Deuterium Oxide Enhances Escherichia coli SOS Response Induced by Genotoxicants.

It has been demonstrated that deuterium oxide enhances the SOS response of Escherichia coli cells induced by chemical genotoxicants and mutagens. This demonstrates that the heavy nonradioactive hydrogen isotope deuterium can be considered to be a comutagen.

[Migration as the main factor of the Russia's urban population dynamics].

This review summarizes the results of the long-term studies performed at the Institute of General Genetics, Russian Academy of Sciences, in the field of genetic demography of migration processes in Russia and its capital. The main population-genetic parameters of migration and their dynamics in Moscow over a hundred years are given. Sociodemographic and population-genetic implications of migration processes are considered. A model predicting the population gene pool dynamics under migration pressure for genes of different localization (autosomal, sex-linked, and mitochondrial), exemplified by predicting the allele frequency dynamics in the Moscow population of some gene markers, including genes accounting for monogenic pathology and genes associated with resistance to socially significant diseases, are presented. The paper discusses the selective character of migration processes, in particular, processes of emigration, with respect to some genetically significant ethnodemographic traits; the problem of adaptation of migrants; and adaptive strategies of consolidation of ethnoconfessional groups in the megalopolis (compact settlement over the urban territory and positive assortative mating with respect to demographic traits). It was shown that, owing to the intense influx of migrants and gene flows between ethnic groups, the population of the megalopolis is of mixed origin in terms of ethnic, anthropologic, and genetic aspects. The results of the study suggest the necessity to develop a specific strategy of genetic database formation for the population of megalopolises for the purposes of medical genetics and forensic medicine.

[Combination of Genetic and Humanitarian (Cross-Cultural) Methods for the Identification of Human Genes Involved in the Process of Adaptation to Evolutionary New Environmental Factors].

Human settlement from the African ancestral home was accompanied by cultural and genetic adaptation to new habitat conditions (climate, infections, diet, etc.). We previously suggested for the first time an approach to the identification of human genes presumably involved in adaptation to evolutionary new environmental factors based on a combination of genetic and humanitarian methods of study. In order to search for the genes involved in adaptation and for environmental factors (to which this adaptation occurs), we attempted to find correlations between the population allele frequencies of the studied gene and formalized descriptions of peculiarities of the habitat of ethnic groups given in "Ethnographic Atlas" by G. P. Murdock. In the presented review, we summarized our own data on an experimental determination of the allele frequencies for lactase (LCT*), apolipoprotein E (APOE), and alcohol dehydrogenase (ADH1B) genes in populations of Russia. Based on these data and available materials of other investigators, we developed maps of worldwide allele frequency distribution for these genes. We detected a correlation of allele frequencies of these genes in populations with the presence of certain factors of the environment that these populations inhabit. It was also confirmed that the evolutionarily young LCT*-13910T allele, which determines lactase persistence and the possibility of milk consumption in adults, is distributed in populations for which dairy animal husbandry is typical. During the analysis of 68 populations, we for the first time demonstrated that the frequency of the APOE e4 allele (which is ancestral for humans and influences the lipid metabolism) is higher in groups with a high contribution of hunting and gathering. Our data are in favor of the hypothesis that it was exactly the e4 allele that was a subject for selection, while the e3 allele was less important for adaptation. We also for the first time demonstrated that the evolutionarily young ADH1B*48His allele (which determines a high rate of ethanol metabolism into acetaldehyde) is presented with a large frequency in those populations where filariasis is endemic. The obtained data indicate the possible involvement of endogenous ADH1B gene substrates or their metabolites in the resistance to filaria and open a new path to the development of drugs for this widespread human disease.

[Analysis of genetic diversity of Russian regional populations based on common STR markers used in DNA identification].

We conducted the first genetic analysis of a wide a range of rural Russian populations in European Russia with a panel of common DNA markers commonly used in criminalistics genetic identification. We examined a total of 647 samples from indigenous ethnic Russian populations in Arkhangelsk, Belgorod, Voronezh, Kursk, Rostov, Ryazan, and Orel regions. We employed a multiplex genotyping kit, COrDIS Plus, to genotype Short Tandem Repeat (STR) loci, which included the genetic marker panel officially recommended for DNA identification in the Russian Federation, the United States, and the European Union. In the course of our study, we created a database of allelic frequencies, examined the distribution of alleles and genotypes in seven rural Russian populations, and defined the genetic relationships between these populations. We found that, although multidimensional analysis indicated a difference between the Northern gene pool and the rest of the Russian European populations, a pairwise comparison using 19 STR markers among all populations did not reveal significant differences. This is in concordance with previous studies, which examined up to 12 STR markers of urban Russian populations. Therefore, the database of allelic frequencies created in this study can be applied for forensic examinations and DNA identification among the ethnic Russian population over European Russia. We also noted a decrease in the levels of heterozygosity in the northern Russian population compared to ethnic populations in southern and central Russia, which is consistent with trends identified previously using classical gene markers and analysis of mitochondrial DNA.

The Impact of ADH1B Alleles and Educational Status on Levels and Modes of Alcohol Consumption in Russian Male Individuals.

Alcohol abuse is one of the main reasons behind the low life span in Russia. Both social and genetic factors affect the alcohol consumption level. The genetic factors are alleles of the alcohol dehydrogenase ADH1B and aldehyde dehydrogenaseALDH2 genes. We have typed and found frequencies for the alleles in a cohort of 642 men, ethnic Russians. The individuals of the cohort were asked to complete a questionnaire in the framework of the Izhevsk Family Study (Leon et al., 2007, 2009) regarding the amount of alcohol consumed and on the type of hazardous alcohol consumption (nonbeverage alcohol consumption and the so-called "zapoï" which is a Russian term for a heavy drinking bout lasting for at least 2 days, when an individual is withdrawn from the normal social life). The ADH1B*48His allele was found among heterozygous individuals only (N=68, 10.6% of the cohort). The ALDH2*504Lys allele was also found among heterozygous individuals only (N=2, 0.3%) The effect of ADH1B alleles and the influence of the education level on the amount and type of alcohol consumed had not previously been studied in Russians. We have found that the amount of consumed alcohol is 21.6% lower (1733 g of ethanol per year) for ADH1B*48His allele carriers in the cohort of Russian men. The amount of consumed alcohol was found to be 9.8% lower (793 g of ethanol per year) in the case when individuals had a higher education as compared to those who had a secondary- or elementary school education level in the same cohort. Hence, the protective effect of the genetic factor (ADH1B*48His allele carriage) has proven to be more pronounced than the influence of the social factor (education level) at the individual level in the cohort of Russian men. Both factors have also proven to have a protective effect against hazardous types of alcohol consumption. Zapoï was not scored among individuals of the cohort with ADH1B*48His allele carriage (OR=12.6, P=0.006), as compared to 8.4% of "zapoï" individuals who did not carry the ADH1B*48His allele (genotype Arg/Arg).The percentage of individuals who consume non-beverage alcohol is lower (0.6%) in the subcohort of people with a higher education degree. This percentage is higher (6.0%, OR=10.0, P=0.004) in the subcohort of people without a higher education degree.

[Allele frequency distributions of -174G/C polymorphism in regulatory region of interleukin 6 gene (IL6) in Russian and worldwide populations].

Allele and genotype frequencies of the -174G/C polymorphism (rs1800795) in the regulatory region of the IL6 gene, which encode anti-inflammatory cytokine interleukin 6, were determined in seven populations representing five ethnic groups from the European part of Russia (440 individuals), as well as in small cohorts that represent populations from 24 countries of Africa and Eurasia (365 individuals). The maps of the geographic distribution of the -174G/C allele frequencies were constructed based on personal (22 populations) and the literature data (66 populations), and the data from dbSNP database obtained by the HapMap project (10 populations). The frequency of the -174G allele varied from 45 to 100% and was characterized by nonrandom geographic distribution. These data could reflect the adaptive load of the alleles examined, which was different in different regions of the world. It is suggested that the level of pathogen prevalence is one of the environmental factors that determine different adaptive values of the IL6*--174G/C alleles. This suggestion is supported by a positive correlation between the -174G allele frequency and level of pathogen prevalence calculated based on historical data (R = 0.768; p < 0.0001).

Risk of HIV Infection and Lethality Are Decreased in CCR5del32 Heterozygotes: Focus Nosocomial Infection Study and Meta-analysis.

CCR5del32 Homozygous deletion in the chemokine receptor R5 gene provides almost complete protection to individuals against HIV infection. However, data relating to the protective effect forCCR5del32 heterozygous individuals have been contradictory. The frequency of theCCR5del32allele in population control cohorts was compared with that of a group of children (27 Kalmyks and 50 Russians) infected by G-subtype HIV-1 in a nosocomial outbreak. The frequency of theCCR5del32allele was shown to be lower among the infected children in comparison with that of the control group; however, the difference was small and statistically insignificant. Similar results were obtained in a number of earlier studies. The insignificance of the small differences could be a result of one of two reasons. (i) The fact that there is no protective effect of the heterozygous state, and that the phenomenon depends only on the fluctuation of allele frequencies. In this case, there would be no differences even if the infected cohort is enlarged. (ii)The protective effect of the heterozygous state is real; however, the size of the studied cohort is insufficient to demonstrate it. In order to discern between these two reasons, a meta-analysis of data from 25 published articles (a total of 5,963 HIV-infected individuals and 5,048 individuals in the control group, including the authors' own data) was undertaken. A conclusion was drawn from the meta-analysis that theCCR5del32 allele protects individuals against the HIV infection even in a heterozygous state (OR=1.22, 95%CI=1.10-1.36). The risk of HIV infection forCCR5 wt/del32 heterozygotes was lower by at least 13% as compared to that for wild typeCCR5 wt/wthomozygotes. Prior to this study, no data of the type or any conclusions had been published for Caucasians. The mortality rate in the 15 years following the infection was found to be approximately 40% lower forCCR5del32 heterozygotes in comparison with that for the wild type homozygotes in the studied group. The size of the studied group was insufficient to claim difference validity (OR=2.0;p= 0.705), even though the effect quantitatively matched the published data. The features of the meta-analysis influencing the threshold level and the statistical validity of the effects are being discussed. The level of theCCR5del32 protective effect on the chances to be infected with HIV and on the outcome of the HIV infection was assessed for various ethnic groups.

Association of cytokine gene alleles with the inflammation of human periodontal tissue.

Gingivitis and periodontitis are chronic inflammatory diseases of the periodontal tissue in humans caused by both environmental and genetic factors. The human cytokine genes that regulate the immune response may play an important role in the development of these chronic inflammatory diseases. The aim of this study is to analyze the allele status of eight human cytokine genes and to associate it with the inflammation of periodontal tissue in humans. A total of 296 unrelated males of Russian origin were studied. A significant association of theIL1BandIL6 minor alleles and gingivitis was found. In addition, we found a significant association of the OHI-S index with theIL18gene alleles. The influence of genetic factors on gingivitis may contribute to the understanding of the mechanisms of interaction between genetic and environmental factors in periodontal conditions, and to the identification of risk groups for effective prevention and treatment.

Characteristics of Populations of the Russian Federation over the Panel of Fifteen Loci Used for DNA Identification and in Forensic Medical Examination.

Seventeen population groups within the Russian Federation were characterized for the first time using a panel of 15 genetic markers that are used for DNA identification and in forensic medical examinations. The degree of polymorphism and population diversity of microsatellite loci within the Power Plex system (Promega) in Russian populations; the distribution of alleles and genotypes within the populations of six cities and 11 ethnic groups of the Russian Federation; the levels of intra- and interpopulation genetic differentiation of population; genetic relations between populations; and the identification and forensic medical characteristics of the system of markers under study were determined. Significant differences were revealed between the Russian populations and the U.S. reference base that was used recently in the forensic medical examination of the RF. A database of the allelic frequencies of 15 microsatellite loci that are used for DNA identification and forensic medical examination was created; the database has the potential of becoming the reference for performing forensic medical examinations in Russia. The spatial organization of genetic diversity over the panel of the STR markers that are used for DNA identification was revealed. It represents the general regularities of geographical clusterization of human populations over various types of genetic markers. The necessity to take into account a population's genetic structure during forensic medical examinations and DNA identification of criminal suspects was substantiated.

Achievements and peculiarities in studies of ancient DNA and DNA from complicated forensic specimens.

Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in "postmortem" mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems.. Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.

RFP2, c13ORF1, and FAM10A4 are the most likely tumor suppressor gene candidates for B-cell chronic lymphocytic leukemia.

Occurrence of 13q14 deletions between D13S273 and D13S25 in B-cell chronic lymphocytic leukemia (B-CLL) suggests that the region contains a tumor suppressor gene. We constructed a PAC/cosmid contig largely corresponding to a 380-kb 13q14 YAC insert that we found deleted in a high proportion of B-CLL patients. We found seven genes by exon trapping, cDNA screening and analysis/cDNA extension of known expressed sequence tags. One appeared to originate from another region of 13q. Recent publications have focused on two of the genes that most likely do not have a tumor suppressor role. This study evaluates the remaining four genes in the region by mutation scanning and theoretical analysis of putative encoded products. No mutations suggestive of a pathogenic effect were found. The 13q14 deletions may be a consequence of an inherent instability of the region, an idea supported by our finding of a considerable proportion of AluY repeats. Deletion of putative enhancer sequences and/or genes in the region may result in an inactivation of tumor suppression by a haploinsufficiency mechanism. We conclude that RFP2, c13ORF1, and a chromosome 13-specific ST13-like gene, FAM10A4, are the most likely candidates for such a type of B-CLL TSG.

A new human gene KCNRG encoding potassium channel regulating protein is a cancer suppressor gene candidate located in 13q14.3.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K+ channels. Using the patch-clamp technique we determined that KCNRG suppresses K+ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K+ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

In silico screening for tumour-specific expressed sequences in human genome.

A computer-based differential display tool named HsAnalyst has been developed and successfully used for the comparison of expression patterns in a set of tumours versus a set of normal tissues. A list of EST clusters highly represented in tumours and rarely observed in normal tissues has been developed as a resulting output file of the program. These differentially expressed EST clusters (genes) can be useful for developing new tumour markers and prognostic indicators for a wide set of human malignancies. Tumour-specific protein-coding genes may be considered a manifestation of tumour-specific gene expression.

Recombinant Listeria strains producing the nontoxic L-chain of botulinum neurotoxin A in a soluble form.

Fragments of Clostridium botulinum neurotoxin A (BoNT/A) gene (botA) were expressed in Listeria monocytogenes ATCC10527 to produce the L-chain of the toxin in a soluble form. A shuttle vector pAT19 (EmR) was used to make plasmid pAT-RL containing a botA gene fragment placed under C. botulinum ntnH-gene promoter control. The plasmid also contained a C. botulinum botR/A gene, a positive transcriptional regulator of botA. The cytoplasmic fraction of the L. monocytogenes (pAT-RL) strain was found to contain up to 3 mg/L of a soluble protein of expected size and immunologically positive towards BoNT antibodies. This is the first evidence of heterologous botA gene expression producing a soluble safe derivative of botulinum neurotoxin A needed as a molecular tool for exploratory research in neurosciences as well as a basis for raising protective immunity in humans.

Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae.

Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae. where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.

Diphtheria toxin NAD affinity and ADP ribosyltransferase activity are reduced at tryptophan 153 substitutions for alanine or phenylalanine.

Previous studies on chemical modifications of diphtheria toxin (DT) fragment A have suggested that the Trp153 amino acid residue is essential for the ADP ribosylation of elongation factor 2. We verified this experimentally after replacing Trp153 by Phe or Ala residues through in vitro mutagenesis of a cloned toxin gene fragment. Each of the mutant fragment A forms were found to reveal a reduced ADP ribosyl transferase (ADPRT) activity as well as lower affinity for NAD. Both ADPRT activity and NAD affinity of DT fragment A were only partially destroyed by nearly synonymous Trp153 ==> Phe153 substitution, but dramatically destroyed by Ala153 substitution. At the same time, each of the mutant fragment A forms appeared to be thermostable, suggesting that the mutations do not dramatically destroy the structure of the protein. These results clearly demonstrate that Trp153 is not highly specific for DT fragment A structure maintenance, but is highly specific for the key toxin functions such as ADP ribosylation of elongation factor 2 and NAD binding. We suggest that the Trp153 role in DT functioning may be that of binding the ribose moiety of NAD, which is crucial for DT catalytic activity and hence for toxicity.

Comparative sequence analysis of a region on human chromosome 13q14, frequently deleted in B-cell chronic lymphocytic leukemia, and its homologous region on mouse chromosome 14.

Previous studies have indicated the presence of a putative tumor suppressor gene on human chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have recently identified a minimally deleted region encompassing parts of two adjacent genes, termed LEU1 and LEU2 (leukemia-associated genes 1 and 2), and several additional transcripts. In addition, 50 kb centromeric to this region we have identified another gene, LEU5/RFP2. To elucidate further the complex genomic organization of this region, we have identified, mapped, and sequenced the homologous region in the mouse. Fluorescence in situ hybridization analysis demonstrated that the region maps to mouse chromosome 14. The overall organization and gene order in this region were found to be highly conserved in the mouse. Sequence comparison between the human deletion hotspot region and its homologous mouse region revealed a high degree of sequence conservation with an overall score of 74%. However, our data also show that in terms of transcribed sequences, only two of those, human LEU2 and LEU5/RFP2, are clearly conserved, strengthening the case for these genes as putative candidate B-CLL tumor suppressor genes.

Mitochondrial DNA sequence diversity in Russians.

The article presents the results of the first regular study of Russian populations by sequencing the control region of mitochondrial DNA (mtDNA). The sequenced region is the most variable on mtDNA molecule and is commonly used for population and evolutionary studies. Russians form one of the largest ethnic groups (more than 129 million). However, their genetic diversity had only been characterized with RFLP and biochemical markers, although there are already established mtDNA sequence databases for many ethnic groups of the world. We have obtained sequence data from 103 individuals living in three Russian regions: Kostroma, Kursk, and Rjazan. The sequenced fragment analyzed is 360 bp in length (positions from 16024 to 16383). Fifty nine nucleotide positions have been found polymorphic in Russians, among those were 57 transitions and two transversions. One individual is found having two insertions of two cytosines between positions 16184 and 16193. Among 64 different mitotypes identified in the study 52 were unique in these samples. The index of genetic diversity (Nei, 1987) for Russians is 0.96. This value is within the established range for European populations (0.93 to 0.98). Genetic distances calculated from our data show that Russians form a cluster with Germans, Bulgarians, Swedes, Estonians, and Volgo-Finns are more distant from Karelians and Finns, and much more differ from Turks and especially Mongolians.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5.

B-cell chronic lymphocytic leukemia (B-CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2-42), which corresponds to the minimal region shared by B-CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc-finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B-CLL rearrangements.

Detailed molecular delineation of 13q14.3 loss in B-cell chronic lymphocytic leukemia.

A region of chromosome 13q14.3, telomeric to the Retinoblastoma gene RB-1 is frequently deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). A cosmid and P1-derived artificial chromosome (PAC) contig spanning over 600 kb has been constructed, which encompasses this locus. The contig clones have been used to order a number of markers along the minimally deleted region and to localize a series of CpG islands corresponding to possible candidate genes. A novel polymorphic dinucleotide repeat, 6E3.2, present in one of the ordered cosmid clones has been isolated for use in deletion mapping studies of patient DNA. Leukemic samples from 229 CLL patients have been screened for loss of heterozygosity using microsatellite markers and analyzed for hemizygous and homozygous deletions by Southern blot techniques using genomic probes selected from cosmids across the region. Hemizygous deletions were found in 31% of cases with an additional 10% showing homozygous loss. The use of these probes has defined the commonly deleted area to less than 130 kb, centromeric to the locus D13S272.