The question of DNA repair in hyperthermophilic archaea

Hyperthermophilic archaea grow at temperatures that destabilize the primary structure of DNA and in evolutionary terms they are highly divergent from other well studied microorganisms. These prokaryotes should therefore require DNA damage repair to be unusually effective, and could employ novel mechanisms for this repair. Recent genome sequence analyses and biochemical and genetic assays suggest a distribution of DNA repair strategies that raises intriguing questions for future study.     

On the Organization of Higher Chromosomes

OHTA and Kimura¹ have argued that only about 6% of the sequences in mammalian DNA can be under the intense selection that has characterized the evolutionary history of the cytochromes c, the globin chains and the histones. From the calculated mutation rate of fibrinopeptides A and B they show that if all genes are subjected to the same mutation rate 8.3 mutations would accumulate per genome per generation. Because 0,5 deleterious mutations per genome per generation is the maximum allowable in an equilibrium population², they conclude that the amount of DNA that codes for informational sequences such as the cytochromes, globins and histones must be no more than 0.5/8.3, or 6%. We are therefore left with the interesting observation that 94% of mammalian nuclear DNA serves a function not under strong selection. These authors make several assumptions, one of which is that the spontaneous mutation rate characteristic of a species is constant over all nucleotide sequences. I suggest here that this assumption is incorrect, for a variety of reasons and that by assuming that spontaneous mutation rates vary sequence by sequence, one can arrive at a plausible organizing principle for the structure of higher chromosomes.     

Cloning and characterization of a highly conserved satellite DNA from the mollusc Mytilus edulis.

Sperm DNA of the common mussel, Mytilus edulis, has been found to contain a highly repeated sequence identifiable upon restriction with the endonuclease ApaI. The repetitive nucleotide (nt) sequence amounts to 0.63% of the mollusc genome with an estimated copy number of 5.4 x 10(4) copies per haploid complement. The monomer unit with a 173-bp repeat length has been cloned. Progressive DNA digestions with ApaI yield ladder-like banding patterns on agarose gels, indicating that the repeated elements are tandemly arranged in the genome and therefore represent a sequence of satellite DNA. The degree of internal redundancy of the reiterated sequence is deemed negligible, since nt sequence analysis of a random set of cloned monomers has detected the presence of only a few direct repeats while inverted repeated motifs or any other internal substructures appear absent. The homologies found among cloned monomers are strikingly high, averaging 95%. The results suggest that the exceptional sequence homogeneity of this satellite DNA may be attributed either to some homogenizing mechanism or to evolutionary conserved trends.     

The genetic code in bovine mitochondria: sequence of genes for the cytochrome oxidase subunit II and two tRNAs

Bovine-heart mitochondrial DNA from a single animal was isolated and fragments representative of the entire genome cloned into multicopy plasmid vectors to facilitate determination of its complete nucleotide sequence. We present here the sequence of the region covering the gene for cytochrome oxidase subunit II. Comparison of this sequence with the amino acid sequence of the homologous beef-heart protein has enabled the determination of most of the bovine mitochondrial genetic code. The code differs from the "universal" genetic code in that UGA codes for tryptophan and not termination, and AUA codes for methionine and not isoleucine. The only codon family not represented is the AGA/AGG pair normally used for arginine; evidence from other genes suggests that these code for termination in bovine mitochondria. The sequence presented also includes the adjacent tRNAAsp and tRNALys genes. The tRNAAsp gene is separated by one nucleotide from the 5' end of the COII gene and only three bases separate the 3' end of this gene and the adjacent tRNALys gene. This highly compact gene organisation is very similar to that found in the corresponding region of the human mitochondrial genome and the gene arrangement is identical. The structure of the respective bovine and human tRNAs vary primarily the "D-" and "T psi C-loops".     

Recombination in simian virus 40-infected cells. Structure of naturally arising variants ev-2114, ev-2102, and ev-1110

The complete nucleotide sequence has been determined for three newly cloned evolutionary variants from two different independently generated evolutionary series (1100 and 2100 series) of simian virus 40 (SV40). These naturally arising variants, designated ev-1110, ev-2102, and ev-2114, were isolated after five high multiplicity serial passages. The structure of the variants consists of a monomeric unit tandemly repeated four times (ev-2102 and ev-2114) or six times (ev-1110) in the variant genome; the variants have four or six copies, respectively, of the viral origin signal for DNA replication. The DNA content in the three variants is vastly different in that the genome of variant ev-2114 contains only rearranged viral sequences, while variant ev-2102 contains a substitution with monkey DNA sequences consisting of a nearly complete dimeric unit of Alu family sequences as well as less repetitive sequences and variant ev-1110 contains monkey DNA sequences derived solely from repetitive alpha-component DNA. Recombination events, cellular sequences, and structural features of these and other naturally arising SV40 variants are compared.     

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of a Xenopus laevis mitochondrial DNA fragment containing the D-loop, flanking tRNA genes and the apocytochrome b gene

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial (mt) displacement (D) loop and surrounding genes [Wong et al., Nucl. Acids Res. 11 (1983) 4977-4995] are reported, including addition of two stretches of nucleotides and 60 scattered modifications. The additional sequences presented here correspond to the apocytochrome b gene, the tRNAGlu gene and part of URF6. This allows us to propose a conformational model for the X. laevis apocytochrome b protein and also permits comparisons with mammalian mtDNA. The D-loop sequence is poorly conserved except for sequences involved in the regulation of the mt genome (conserved sequence blocks and the DNA polymerase stop sequences). On the other hand, all genes show marked conservation both of their nucleotide sequence and their respective location on the mt genome. Organization of the genetic information described for mammalian mtDNA also holds for the X. laevis mtDNA. This result strongly suggests that all animal vertebrate mtDNAs have followed the same evolutionary pathway.     

Gradual evolution of a specific satellite DNA family in Drosophila ambigua, D. tristis, and D. obscura

The highly repetitive satellite DNA family "ATOC180" is specific for the three closely related species Drosophila obscura, D. ambigua, and D. tristis but does not occur in their closest relatives D. subsilvestris and D. bifasciata. Approximately 10,000 copies/haploid genome of approximately 180-bp repetition units are tandemly arranged in the centromeric heterochromatin of all chromosomes of all three species. Molecular analysis of 29 cloned repeats shows much intra- and interspecific sequence homogeneity. Single nucleotide changes are the main source of variability and distinguish the sequence-, subfamily- and species-specific ATOC180 repeats from each other. Based on these nucleotide differences, phylogenetic dendrograms were constructed and compared with published trees for other traits. The data indicate that the sequences of the ATOC180 satellite DNA family probably arose in a phylogenetically "short period" during the anagenetic evolution of the common ancestor of D. obscura, D. tristis, and D. ambigua, as a consequence of a process of genome reorganization, followed by a "long period" of entirely gradual sequence evolution. For the latter period, an evolutionary rate of 3 x 10(-8) substitutions/site/year was calculated.      

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.     

Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus.

Avian myeloblastosis virus contains a continuous sequence of approximately 1,000 nucleotides which may represent a gene (amv) responsible for acute myeloblastic leukemia in chickens. This sequence appears to have been acquired from chicken DNA and to be substituted for the envelope gene in the viral genome. We used hybridization probes enriched for the amv sequences and conditions that facilitate annealing of partially homologous nucleotide sequences to show that cellular sequences related to amv are present in the genomes of all vertebrates ranging from amphibians to humans but were not detected in fish, sea urchins, or Escherichia coli. In contrast to the preceding findings, nontransforming endogenous proviral nucleotide sequences closely related to the remainder of the avian myeloblastosis virus genome and to the entire myeloblastosis-associated helper virus are present only in chicken DNA. The amv-related cellular sequences appear to be highly conserved during evolution and to be contained at only one or a few locations in the genome of vertebrates. Within closely related species, they appear to share common evolutionary genetic loci. These findings and similar ones obtained with other highly oncogenic retroviruses containing a transforming gene suggest a general mechanism for acquisition of viral oncogenic sequences and an essential role for these sequences in the normal cellular state.     

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

Is mitochondrial DNA a strictly neutral marker?

Variation and change in mitochondrial DNA (mtDNA) is often assumed to conform to a constant mutation rate equilibrium neutral model of molecular evolution. Recent evidence, however, indicates that the assumptions underlying this model are frequently violated. The mitochondria) genome may be subject to the same suite of forces known to be acting in the nuclear genome, including hitchhiking and selection, as well as forces that do not affect nuclear variation. Wherever possible, evolutionary studies involving mtDNA should incorporate statistical tests to investigate the forces shaping sequence variation and evolution.     

Clusters of nucleotide substitutions and insertion/deletion mutations are associated with repeat sequences

The genome-sequencing gold rush has facilitated the use of comparative genomics to uncover patterns of genome evolution, although their causal mechanisms remain elusive. One such trend, ubiquitous to prokarya and eukarya, is the association of insertion/deletion mutations (indels) with increases in the nucleotide substitution rate extending over hundreds of base pairs. The prevailing hypothesis is that indels are themselves mutagenic agents. Here, we employ population genomics data from Escherichia coli, Saccharomyces paradoxus, and Drosophila to provide evidence suggesting that it is not the indels per se but the sequence in which indels occur that causes the accumulation of nucleotide substitutions. We found that about two-thirds of indels are closely associated with repeat sequences and that repeat sequence abundance could be used to identify regions of elevated sequence diversity, independently of indels. Moreover, the mutational signature of indel-proximal nucleotide substitutions matches that of error-prone DNA polymerases. We propose that repeat sequences promote an increased probability of replication fork arrest, causing the persistent recruitment of error-prone DNA polymerases to specific sequence regions over evolutionary time scales. Experimental measures of the mutation rates of engineered DNA sequences and analyses of experimentally obtained collections of spontaneous mutations provide molecular evidence supporting our hypothesis. This study uncovers a new role for repeat sequences in genome evolution and provides an explanation of how fine-scale sequence contextual effects influence mutation rates and thereby evolution.     

Evolution and Phylogenetic Analysis of Full-Length VP3 Genes of Eastern Mediterranean Bluetongue Virus Isolates

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979–2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an ‘eastern’ (BTV-9, -16 and -1) and a ‘western’ (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.