric and telomeric constitutive heterochromatic regions of onager chromosomes. These studies have suggested that satellite DNA's with similar sequences are present in the centromeric, as well as telomeric, heterochromatic regions of some onager chromosomes. The centromeric region of the fusion metacentric t(23;24) of the onager is deficient in sequences homologous to the onager 1.716 g/ml satellite DNA, indicating a loss of satellite DNA during fusion or an amplification of the satellite DNA in the centromeric regions of the acrocentric chromosomes 23 and 24 subsequent to fission. Sequences complementary to onager 1.716 g/ml satellite DNA show extensive hybridization to the constitutive heterochromatin of the feral donkey (E. asinus) karyotype, consistent with a view of conservation and amplification of similar or identical sequences in the two species.     

Surrogate origins of replication in the mitochondrial genomes of ori-zero petite mutants of yeast.

We have investigated the mitochondrial genome of eight ori-zero spontaneous petite mutants of Saccharomyces cerevisiae. The tandem repeat units of these genomes do not contain any of the seven canonical ori sequences of the wild-type genome. Instead, they contain one, or more, ori-S sequences. These 44-nucleotide long surrogate origins of replication are a subset of GC clusters characterized by a potential secondary fold with two sequences ATAG and GGAG , inserted in AT spacers, two AT base pairs just following them, a GC stem (broken in the middle, and, in most cases also near the base, by non-paired nucleotides), and a terminal loop. This structure is reminiscent of that of GC clusters A and B from canonical ori sequences and supports the view (Bernardi, 1982a ) that the GC clusters of the mitochondrial genome arose, by an expansion process, from the canonical ori sequences. Like the latter, ori-S sequences are present in both orientations, are located in intergenic regions, and can be used as excision sequences when tandemly oriented. Again as in the case of canonical ori sequences, the density of ori-S sequences on the repeat units of petite genomes are correlated with the replication efficiency of the latter, as assessed by the outcome of crosses with wild-type or petite tester strains.     

CpG deficiency, dinucleotide distributions and nucleosome positioning

The dinucleotide CpG is deficient in (A + T)-rich regions of vertebrate DNA in both coding and non-coding sequences and there is a corresponding increase above expectation in the occurrence of TpG and CpA. By contrast in (G + C)-rich regions no deficiency of CpG is found. Such (G + C)-rich sequences, containing the expected number of CpG dinucleotides, alternate along the genome with (A + T)-rich sequences which have a lower than expected CpG content. The G + C content of vertebrate DNA can oscillate with a period of 150-200 bp and this may be a factor in positioning nucleosomes. The role of mutagenesis in loss of CpG and increase of A + T, particularly in non-coding regions, is discussed.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA

The presence of deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA has been demonstrated by hybridization with ³H-labelled poly(U) and ³H-labelled poly(C). For hamster BHK-21/C13 cells, the dA-rich regions are up to 130 nucleotides long and comprise up to 0.4% of the DNA. Those dA-rich regions which comprise 0.13% of the DNA contain 2 to 6% of bases other than adenine. The dG-rich regions, in which 10 to 30% of the bases are other than guanine, are less than 40 nucleotides long and are present at a level of about 0.1% of the DNA. Exhaustive digestion of the hybrids with RNAase enables detection of deoxyhomopolymeric regions in the DNA, poly (dA) sequences of an average size of about 30 nucleotides long accounting for 0.008% of the DNA, and poly(dG) sequences, 17 nucleotides long, comprising 0.0016% of the DNA.Both dA-rich and dG-rich regions are found in DNA sequences with a wide variety of base composition. Extensive shearing of the DNA is required to produce some enrichment for dA-rich sequences in the (A + T)-rich fraction, although dG-rich sequences are slightly enriched in the (G + C)-rich fraction of even unsheared DNA. The buoyant density of hybrid molecules was found to be significantly greater than that of unhybridized DNA only when highly sheared DNA was used. These findings suggest that the dA-rich and dG-rich regions have a widespread distribution throughout DNA molecules. In situ hybridization studies with ³H-labelled poly(U) further suggest that the dA-rich regions are not localized to any particular chromosome or to any specific region of the chromosomes. Analysis of DNA from a number of different species has shown that, in general, the dA-rich and dG-rich regions are present at a much higher level in mammalian DNA than in bacterial, bacteriophage or mammalian virus DNA.Possible functions of these unusual deoxynucleotide sequences are discussed.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.     

Site-specific deletions in the recombinant plasmid pSC101 containing the redB-ori region of phage lambda

Large deletions occur in the hybrid plasmid formed by pSC101 and the EcoRI fragment f2 of phage lambda (redB-ori region) under well defined growth conditions (Bernardi and Bernardi, 1980). We have sequenced the novel joints of the four deletions so obtained and shown that they have one endpoint in pSC101, identical in all four cases, the other endpoint being located in four different lambda sequences. Furthermore, the nucleotide sequences of the novel joints show homologies between the conserved pSC101 sequence and the lambda sequences both conserved and deleted. The presence of an IS-type element in pSC101 is postulated; however, this element is unrelated to the 200 bp element already described in pSC101 (Ravetch et al., 1976).     

Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.