Thermostable Lichenase from Clostridium thermocellum as a Host Protein in the Domain Insertion Approach.

Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.

Expression of Soluble Active Interferon αA in Escherichia coli Periplasm by Fusion with Thermostable Lichenase Using the Domain Insertion Approach.

A recombinant DNA in which the interferon αA (IFN-αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN-αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN-αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.

Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability.

The Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins.

[Morphophysiological and biochemical characteristics of potato plants with various expression rates of the Δ12 acyl-lipid desaturase gene].

This paper reports on morphophysiological and biochemical characteristics of control and potato plants (Solarium tuberosum L., Skoroplodnyi cultivar) transformed with the Δ12 acyl-lipid desaturase gene (desA) grown long-term in vitro. The transformed plants showed faster growth and faster ontogenesis as compared to controls, which was accompanied with changes in the accumulation of photosynthetic pigments (chlorophylls a and b, carotenoids) and phenolic compounds, including flavonoids in the leaves. These characteristics were pronounced to a high degree in Line II plants with high expression rates of the desA gene, whereas Line I plants (moderate expression rate) were similar to control plants in many parameters.

[Range of Gene candidates involved in biosynthesis of laccase from basidiomycete Trametes hirsuta].

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.

[Set of module vectors for stable or transient expression of heterologous genes in plants].

A set of module vectors for stable or transient gene expression in plants was constructed with regard to the majority of factors ensuring efficient heterologous gene expression in plants. The vectors are convenient to clone new regulatory elements and genes of interest via simple molecular cloning procedures. The vectors can be used to obtain transgenic plants with stable heterologous gene expression as well as to achieve transient expression because one vector includes the gene for the tomato bushy stunt virus p19 protein, which acts as a suppressor of posttranscriptional gene silencing.

[Expression of heterologous genes in plant systems: new possibilities].

The basic methods used in current practice for stable and transient expression of heterologous genes in plants are presented and compared. The key areas of research in the heterologous expression of genes in plants have been identified by analyzing literature and experimental data: modeling of metabolic pathways; creation of marker-free transgenic plants; the search for new regulatory elements and plant genes influencing the efficiency of expression of heterologous genes in plants; development of new methods for analyzing of transgenic plants and new approaches to the expression of heterologous genes in plants.

[The Dagestan gene pool: polymorphism of immunogenetic and biochemical markers in Kumyks].

This study is a part of long-term investigations devoted to the analysis of the gene pool of Dagestan ethnic groups. The phenotype (in %), gene, and haplotype frequencies in Kumyk ethnic group are reported. A total of 39 alleles and six haplotypes of 14 loci (AB0, Rhesus, P, Levis, Kell, HP, GC, C'3, TF, 6PGD, GLO1, ESD, ACP, and PGM1) of immunobiochemical genetic marker systems were examined. Rare haplotypes of the Rhesus system were identified, including CDE in the Karabudakhkent population with the frequency of 0.030, and Cde and cdE in the Dorgeli population with the frequencies of 0.034 and 0.38, respectively. Similarly to the other ethnic populations of Dagestan examined, Kukyk populations carried rare, albeit typically "Caucasoid" gene ACP1(c) of the AcP1 locus. The frequency of this allele in the two populations was similar, constituting 0.031 for Karabudakhkent and 0.032 for Dorgeli. In Kumyks, allele frequencies of the AB0, Rhesus, P, Lewis, Kell, HP, GC, C'3, TF, 6PGD, GLO1, ESD, ACP, but not PGM1, systems were similar to the mean allele frequencies at these loci observed in the other ethnic groups from the Dagestan, Caucasus, and the whole European historical ethnographic province. At the same time, the allele frequency values obtained were different from those for the populations of Kazakhstan, Central Asia, Siberia, and the Ruswsian Far East. Thus, the results obtained for classical genetic markers indicate that Kumyks are genetically closer to the indigenous populations of Dagestan than to Turkic-speaking populations. Analysis of the fit of the observed phenotype frequencies to the Hardy-Weinberg expectations showed that compared to other indigenous populations of Dagestan examined, in Kumyks the genetic state of the population upon random allele association was close to equilibrium. Probably, this state was determined by practical absence of the consanguineous marriages upon preservation of intra-aul endogamy.

[Analysis of clusterin gene (CLU/APOJ) polymorphism in Alzheimer's disease patients and in normal cohorts from Russian populations].

Three genes mutations in which cause familial forms of Alzheimer's disease are known to date:PSEN1, PSEN2 and APP; and APOE gene polymorphism is a strong risk factor for Alzheimer's disease. We have evaluated allele and genotype frequency distribution of rs11136000 polymorphism in clusterin (CLU) gene (or apolipoprotein J, APOJ) in populations of three Russian regions and i nAlzheimhner's diseasepatients. Genome-wideassociation studies in samples from several European populations have recently revealed highly significant association o fCLU gene with AD (p = 8.5 x 10(-10)). We found no differences in allele and genotype frequencies of rs11136000 between populations from Moscow, Ural and Siberia regions. The allele frequencies are close to those in European populations. The genetic association analysis in cohort of Alzheimer's disease patients and normal individuals (>500 individuals ineach group) revealed no significant association of the rs11136000 polymorphism in CLU with Alzheimer's disease in Russian populations. Although our resultsdo not confirm the role of CLU gene as a majorgenetic factor forcommon form of Alzheimer's disease, the data do not rule out the possibility of modest effect of CLU and interaction between CLU and APOE genotypes in etiology of Alzheimer's disease.

[Transgenic tomato plants expressing recA and NLS-recA-licBM3 genes as a model for studying meiotic recombination].

Homologous DNA recombination in eukaryotes is necessary to maintain genome stability and integrity and for correct chromosome segregation and formation of new haplotypes in meiosis. At the same time, genetic determination and nonrandomness of meiotic recombination restrict the introgression of genes and generation of unique genotypes. As one of the approaches to study and induce meiotic recombination in plants, it is recommended to use the recA gene of Escherichia coli. It is shown that the recA and NLS-recA-licBM3 genes have maternal inheritance and are expressed in the progeny of transgenic tomato plants. Plants expressing recA or NLS-recA-licBM3 and containing one T-DNA insertion do not differ in pollen fertility from original nontransgenic forms and can therefore be used for comparative studies of the effect of bacterial recombinases on meiotic recombination between linked genes.

[Gene pool of Dagestan ethnic groups: polymorphism of classical gene markers in the Darghin ethnic group].

The work is part of a study of the gene pool for Daghestan ethnic groups. In total, 38 alleles and eight genotypes were studied at 14 loci (AB0, Rhesus, P, Lewis, Kell, HP, GC, C'3, TF, 6-PGD, GLO1, ESD, ACP, and PGM1) of immunogenetic and biochemical polymorphic gene systems. A high frequency of allele d of the Rhesus system was observed in all populations examined (0.399-0.474). Among the rare haplotypes of the Rhesus system, we observed CDE in the Degva population, Cde in the Sergokala and Degva populations, and cdE in the Sergokala and Vanashimakhi populations. The typical Caucasian ACP1c allele of the ACP1 locus, which is rather uncommon, was observed at a relatively high frequency in three (Segokala, Vanashimakhi, and Gubden) of the four local populations under study. In the Lewis system, a high frequency of the Le(a+b+) phenotype, which is characteristic of early childhood, was detected in the adult populations of Sergokala and Degva. The rare PGM1v allele of the phosphoglucomutase 1 system (PGM 1) was additionally observed in the Sergokala population. Statistical analysis identified 19 cases where the observed phenotype frequencies significantly differed from the frequencies expected from the Hardy--Weinberg equilibrium.

[Association of NAT2 polymorphism with risks to develop psoriasis and various dermatological diseases in Moscow population].

Ruacetyltransferase 2 (NAT2) is one of key enzymes of the second phase of biotransformation that metabolize genotoxic compounds such as carcinogens and mutagens in different types of cells. There is a correlation between the decreasing activity of NAT2 gene product and the sensitivity to harmful environmental factors that increase the risk of occurrence of different multifactorial diseases, including dermatological ones like psoriasis. We developed the NAT2-biochip for 17 SNPs. The biochip was been tested on 279 clinical DNA samples from 180 patients with psoriasis and 99 healthy individuals, residents of Moscow. We found only six SNPs that were significant for European populations (282C > T, 341T > C, 481C > T, 590G > A, 803A > G and 857G > A). The analysis in psoriasis group did not show any genotype association. The increase in frequency of a slow acetylation phenotype in group of patients with type II psoriasis and in group of patients with normosthenic constitution, in comparison with control group (OR = 1.76,p = 0.177 and OR = 2.07,p = 0.050, respectively) has been revealed. The results for patients smoking one or more pack of cigarettes per day, and daily alcohol drinking in comparison with the control showed an increase in frequency for the genotype 341C/C, 481T/T, 803G/G (OR = 7.42, p = 0.008 and OR = 106.11, p = 0.003, respectively). We also found an increase of frequency of genotype 341T/T, 481C/C, 590A/-, 803A/A in patients with side reactions to medical products comparing with group of healthy donors (OR = 2.05, p = 0.099). Thus, the present data show that the certain NAT2 genotypes and some styles of life can be considered as risk factors of psoriasis development in this muscovite population.

[Association of xenobiotic-metabolizing gene polymorphisms with childhood atopic diseases in Russian patients from the Republic of Bashkortostan].

Enzymes of biotransformation system involved in the metabolism of exogenous and endogenous compounds are effective mechanism of protection from negative environmental factors. Decreasing activity or insufficient synthesis of biotransformation system enzymes caused by genetic polymorphism form the risk of various complex diseases, including atopic. Using allele-specific hybridization on the biochip the frequencies of xenobiotic-metabolizing gene polymorphisms in Russian children with bronchial asthma, allergic rhinitisand healthy donors from the Republic of Bashkortostan have been determined. The analysis of polymorphisms in CYP1A1, GSTT1, GSTM1, NAT2, MTHFR, CYP2C9 and CYP2C19 genes didn't reveal any association with atopic diseases. The frequencies of CYP2D6*1934G/G genotype and CYP2D6*1934G allele were significantly higher among boys with rhinitis symptoms than in control group.

[Identification of functionally significant mutations in NAT2 gene using biological microchips].

The product of gene NAT2 (N-acetyltransferase 2) is involved in the biotransformation system and participates in detoxication of some arylamine derivatives (in particular 2-aminofluorene, 4-aminobiphenyl and 4-naphthylamine) which are strongly mutagenic and carcinogenic. It also renders toxicological and pharmacological influence on a metabolism of medical products metabolized by the enzyme. We developed a microchip for detection of 16 functionally significant mutations coding 36 alleles of gene NAT2. Combinations of these alleles allow us to reveal more than 660 genotypes, which can be divided into four groups according acetylation phenotype: "fast" (R/R), "intermediate" (R/S), "slow" (S/S) and group with average or slow acetylating (R/S or S/S) alleles. The groups "R/S or S/S" include alleles, formed by a combination of 7 mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, 857G/A), theirs cis-trans position can be revealed by restriction analysis. In 37 of 71 DNA samples we unequivocally defined NAT2-genotypes, and other 34 samples have been characterized by more than two genotypes. 16 samples out of 34 had acetylation phenotype of group "R/S or S/S", which is characterized by the following combination of mutations: 282C/T, 341T/C, 481C/T, 590G/A and 803A/G. Thus, the developed biochip is a convenient screening method for primary detection of the majority of polymorphic replacements in gene NAT2.

[Experimental models for creation of transgenic plants resistant to stress factors].

Experimental models of the potato primary transgenic plants which express the hybrid gene cry3aM-licBM2 have been created. Modecular analysis and the biotests of the experimental models allow proposing a new system of cry genes expression in plants. The system is based on the expression of hybrid genes possessing the sequence of reporter lichenase gene and the use as a regulator element of a light-induced promoter providing preferential expression of the controled genes only in green plant tissues (leaves)--the target tissues for pests. The lichanase presence in hybrid proteins facilitates selection and analysis of the expression level of the hybris proteins in transgenic organisms. Basing on the lichenase properties in hybrid proteins it seems possible to use this reporter system for transgene monitoring in agrocoenosis as this system is rather simple and precise and does not need large material and time expenses.

[Experimental models of transgenic plants promising for the modern technologies].

Transgenic popato plants have been created which express recombinant proteins, analogues of spidroin 1, the protein of the cobweb skeleton thread. Expression of the hybrid spidroin 1 genes possessing some repeated sequences retains both in the model test-tube-growing plants and in the crops. Expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity and plant species but not on the duration of plant material storage. The results show that the strategy based on constuction and expression of hybrid proteins which include the reporter protein makes it easier to select and analyse expression of hybrid proteins in transgenic organisms.

[Comparative expression in Escherichia coli of the native and hybrid genes for acyl-lipid delta(9) desaturase].

Expression of the desC gene coding for acyl-lipid delta(9) desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid delta(9) desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acids.

[Population analysis and determination of the ethnic background are necessary in the study of multifactorial diseases: a study using the Dagestan population as a model].

Psoriasis, a multifactorial disease with genetic predisposition, has been used as an example to study the role of the ethnic background in multifactorial diseases in the Dagestan population. The individual information card (IIC) is proposed as the main tool for correct collection and processing of information. The results of the study demonstrate that the Dagestan population is a convenient and adequate model population for studying multifactorial diseases, such as psoriasis, and may serve as an object for studying the role of heredity in the etiologies and pathogeneses of this and other multifactorial diseases.

[Comparative analysis of N-acetylation polymorphism in humans as determined by phenotyping and genotyping].

The N-acetylation polymorphisms of volunteers from the Moscow population analyzed by phenotyping and genotyping have been compared. The ratios between the proportions of fast acetylators (FAs) and slow acetylators (SAs) estimated by phenotyping and genotyping do not differ significantly from each other (47 and 44%, respectively). The absolute acetylation rate widely varies in both FAs and SAs. The NAT2 genotype and allele frequencies in the population sample have been calculated. The most frequent alleles are NAT2*4 (a "fast" allele), NAT2*5, and NAT2*6 ("slow" alleles); the most frequent genotypes are NAT2*5/*5, NAT2*4/*6, and NAT2*4/*5. Comparative analysis of N-acetylation polymorphism estimated by phenotyping and genotyping in the same subjects has shown a complete concordance between the phenotype and genotype in only 62 out of 75 subjects (87%). Comparative characteristics and presumed applications of the two approaches (quantitative estimation of acetylation rate and qualitative determination of the acetylator genotype) to the identification of individual acetylation status are presented.