Nonstructural p26 proteins encoded by the 3'-proximal genes of velariviruses and criniviruses are orthologs.

The 3'-most genes in RNA-2 of the Crinivirus genus members (family Closteroviridae) code for non-structural p26 proteins that share amino acid sequence similarity [Stewart LR, Hwang MS, Falk BW (2009) Virus Res 145:293-299]. In this study, sensitive bioinformatic tools have been used to identify the homologous p26 proteins encoded by the 3' genes in monopartite genomes of the members of Velarivirus, another Closteroviridae genus, and mint vein banding-associated virus, an unassigned member of the family. The p26 proteins showed similarity in their predicted secondary structures, but an amino acid sequence alignment showed no strictly conserved positions, thus indicating a high plasticity of these non-structural proteins. The implications of the sequence analysis for possible functions of the crinivirus and velarivirus p26 proteins are discussed.

Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast.

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.

Analysis of mitochondrial DNA somatic mutations in OXYS and Wistar strain rats.

Rats of the OXYS strain are sensitive to oxidative stress and serve as a biological model of premature aging. We have compared spectra of somatic mutations in a control region of mtDNA from the liver of the OXYS rat strain and of Wistar rats as a control. The majority of nucleotide substitutions in the mutation spectra were represented by transitions: 94 and 97% in the OXYS and Wistar rats, respectively. It was shown that 40% of somatic mutations in the control region of mtDNA from Wistar rats were significantly consistent with the model of dislocation mutagenesis. No statistical support for this model was found for mutations in the control region of mtDNA from OXYS rats. The mutation frequency in the ETAS section was higher in the OXYS strain rats than in Wistar rats. These results suggest different mechanisms of mutagenesis in the two rat strains under study.

[Vertebrate immunity: mutator proteins and their evolution].

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes.

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.

Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis.

A new approach for the analysis of hotspots of mutations is described. It is based on the classification of hotspot site sequences. Using this approach, the consensuses RGYW and TAA of hotspot sites were revealed in the V gene. Correlation between somatic mutations and these consensuses is investigated by the statistical weight method in 323 somatic substitutions in 14 V genes. Assuming the absence of any correlation, the probability of observing such data in the sample would be very low (0.0003). These results support the idea that emergence of somatic mutation is significantly influenced by neighbouring base sequences. This idea was also supported by the analysis of 296 somatic mutations in flanking sequences of V genes. It is supposed that this influence is an important feature of somatic hypermutagenesis.

Somatic hypermutagenesis in immunoglobulin genes. I. Correlation between somatic mutations and repeats. Somatic mutation properties and clonal selection.

Based on the analysis of some immunoglobulin V-gene sequences, somatic mutations are assumed to occur by correction of complementary violations in heteroduplexes formed by direct or inverted repeats. Correlation between somatic mutations and repeats is investigated by a statistical weights method in 323 somatic substitutions in 14 V-genes. Assuming absence of correlation, the probability of observing data in the sample would be very low (0.00004). This result supports the idea that somatic mutations may arise from heteroduplex repair. The high frequency of these mutations in complementarity-determining regions (CDRs) of V-genes may be due to a high concentration of repeats in these regions. Analysis of somatic substitutions has revealed that stabilizing selection seems to provide conservation of framework regions (FRs) (which leads to preservation of the protein's three-dimensional structure). Positive selection may be provided by B-lymphocyte proliferation with large changes in CDRs.

Somatic hypermutagenesis in immunoglobulin genes. III. Somatic mutations in the chicken light chain locus.

We report a new approach based on the Monte Carlo method, to analyze gene conversion. With this, we have examined 235 somatic mutations in the chicken Vlambda gene and found that about 75% of somatic mutations significantly correlated with donor sequences in 25 pseudo Vlambda genes (set C) versus about 25% that did not (set N). The RGYW and TAA consensus sequences of mutational hotspots were earlier revealed in mammalian V genes. Analysis for correlation between somatic mutations in the Vlambda gene and the consensus sequences showed that the somatic mutations of set N were correlating with the consensus sequences (P(W < Wrandom) < 0.01) and the somatic mutations of set C were not. Based on further statistical analysis, we suggest that there must be at least two mechanisms responsible for somatic hypermutagenesis in the Vlambda gene: gene conversion and another, which accounts for the elevated frequencies of somatic mutations at the RGYW and TAA consensus sequences.

Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix.

Scaffold/matrix-associated region (S/MAR) sequences are DNA regions that are attached to the nuclear matrix, and participate in many cellular processes. The nuclear matrix is a complex structure consisting of various elements. In this paper we compared frequencies of simple nucleotide motifs in S/MAR sequences and in sequences extracted directly from various nuclear matrix elements, such as nuclear lamina, cores of rosette-like structures, synaptonemal complex. Multivariate linear discriminant analysis revealed significant differences between these sequences. Based on this result we have developed a program, ChrClass (Win/NT version, ftp.bionet.nsc.ru/pub/biology/chrclass/chrclass.zip), for the prediction of the regions associated with various elements of the nuclear matrix in a query sequence. Subsequently, several test samples were analyzed by using two S/MAR prediction programs (a ChrClass and MAR-Finder) and a simple MRS criterion (S/MAR recognition signature) indicating the presence of S/MARs. Some overlap between the predictions of all MAR prediction tools has been found. Simultaneous use of the ChrClass, MRS criterion and MAR-Finder programs may help to obtain a more clearcut picture of S/MAR distribution in a query sequence. In general, our results suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder and MRS.

Error rate and specificity of human and murine DNA polymerase eta.

We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.

Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.

The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

Genome trees constructed using five different approaches suggest new major bacterial clades.

BACKGROUND: The availability of multiple complete genome sequences from diverse taxa prompts the development of new phylogenetic approaches, which attempt to incorporate information derived from comparative analysis of complete gene sets or large subsets thereof. Such attempts are particularly relevant because of the major role of horizontal gene transfer and lineage-specific gene loss, at least in the evolution of prokaryotes. RESULTS: Five largely independent approaches were employed to construct trees for completely sequenced bacterial and archaeal genomes: i) presence-absence of genomes in clusters of orthologous genes; ii) conservation of local gene order (gene pairs) among prokaryotic genomes; iii) parameters of identity distribution for probable orthologs; iv) analysis of concatenated alignments of ribosomal proteins; v) comparison of trees constructed for multiple protein families. All constructed trees support the separation of the two primary prokaryotic domains, bacteria and archaea, as well as some terminal bifurcations within the bacterial and archaeal domains. Beyond these obvious groupings, the trees made with different methods appeared to differ substantially in terms of the relative contributions of phylogenetic relationships and similarities in gene repertoires caused by similar life styles and horizontal gene transfer to the tree topology. The trees based on presence-absence of genomes in orthologous clusters and the trees based on conserved gene pairs appear to be strongly affected by gene loss and horizontal gene transfer. The trees based on identity distributions for orthologs and particularly the tree made of concatenated ribosomal protein sequences seemed to carry a stronger phylogenetic signal. The latter tree supported three potential high-level bacterial clades,: i) Chlamydia-Spirochetes, ii) Thermotogales-Aquificales (bacterial hyperthermophiles), and ii) Actinomycetes-Deinococcales-Cyanobacteria. The latter group also appeared to join the low-GC Gram-positive bacteria at a deeper tree node. These new groupings of bacteria were supported by the analysis of alternative topologies in the concatenated ribosomal protein tree using the Kishino-Hasegawa test and by a census of the topologies of 132 individual groups of orthologous proteins. Additionally, the results of this analysis put into question the sister-group relationship between the two major archaeal groups, Euryarchaeota and Crenarchaeota, and suggest instead that Euryarchaeota might be a paraphyletic group with respect to Crenarchaeota. CONCLUSIONS: We conclude that, the extensive horizontal gene flow and lineage-specific gene loss notwithstanding, extension of phylogenetic analysis to the genome scale has the potential of uncovering deep evolutionary relationships between prokaryotic lineages.

Protein-coding regions prediction combining similarity searches and conservative evolutionary properties of protein-coding sequences.

The gene identification procedure in a completely new gene with no good homology with protein sequences can be a very complex task. In order to identify the protein-coding region, a new method, 'SYNCOD', based on the analysis of conservative evolutionary properties of coding regions, has been realized. This program is able to identify and use the coding region homologies of the non-annotated (unknown) protein-coding sequences already present in the nucleotide sequence databases by using the alignment produced by BLASTN. The ratio of number mismatches resulting in synonymous codons to the number of mismatches resulting in non-synonymous codons is estimated for each open reading frame. Monte Carlo simulations are then used to estimate the significance of the ratio deviation from random behavior. The SYNCOD program has been tested on generated random sequences and on different control sets. The high accuracy of predicting protein-coding regions (the correlation coefficient, CC, varies from 0.67 to 0.79) and the high specificity (the portion of wrong exons, WE, varies from 0.06 to 0.07) have proved to be important features of the suggested approach. The SYNCOD program is resident on the ITBA-CNR Web Server and can be used via the Internet (URL: www.itba.mi.cnr.it/webgene).

Identification of a U7snRNA homologue mapping to the human Xq27.1 region, between the DXS1232 and DXS119 loci.

To contribute to the identification and analysis of novel genes, we undertook the study of a cosmid clone in the Xq27 region of human DNA. The cloned fragment was previously observed to have a high number of evolutionarily conserved sequences. In this genomic stretch of DNA we have identified sequence homologous to the U7 RNA gene including its potential regulatory elements. This paper describes the genomic organisation of this gene and its mapping to the Xq27.1 genomic sub-interval between the DXS1232 and DXS119 loci.

Peculiarities of immunoglobulin gene structures as a basis for somatic mutation emergence.

Using the method of contextual analysis, we have studied the collection of somatic mutations in immunoglobulin genes. It has been found that the emergence of somatic mutations can be based on the reparation of complementarity violations in the heteroduplexes corresponding to complementary palindromes or direct repeats of DNA.

Cloning and functional analysis of SEL1L promoter region, a pancreas-specific gene.

We examined the promoter activity of SEL1L, the human ortholog of the C. elegans gene sel-1, a negative regulator of LIN-12/NOTCH receptor proteins. To understand the relation in SEL1L transcription pattern observed in different epithelial cells, we determined the transcription start site and sequenced the 5' flanking region. Sequence analysis revealed the presence of consensus promoter elements--GC boxes and a CAAT box--but the absence of a TATA motif. Potential binding sites for transcription factors that are involved in tissue-specific gene expression were identified, including: activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF3 beta), homeobox Nkx2-5 and GATA-1. Transcription activity of the TATA-less SEL1L promoter was analyzed by transient transfection using luciferase reporter gene constructs. A core basal promoter of 302 bp was sufficient for constitutive promoter activity in all the cell types studied. This genomic fragment contains a CAAT and several GC boxes. The activity of the SEL1L promoter was considerably higher in mouse pancreatic beta cells (beta TC3) than in several human pancreatic neoplastic cell lines; an even greater reduction of its activity was observed in cells of nonpancreatic origin. These results suggest that SEL1L promoter may be a useful tool in gene therapy applications for pancreatic pathologies.

Similarity pattern analysis in mutational distributions.

The validity and applicability of the statistical procedure - similarity pattern analysis (SPAN) - to the study of mutational distributions (MDs) was demonstrated with two sets of data. The first was mutational spectra (MS) for 697 GC to AT transitions produced with eight alkylating agents (AAs) in the lacI gene of Escherichia coli. The second was a recently summarized data on the distributions of 11562 spontaneous, radiation- and chemical-induced forward mutations in the ad-3 region of heterokaryon 12 of Neurospora crassa. They were analyzed as large two-way contingency tables (CTs) where two kinds of profiles were compared: site (or genotypic class) profiles and origin (or mutagen) profiles. To measure similarity (homogeneity) between any pair of profiles, the relevant sufficient statistics, Kastenbaum-Hirotsu squared distance (KHi(2)), was used. Collapsing the similar profiles into distinct internally homogeneous clusters named 'collapsets' revealed their similarity pattern. To facilitate the procedure, the computer program, COLLAPSE, was elaborated. The results of SPAN for the lacI spectra were found comparable with the results of their previous analysis with two multivariate statistical methods, the factor and cluster analyses. In the ad-3 data set, five collapsets were revealed among origin profiles (OPs): (I) ENU = 4NQO = 4HAQO = FANFT = SQ18506; (II) AF-2 = EI = MMS = DEP; (III) ETO = UV; (IV) AHA = PROCARB; and (V) He ions = protons. Moreover, the previous observation that MDs are dose-dependent was confirmed for X-ray-induced MDs. Profiles induced with the low doses of X-rays are similar to that induced with 85Sr, and profiles induced with the medium X-ray doses to those induced with protons and He ions. Evaluated similarities appear to be rather reasonable: mutagens with similar mode of action induce similar MDs. Similarity pattern revealed among genotypic class profiles (GCPs) seems to be also interpretable. When supplemented with descriptive cluster analysis, SPAN appears to be a fruitful methodology in MS analysis.

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae.

We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast. We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative [PSI]. We have shown that HAP induces predominantly nonsense mutations. While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35. We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene. HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG. Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand. We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.

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.

Sequence and gene content in 35 kb genomic clone mapping in the human Xq27.1 region.

This paper presents detailed analysis of the entire sequence of a cosmid clone, 26H7, containing 35 kb of human DNA. This cosmid resides on the q27.1 region of the human X chromosome between, DXS1232 and DXS119 loci. Novel potential small exons were detected for which conventional gene identification strategies (Northern blot analysis and extensive cDNA library screening) proved to be inefficient. Of the standard repetitive elements we found: 8 Alu's making up 6.2% of the sequence; 10 MIR segments (4.1%); 5 LINE1 elements (4.8%), 3 MIR2 (1.0%); 2 MLT (2.9%), and 1 MSTA (0.7%) representing about 20% of the total sequence. The overall GC content was rather low, only 42% and no CpG island was detected using rare restriction enzymes. However, a CpG-rich region was identified. Computer aided analysis of the sequence inferred the presence of three possible genes: one of them was found to be homologous to the U7 RNA family elements; a second is reported in this paper, however at the moment no significant homology has been found in the data bank. The third predicted gene has not as yet been found to be detectable by RT-PCR. We also report in this paper the identification of X-chromosome specific repeated sequences.