Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.

Gene conversions in genes encoding outer-membrane proteins in H. pylori and C. pneumoniae.

Helicobacter pylori and Chlamydia pneumoniae are both pathogenic to humans. Their genomes have recently been completed, allowing detailed study of their evolution and organization. Here we describe an evolutionary analysis of the H. pylori and C. pneumoniae genes that encode their outer-membrane proteins. By comparing complete genome sequences of two H. pylori strains and two C. pneumoniae strains, we identify multiple independent conversions among these genes. Such recombination events might provide a selective advantage for these bacterial pathogens.

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.

The Zn-peptidase superfamily: functional convergence after evolutionary divergence.

Zn-dependent carboxypeptidases (ZnCP) cleave off the C-terminal amino acid residues from proteins and peptides. Here we describe a superfamily that unites classical ZnCP with other enzymes, most of which are known (or likely) to participate in metal-dependent peptide bond cleavage, but not necessarily in polypeptide substrates. It is demonstrated that aspartoacylase (ASP gene) and succinylglutamate desuccinylase (ASTE gene) are members of the ZnCP family. The Zn-binding site along with the structural core of the protein is shown to be conserved between ZnCP and another large family of hydrolases that includes mostly aminopeptidases (ZnAP). Both families (ZnCP and ZnAP) include not only proteases but also enzymes that perform N-deacylation, and enzymes that catalyze N-desuccinylation of amino acids. This is a result of functional convergence that apparently occurred after the divergence of the two families.

Thermolysin and mitochondrial processing peptidase: how far structure-functional convergence goes.

The structure-functional convergence between two Zn-dependent proteases, namely thermolysin and mitochondrial processing peptidase (MPP), is described. These two families of nonhomologous enzymes show not only functional convergence of several active site residues as in chymotrypsin and subtilisin, but also structural convergence of overall molecular architectures including the beta-sheet arrangement and packing of the surrounding alpha-helices. The major functionally important structural elements are present in both enzymes with different topological connections and often in reverse main-chain orientation, but display similar packing. The structural comparison helps to rationalize sequence "inversion" of the HEXXH thermolysin consensus present as HXXEH in MPP. The described structural convergence may be due to a limited number of alternatives to build a Zn-protease that utilizes hydrogen bonding between a substrate main chain and the enzyme beta-sheet for substrate binding.

A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases.

Computer analysis using profiles generated by the PSI-BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.

Short repeats and IS elements in the extremely radiation-resistant bacterium Deinococcus radiodurans and comparison to other bacterial species.

Computer analysis of the complete genome of Deinococcus radiodurans R1 has shown that the number of insertion sequences (ISs) and small noncoding repeats (SNRs) it contains is very high, and comparable with those of Escherichia coli. IS elements and several families of SNRs are described, together with their possible function in the D. radiodurans genome.

Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast.

Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms. In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog.

[Search for functional domain boundaries and localization of functional sites based on oligopeptide vocabularies]. / Poisk granits funktsional'nykh domenov i lokalizatsiia funktsional'nykh saitov na osnove oligopeptidnykh slovarei.

A new method based on the analysis of oligopeptide composition of the amino acid sequences from different protein families is presented. We assume, that any protein family can be characterized by the set of oligopeptides (oligopeptides vocabulary). We demonstrate, that oligopeptides vocabulary comparison can distinguish different families from each other and from random sequences. It should be noted, that this comparison can be successfully performed on the set of only 25 dipeptides and without preliminary alignment. We demonstrate, that characteristic peptides are localized in the regions of functional significance, as shown on the example of GTP-binding domain of translation elongation factors. We suggest how to use this method to localize the boundaries of functional domains in amino sequences. On the example of few functional domains we demonstrate, that the average error of prediction does not exceed 3-4 amino acid residue.

["DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes]. / "DIROM"--interaktivnaia sistema dlia planirovaniia éksperimentov po napravlennomu mutagenezu i konstruirovaniiu iskusstvennykh genov.

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.

[A small open reading frame of the Stalker retrotransposon reveals a high similarity to the second small frame of the MDG1 retrotransposon]. / Malaia otkrytaia ramka schityvaniia retrotranspozona Stalker obnaruzhivaet vysokoe skhodstvo so vtoroi maloi ramkoi retrotranspozona MDG1.

Sequence analysis of a 5' terminal fragment of stalker retrotransposon, about 1600 bp in length, revealed a number of motifs in the nucleotide sequences of the LTR and other untranslated regions that are common to retrotransposons 412 and MDG1. Taken together, experimental data and computer analysis of this sequence suggest that stalker falls into the gypsy group of retrotransposons and most closely resembles related retrotransposons 412 and MDG1. Stalker bears a small open reading frame (sORF) that shares 24 and 75% identical amino acid residues with the second sORFs of retrotransposons 412 and MDG1, respectively. Thus high similarity between the discovered sORF of stalker and the corresponding frame of MDG1 looks intriguing, since other regions of the sequences display little resemblance. Therefore, the evolutionary proximity of stalker to MDG1 cannot be conjectured solely on the basis of the mentioned similarity. The similarity may be explained by recombination of the two transposons, or, alternatively, the frame can retain its function in the genomes of stalker and MDG1 and lose it in 412.

[Different patterns of molecular evolution of influenza A viruses in avian and human population]. / Razlichie rezhimov molekuliarnoi évoliutsii virusov grippa A v populiatsiiakh ptits i cheloveka.

Patterns of molecular evolution of the influenza virus proteins and genes are discussed. The subsets of all viral genes corresponding to statistically significant clusters on dendrogram were shown to fall into two distinct groups. The first group was characterized by the presence of an exact linear relationship between the year of the strain isolation and the evolutionary distance. The subsets of human influenza virus genes belong to this group. A method for eliminating the "frozen" strains from the subsets and for calculating the evolutionary rates without construction of phylogenetic trees has been elaborated. The substitution rates calculated according to this technique agreed with the data obtained previously. A linear relationship was not observed in the second group. This group was predominantly composed of avian influenza virus genes. The lack of linear correlation pointed to the cocirculation of a large amount of different influenza virus genomic segments in the avian population. An approach for an examination of the role of intragenic recombination in the development of the antigenic subtypes of hemagglutinin is suggested. Our results suggest that recombination did not play a considerable role in this process, and that all modern subtypes of this protein were probably formed before the introduction of the influenza viruses into the human population. These findings are consistent with the hypothesis that influenza viruses penetrated into human population from their pools in avian populations.

[Evolution modes of eukaryote retrotransposons]. / Rezhimy évoliutsii retrotranspozonov éukariot.

A number of general problems of molecular macroevolution of retroposons were examined, including the question of the ratio of the contributions of genomic replication and retrotransposition to mutational variability of retrotransposons, as well as that of the influence of stress-induced transpositional variability on the rate of evolution and the phylogenetic trees of retrotransposons. It is thought that the substitution fixation rate in genes of retrotransposons is determined by the transposition rate and the probability of mutation upon replication of copies of retransposons in the genome and upon retrotransposition, as well as by selection stabilizing the function of proteins of the retroreplicative mechanism. By means of molecular-evolutionary parameters, estimated for Drosophila retrotransposons and animal retroviruses, it was shown that spontaneous retrotransposition makes the dominant contribution to the frequency of mutation, and the expected fixation rate of nucleotide substations is on the order of 2 x 10(-8) per position per year. This version of evolution of retrotransposons agrees with the results of phylogenetic analysis of trees of macroevolution. Another version, associated with stress-induced transpositions, gives a very high rate of evolution, on the order 3 x 10(-6) fixations of nucleotide substitutions per position per year. This version seems improbable, as it leads to a significant hidden genetic load.

[Evolution of gypsy-group retrotransposons: phylogenetic analysis of domains included in the pol-polyprotein]. / Evoliutsiia retrotranspozonov gypsy-gruppy: filogeneticheskii analiz domenov, vkhodiashchikh v sostav pol-poliproteina.

Transposons of gypsy group are assigned to LTR-containing retrotransposons present in the genomes of invertebrates, fungi, and plants. In this work, a theoretical analysis of the potential products of ORFs of these retrotranposons was conducted. Alignments were obtained and trees of similarity were constructed for domains of the POL region. On the basis of the obtained data, two hypothetically monophyletic subgroups of transposons were distinguished within the framework of the gypsy group, settling the genomes of taxonomically related organisms (the subgroup of "true" gypsy of insects and the subgroup of gypsy-like transposons of plants and fungi). A number of peculiarities of the topology of these trees hypothetically indicate cases of genetic conversion and recombination of domains accompanying the evolution of this group. The amino acid substitution fixation rate was evaluated on the basis of comparison of sequences of the protein products of ORFs. Estimates of the time of divergence of subgroups of gypsy-group transposons are significantly less than estimates of the times of divergence of their host species. One explanation for this discrepancy might be the hypothesis of settlement by transposons of the genomes of isolated host species.

[Comparison of the synonymous and non-synonymous substitution rates in various regions of the neuraminidase and hemagglutinin genes of the influenza A virus]. / Sravnenie skorosti nakopleniia sinonimicheskikh i nesinonimicheskikh zamen dlia razlichnykh raionov genov neiraminidazy i gemaggliutinina virusa grippa A.

A new, statistically justified approach was used to estimate the synonymous and non-synonymous substitution rates in several antigenic variants of influenza-virus surface proteins. The rates were compared for antigenic and nonantigenic regions of neuraminidase and hemagglutinin, as well as for neuraminidase surface and internal amino acids identified by X-ray analysis. For neuraminidase, the estimation was performed for the first time. The non-synonymous substitution rate was shown to be significantly higher in antigenic than in nonantigenic sites. However, neither subsample of antigenic sites displayed a fixation rate of non-synonymous substitutions higher than that of synonymous substitutions, which would confirm the effect of positive selection on these sites and argue against a neutral evolution character. Specific features of methods used to estimate the substitution fixation rates and problems in their interpretation are discussed.

[Gene Nc73EF of Drosophila melanogaster encodes a protein highly homologous to E1 subunit of human 2-oxoglutarate dehydrogenase]. / Gen Nc73EF Drosophila melanogaster kodiruet belok, vysoko gomologichnyi E1-sub"edinitse 2-oksoglutaratdegidrogenazy cheloveka.

The primary structure of the cDNA copy of the evolutionary conserved gene Nc73EF from the region 73EF of Drosophila melanogaster was determined. Gene Nc73EF was shown to encode a protein highly homologous to the E1 subunit of human 2-oxoglutarate dehydrogenase, which catalyzes one of the key reactions of the Krebs cycle.

A novel method of protein sequence classification based on oligopeptide frequency analysis and its application to search for functional sites and to domain localization.

A new method for distinguishing among protein families based on the analysis of oligopeptide composition of amino acid sequences is presented. It is assumed that any protein family can be characterized by a set of essential oligopeptides (oligopeptide vocabulary). A simple approach to find such a vocabulary is suggested. It is shown that comparison of the vocabularies can distinguish among different families and the latter from random sequences. This comparison can be successfully made with a small set of frequencies of 25 dipeptides (or tripeptides). No preliminary alignment is necessary. It is established that characteristic peptides are located in the regions of functional value, as shown for GTP-binding domains of the translation elongation factors. It is demonstrated that this method is reasonably efficient for localizing functional domains in the amino acid sequences. The average error of prediction does not exceed three or four amino acid residues as shown for several functional domains.