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.

[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).

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia.

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.

Construction and expression of diphtheria toxin-encoding gene derivatives in Escherichia coli.

We reported earlier that in the periplasmic space of Escherichia coli, truncated derivatives of diphtheria toxin undergo limited proteolysis [Zdanovsky et al., Mol. Biol. 22 (1988) 1037-1293]. Here, we present data indicating that this proteolysis is reduced in cells bearing a mutation in the degP gene. We have also constructed hybrid genes whose products are not secreted into the periplasm. These hybrid genes were expressed in E. coli from both the pR promoter, controlled by the heat-inducible CI857 repressor, and from the P(lac) promoter, controlled by the IPTG-inducible LacI repressor. The latter system proved to be more productive.

Cloning and analysis of structural and regulatory pectate lyase genes of Erwinia chrysanthemi ENA49.

Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

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.

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.

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.

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.

The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation.

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII. The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.

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.

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.

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.

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.

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.