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Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

Studying the structure, functions, and cell physiology of small RNAs remains important. The 4.5SI and 4.5SH small RNAs, which were among the first to be discovered and sequenced, share several features, i.e., they are both approximately 100 nt in size, are synthesized by RNA polymerase III, and are found only in rodents of several related families. Genes coding for these RNAs are evolutionarily related to short interspersed elements (SINEs). However, the two RNAs differ in nucleotide sequence, half-life in the cell, and the organization of their genes in the genome. Although the 4.5SI and 4.5SH RNAs have been identified more than three decades ago, several aspects of their metabolism in the cell are still poorly understood. The 4.5SI and 4.5SH RNA levels were measured in various organs of three rodent species (mouse, rat, and hamster). Both of the RNAs were found to occur at high levels, which were much the same in different organs in the case of the 4.5SI RNA and varied among organs in the case of the 4.5SH RNA. Both 4.5SI and 4.5SH RNAs demonstrated a predominantly nuclear localization with a detectable presence in the cytoplasm. The copy number per cell for the RNAs was estimated at 0.4-2.4 × 10^(6). A quantitative study for the 4.5SI and 4.5SH RNAs was performed for the first time and resolved a number of contradictions in data from other studies.

Analysis of the prevalence of copy number variations of the CNTN6 gene, recently selected as a new candidate gene for intellectual disorders, was performed. Real-time PCR did not detect any change in the number of CNTN6 gene copies in a group of 200 patients with impaired intellectual development. However, taking into account our data from the previous aCGH analysis and published data, the overall frequency of microdeletions and microduplications of CNTN6 was estimated as 1: 265 (0.4%). The common phenotypic features of 40 patients with microdeletions and microduplications of CNTN6 appeared to be the autism spectrum disorders, developmental delay, intellectual disability, seizures, cognitive impairment, cardiological defects, and behavioral problems.

Transgenic clones of mouse embryonic stem cells of the RI line were -received by transfection of plasmid linear vectors. The changes in the transgene structure during its integration into the genome of the target cells were investigated. Displacements were found on the flanks of the integrated transgene. It was found that multicopy tandem structures are formed in head- -tail orientation at the transgene integration. It was noted that the number of copies of the integrated transgenes varies considerably, but the introduction of DNA fragments from the nuclear shells into the flanks of the transgene normalizes the number of its copies.

The clinical isolates of Staphylococcus aureus (n = 102) were analyzed on sensitivity and to gentamicin, tobramicin, netimicin and amikacin. The disc diffusing technique was applied. The technique ofpolymerase chain reaction was applied to analyze all strains establishing presence in their genomes genes aac (6'-Ie/aph(2"), ant1, aac, ant(6)-Ia, aph (3')-IIIa and ant(4')-Ia coding amino-glycoside-modifying enzymes. The strains sensitive to amino-glycosides had no the given genes in genome. The genome of all strains resistant to amino-glycosides included no less than two of enumerated genes. The 100% correlation was established between phenotypic resistance of analyzed strains to amino-glycosides and availability in them of gene aac(6')-Ie/aph(2").

Type I-IV proximal spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseas- es, which are characterized in the majority of cases by a severely disabling course. Proximal SMA results from mutations in the telomeric copy of SMN-SMN1 gene. Major SMN1 gene mutation types are deletions in the exons 7 and/or 8, which were revealed to be in the homozygous state in 95% of patients. Deletions in the in- dicated exons of SMN1 gene were revealed in a compound-heterozygous state in combination with intragenic point mutations in the remainder 5% of proximal SMA cases. In the present study, we conducted an analysis of point mutations in eight patients with type I-III proximal SMA phenotype, which had a deletion in 7-8- exons of SMN1 gene in the heterozygous state. We revealed seven different mutations, two of which (c.824G > C (p.Gly275A1a) and c.825-2A > T) are described here for the first time. In addition, mutation c.824G > C (p.Gly275A1a) was observed twice in the examined sample. In seven cases a heterozygous carrier of point mutations was one of the parents of the affected children (in six cases, the father; in one case, the mother). Only one mutation, c.43C > T (p.Gln15X), emerged de novo in a genital cell of the child's father.

Cloning and sequencing of the partial reverse transcriptase gene (750 bp) of the Bov-B LINE retrotransposon have been held in parthenogenetic lizards Darevskia unisexualis and its assumed parental bisexual species D. nairensis and D.valentini. It was shown that the percentage of transcriptionally active copies of this gene, which does not contain a stop codon, is almost the same in the three species and is about 75%. The intragenomic divergence level of these sequences is low and was found to be 2.6% in D. unisexualis, 1.9% in D. nairensis, and 1.6% in D. valentini. The phylogenetic analysis shows the distribution of copies of D. unisexualis in each of the two clusters of RT sequences characteristic of D. nairensis and D. valentini. This result supports the view of the hybrid origin of D. unisexualis and does not exclude intraspecific hybridization between D. nairensis and D. valentini.

In the review of literature the structure, localization and a physiological role of efflux biobiotics and xenobiotics transporter - P-glycoprotein - is characterized, molecular mechanisms of induction and inhibition of its functional activity are systematized.

The first data on the existence of multiple genomic rearrangements, such as copy number variation (CNV) and copy neutral loss of heterozygosity, in vascular tissues and peripheral blood leukocytes from patients with atherosclerosis, are presented. Compared to internal mammary arteries and peripheral blood leukocytes, right coronary arteries in the atherosclerotic plaque area presented with a higher CNV length and number of genes located in their vicinity. In each of the patients, 6-16% of CNVs were common to the three types of tissues examined. Therefore, most of the copy number variations in the tissues affected by atherosclerosis (from 68 to 91% in each of the patients) were of somatic origin. The gains in 3p21.31 (CACNA2D2), 7q32.1 (FLNC), 19p13.3 (C19orf29, PIP5K1C), and 21q22.3 (COL6A1) were detected in vascular tissues but not in peripheral blood leukocytes. Moreover, the gain in 7p15.2 (SKAP2), detected in the patients with atherosclerosis, did not overlap with any CNV regions currently reported in The Database of Genomic Variants. The loss of heterozygosity in 12 out of 13 chromosomal regions was copy neutral and covered tumor suppressor genes (SFRP1, CEBPD, RB1CC1, DIRAS3, TUSC3, and ZDHHC2).

Objective: Spinal muscular atrophy (SMA) is currently untreatable hereditary disorder caused by few types of mutations in the SMN1 gene and respective lack of gene's product - survival motor neuron protein (SMN). Last decade studies have shown that phenotype of the disorder is substantially influenced by copy numbers of homologous SMN2 gene; also, an ability of valproic acid to increase the level of SMN in vitro and in vivo has been shown. We investigated an effect of valproic acid on SMN level in peripheral blood mononuclear cells derived from patients with SMA and their parents and sought for possible predictors for treatment efficacy., Material and Methods: We examined 10 children with SMA and 6 their parents as heterozygous carriers of the mutation using appropriate molecular-genetic techniques. Valproic acid was prescribed in 20mg/kg/day during 2 weeks., Results and Conclusion: There were no correlation between baseline SMN level and SMN2 copy number; both of the markers do not predict SMN level after the treatment with valproic acid. However, all of patients responded to valproic acid treatment with different grades of SMN level increase. Strong intrafamilial correlation was found for the SMN/Β2-microglobulin ratio.

In this study we analyzed how a dosage decrease in mono- and diheterozygotes on both lethal alleles of the lgl-gene and hsfheat shock regulator influences viability and life span at optimal and high temperature 29 degrees C conditions. We found that hsf(1) (1 dosage of active hsf-factor) heterozygote animals had significantly increased viability (up to 30-39%) in case of its development from egg to imago under the stress of 29 degrees C. However, this stress-protective effect of a decreased dosage of HSF1 was suppressed in diheterozygotes, while the dosage of tumor suppressor lgl was simultaneously decreased. Under stress temperature conditions, a decrease in dosage of one of the alleles also increased the average life span and delayed aging, especially in the case of maternal inheritance of each of the loss-of-function mutations. In diheterozygotes the average life span had intermediate meanings. However, in diheterozygote males under stress conditions the positive longevity effect of hsf was suppressed in the presence of the lgl-mutation. Paradoxically, that decrease of expression of each of the studied vital genes provided a positive effect on both viability and life span under stress conditions. However, a simultaneous dosage decrease of two loss-of-function alleles in diheterozygotes resulted in disbalanced effects on the organism level. The received data indicate interaction between HSF1 and LGL gene products during ontogenesis and stress-defending processes.

Aim: Determination of copy number dynamics of plasmid DNA and anti-lysozyme activityunderthe influence of chloramphenicol in cells of Escherichia coli 252 and 242 strains (ICIS), that have identical biochemical profile and differ by antibiotic resistance, plasmid profile and level of anti-lysozyme activity., Materials and Methods: E.coli strains were isolated during screening of antibiotic resistant enteric bacteria of human intestinal biotope. The selected strains were cultivated in medium with 0.17; 0.5; 1 and 1.5 MIC of chloramphenicol by nonspecific amplification ofplasmid DNA in medium with chloramphenicol (technique by T. Maniatis et al.). Plasmid DNA was recovered by alkaline lysis method with subsequent electrophoresis in agarose gel. The quantity ofplasmid DNA was evaluated by comparison of luminescence intensity of marker and experimental DNA sample bands. Anti-lysozyme activity was measured photometrically (O.V.Bukharin et al.)., Results: E. coli 252 strain had higher direct correlation of bacteria anti-lysozyme activity and increase of plasmid copy number per cell (r > or = 0.9) during increase of chloramphenicol concentration in sub-bacteriostatic range. Dynamics of these parameters in E. coli 242 control strain were not detected., Conclusion: The data obtained give evidence in favor of the action of plasmid localized "gene dose" effect as a mechanism of anti-lysozyme activity increase of bacteria under the influence of chloramphenicol.

A microarray analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (RT-PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microarray. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 x 10(4) copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model probes. The abilities of the created system were demonstrated on the example of microarray analysis of samples with different content of DNA, low absolute limits of identification (20 DNA copies in microreactor), and high reproducibility of the analysis.

Effective recombinant strains Pichia pastoris that produce functionally active hybrid of Trigonopsis variabilis D-aminoacids bond with chitin-connecting domain of chitinase A1 of Bacillus circulans (DAOcbd) were obtained. The dependence of DAOcbd production levels from production of the number of copies of "expression cassette" integrated in the AOX1 locus of recombinant strains was studied. It was indicated that synthesized DAOcbd may be easily purified and immobilized on chitin sorbents and possessed high specific activity. Produced strains and methods of their cultivation and DAOcbd extraction may be used for development of technologies of obtaining of biocatalyzers in technological processes of obtaining of 7-aminocephalosporane acid.

Genes of box C/D small nucleolar RNAs (snoRNAs) were searched for in the genomes of members of all classes of vertebrates that do not belong to placental mammals. A tendency for an increase in the number of copies of snoRNA genes was observed in such vertebrates. This trend was most pronounced in anamnia (amphibians and fish). Box C mutations were found in 14 snoRNAs in all gene copies among all species studied. The role of the described events is discussed.

Completion of human genome reading stimulated intense studies in the field of functional genomics and characterization of individual genomes. Of considerable importance is the study of the complex of multicopy ribosomal genes (RGs), but its thorough analysis was not a task of the "Human Genome" program. In this short review we present our data on the copy number of rRNA genes in individual human genomes and on their heterogeneity in the functional respect. Fractions of active and potentially active RGs as well as fractions of inactive and silent RGs intensively methylated in the transcribed region are characterized. Their location in the nucleolus structures and in metaphase chromosomes is discussed.

Twenty samples of benign pigmented neoplasms of the skin, including 9 intradermal nevi and 11 complex ones, were investigated. Fluorescence in situ hybridization was used to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes. Analysis of the findings revealed no significant changes characteristic of melanoma in the nevi. However, the authors established that there was a direct correlation between the copy number of the MYB and RREB1 genes and that the amount of the MYB gene most frequently deviated from the normal values. In addition, a relationship was found between the number of MYB gene copies and the depth of the epidermal layer. In cases of an intradermal nevus, the copy numbers of the CCND1 and MYB genes were shown to vary more greatly than in cases of a complex nevus.

Different exogenic antioxidants and geroprotectors are used to decrease age abnormalities and enhance the human life span. However, the antioxidant effect on lifespan is variable and requires detailed analysis. In the present report, we modeled in Drosophila the peculiar character of action of various doses of a new phenol antioxidant TC-13. We studied the TC-13 effect on aging of two Drosophila lines with genetically determined contrast lifespan dynamics. In addition, we tested the TC-13 antioxidant influence on the background of heterozygozity on the loss-of-function mutation of the l(2)gl tumor suppressor. The differing effect of TS-13 on LS, the character of which depends on the antioxidant dosage, genotype of line, and sex of Drosophila, was found. TS-13 in the concentration 0.2% did not affect the lifespan in all studied lines and decreased survival, whereas the antioxidant in a concentration of 1% positively affected the lifespan in both males and females of long-living lines. The antioxidant effect on animal lines with a smaller dose of tumor suppressor l(2)gl resulted in a decrease of the lifespan.

Fifty-two patients that had ParkinsonTs disease with autosomal dominant type of inheritance were analyzed for the presence of duplications and triplications in exons 4--6 of alpha-synuclein gene using real-time PCR with Taq-Man probes. No mutations involving the examined exons dosage were revealed in alpha-synuclein gene. Thus, mutations modifying copy number of alpha-synuclein gene do not significantly affect the pathogenesis of the autosomal dominant form of ParkinsonTs disease in patients from Russia.

Two members of the Baikal sig family, a lake sig (Coregonus lavaretus baicalensis Dybovsky) and omul (C. autumnalis migratorius Georgi), are close relatives that diverged from the same ancestor 10-20 thousand years ago. In this work, we studied genomic polymorphism of these two fish species. The method of subtraction hybridization (SH) did not reveal the presence of extended sequences in the sig genome and their absence in the omul genome. All the fragments found by SH corresponded to polymorphous noncoding genome regions varying in mononucleotide substitutions and short deletions. Many of them are mapped close to genes of the immune system and have regions identical to the Tc-1-like transposons abundant among fish, whose transcription activity may affect the expression of adjacent genes. Thus, we showed for the first time that genetic differences between Baikal sig family members are extremely small and cannot be revealed by the SH method. This is another endorsement of the hypothesis on the close relationship between Baikal sig and omul and their evolutionarily recent divergence from a common ancestor.

Human populations are polymorphic for risk factor mutations predisposing to diseases and cancer. The evolutionary conservative gene, tumor suppressor lethal (2) giant larvae (lgl), whose null variants are widespread in natural Drosophila populations, can be used as a model to study this phenomenon. We studied the effect of deletion and insertion mutant alleles of the lgl gene on the survival and longevity of their carriers depending on the genotype, cross direction, and action of permanent or impulse temperature stress. Under constant temperature stress, the viability and longevity of lgl/+ deletion heterozygotes increased compared to the control flies carrying two alleles of this gene. This haploid effect was maternally mediated. Exposure of deletion heterozygotes at successive oogenesis stages to moderate impulse temperature stress affected progeny survival in a similar way. The effect of impulse heating at the preembryonal stage was bidirectional: positive in the case of impulse warming of mature eggs and negative during egg differentiation. However, in both cases, deletion of one allele of the tumor suppressor gene in lgl/+ females increased survival of the next generation and delayed senescence. These data were compared to similar epigenetic transgeneration effects reported earlier for humans.

This is a short review of recently described copy number variation of some segments of DNA. The origin of this phenomenon, the localization of these segments and their possible role in regulation of gene expression are discussed. The copy number variation should be regarded as a new form of genomic diversity.

The goal of this study is to understand the evolution relationship of the members of B-hordein gene family in hull-less barley by analysis of their structure and to explore their utility in grain quality improvement. Six copies of B-hordein gene (Hn1-Hn3, Hn7-Hn9) were cloned from six hull-less barley cultivars collected from Qinghai-Tibet Plateau and molecularly characterized. Comparison of their predicted polypeptide sequences with the published suggested that they all share the same basic protein structures. In addition, we found that the C-terminal end sequences of all B-hordeins shared a similar feature. In the six clones and the other three published (Hn4, Hn5 and Hn6) from hull-less barley, Hn2 and Hn7 contained identical C-terminal end sequence DIMPVDFWH, Hn3, Hn4, Hn5, Hn8 and Hn9 also shared the common sequence DIMPPDFWH, which was similar to that of a B-hordein reported previously. Both Hnl and Hn6 exhibited differences in their C-terminal end sequences, and they clustered into different subgroups. The B-hordeins with identical C-terminal end sequences were clustered into a same subgroup, so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide. Phylogenetic analysis also indicated that there is a relatively weak identity between our predicted B-hordeins and those reported from H. chilense and H. brevisubulatum. All of our nine predicted B-hordeins were clustered together and other B-hordeins formed another cluster. The possible use of these genes in relation to the barley quality is discussed.

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9-12 mice aged 1-2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105-181, mean +/- SD 136 +/- 27) and NZB (129-169, 148 +/- 12) was significantly lower than in strains CBA (172-267, 209 +/- 31), 101 (179-270, 217 +/- 30), and 129 (215-310, 264 +/- 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.

The results of four selection-genetic experiments aimed at the genetic transformation of the quantitative trait controlled by the radius incompletus gene of Drosophila melanogaster are given. Directional (s+) and (s-)-selection was conducted. At the of end all the experiments, in (s+)-selection the radial vein of the wing was restored to the wild phenotype, in (s-)-selection complete elimination of the radial vein took place. In four variants of selection, different TE412 pattern was formed under (s+)-selection and (s-)-selection in final generations. Correlation coefficient between (s+)-selection and (s-)-selection is -0.576, p < 0.001. At the same time, correlation coefficient between two independent replications of (s+)-selection is 0.912, p < 0.001, and of (s-)-selection, 0.946, p < 0.001. Thus, the availability of associated response to the selection of a quantitative trait and to the TE412 pattern is experimentally proved. Three hypotheses of the possible TE behaviour under selection are discussed.

The interactions between mouse angora-Y (Fgf5go-Y) and hairless (hr) genes have been studied. Homozygous mutant gene Fgf5go-Y increases hair length starting on day 14 after birth. We obtained mice with genotypes +/+ hr/hr F2, +/Fgf5go-Y hr/hr and Fgf5go-Y/Fgf5go-Y hr/hr. Both +/Fgf5go-Y hr/hr and +/+ hr/hr mice began to loose hair from their heads on day 14. This further extended on the whole body. On day 21 the mice were completely deprived of hair. Therefore a single dose of gene Fgf5go-Y does not affect alopecia mice homozygous for hr. However in double homozygotes Fgf5go-Y/Fgf5gO-hr/hr alopecia started 4 days later, namely on day 18. It usually finished 10-12 days after detection of first bald patches. On days 28-30 double homozygotes have lost all the hair. Hair loss in double homozygous mice was 1,5-fold slower than in +/+ hr/hr mice. This resulted from a significant extension of anagen phase induced by a mutant homozygous gene Fgf5go-Y in morphogenesis of the hair follicle. In contrast, hr gene was expressed only at the transmission phase from anagen to catagen. Our data shows that the angora gene is a modifier of the hairless gene and this results in a strong repression of alopecia progression in double homozygous mice compared to +/+ hr/hr animals.

In this work for the first time copy number and expression changes of the tumor suppressor gene RBSP3 (3p21.3) were investigated. The study was performed on HPV-positive squamous cervical carcinoma (SCC) using real-time PCR. Deletions were detected in 42% of cases (19 of 45 studied biopsies). Frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P < 0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). Expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P < 0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparison of copy number and expression changes showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes in copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC and complex mechanisms for inactivation of RBSP3. We also hypothesize that these data indicate that RBSP3 in addition to dephosphorylation of pRb has other functions important for malignant transformation because pRb is almost absent in HPV-positive SCC.

Mutations of tumor suppressor lgl induce neuroblastoma and malignant transformation of epithelial larval tissues in Drosophila. We have already shown that heterozygotes for lethal null variants lgl/+ are widespread in natural populations. In order to elucidate this paradox, we analyzed the parameters of biological adaptation of the carriers of one dose of the tumor suppressor. We studied the patterns of embryonic survival rate of lgl/+ flies under the conditions of competition for life resources and development at elevated and lowered temperatures (29 and 16 degrees C), influence of stress thermal conditions on life span, influence of short-term temperature stress during prezygotic period in the course of oogenesis of mothers on survival rate of F1 progenies, and resistance of heterozygotes for different lethal lgl alleles against RNA virus DCV. The loss of one dose of tumor suppressor lgl+ provided for increased survival rate and life span of lgl/+ heterozygotes under stress conditions. This phenomenon was called haploadaptivity. Important features of adaptogenesis were established in lgl/+ heterozygotes: dependence on the maternal genotype and critical periods in development. The increased survival rate of F1 progenies was determined already during early oogenesis of their lgl/+ mothers at the proembryo stage. With respect to humans, this conclusion draws attention to the oogenesis-dependent transgeneration aspect of determination and expression of mutant factors of risk, including tumor suppressors. The data obtained are essential for understanding of the causes of spreading null variants for the genes related to multiple pathologies, including cancer, in human populations.

A fragment of Penicillium canescens genomic DNA carrying the xlnR gene coding for a translational activator of xylanolytic genes was isolated. It was demonstrated that a loss of this function in genetically modified transformants resulted in a drastic decrease in the production of P. canescens major xylanases and had a negative effect on the syntheses of several other extracellular xylanases. An increase in the dose of the xlnR gene elevated the xylanolytic activity as well as the activities of a number of other auxiliary enzymes involved in xylan degradation. The activities of two P. canescens major secreted enzymes--beta-galactosidase and alpha-L-arabinofuranosidase-appeared weakly dependent on the translational activator xlnR.

RHOA protein, a member of small GTPases family, is implicated in cell morphogenesis, adhesion, and in cell cycle regulation. RHOA gene (3p21.31) exhibits cell transformation activity, and therefore gene is considered as a potential oncogene. The aim of this study was to investigate RHOA transcription and copy number changes in three epithelial tumors (breast, renal cell and epithelial ovarian carcinomas, 45 tumor/normal pairs altogether). EII, HhaI, AciI n Bsh1236I). Hypomethylation of the RHOA promoter region in tumor DNA was observed two times more frequently than increased methylation. Moreover, all (15) cancer cases with hypomethylation of the RHOA gene showed a 2-10 fold increased expression of RHOA. It was concluded that gene copy multiplication and demethylation of the RHOA promoter region can contribute to transcription activation of this gene in epithelial tumors.

Serotonin 5-HT(1A) receptors participate in the regulation of many kinds of behavior and are implicated in the mechanism of action of anxiolitics and antidepressants. The investigation of 5-HT(1A) receptor gene expression is complicated by low concentration of the receptor mRNA. Our method of quantification of the receptor gene expression in brain structures includes estimation of the concentration of genomic DNA contamination, the number of cDNA copies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)--one of the "housekeeping genes", and the number of cDNA copies of 5-HT(1A) receptor in the sample. To evaluate the number of cDNA copies of the receptor and GAPDH, the fluorescence intensity of PCR-product was calibrated using genomic DNA-standard of a known concentration. The intensity of 5-HT(1A) receptor gene expression was corrected by genomic DNA contamination and was evaluated as a number of copies of 5-HT(1A) receptor cDNA per 100 copies of GAPDH cDNA. Using this method an increase of 5-HT(1A) receptor gene expression in the frontal cortex and amygdala in monoamine oxidase A knockout mice was shown.

PCR assay with internal control was developed to quantify the her-2 gene dosage in human breast cancer tumor samples. Recombinant plasmid with a fragment of the her-2 gene containing the fragment of T7 phage DNA was designed especially to be used as an internal control in the PCR her-2 gene dosage assay. PCR conditions were optimized for the simultaneous usage of two templates--human genomic DNA and DNA of recombinant plasmid (internal control). The ability of the technology to discriminate a twofold increase of the her-2 gene dosage was demonstrated. Increased level of her-2 gene amplification was observed in 6 of 38 samples investigated. This new simple rapid assay can be an alternative to fluorescence in situ hybridization (FISH) and immunochemistry (ICH) tests for the detection of her-2 amplification in human tumors. This technique may be a useful tool for large randomized, prospective cooperative group trials and may support selection of optimal therapy for breast cancer patients in the future.

The study of heterotrophic bacteria isolated from the brackish waters of Lake Shira has shown that some of them contain plasmid pSH1 of approximately 2.7 kb in size. The number of plasmid copies in plasmid-containing strains cultivated at a minimal concentration of sodium chloride is found to be low, whereas the subculturing of these strains at high salt concentrations increases the plasmid number. The role of natural pSH1 plasmid in the osmotolerance of host bacteria is discussed.

A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.

Genomic dosage (copy number) of active ribosomal genes was evaluated using visual semi-quantitative method determining the sizes of Ag-NORs in acrocentric chromosomes after selective silver nitrate staining. A relationship between the length of service and the active ribosomal gene copy number was established: the highest numbers of active rRNA genes were observed in coke-oven workers with a length of service exceeding 20 years. An inverse relationship between the individual doses of active ribosomal genes and toxicogenetic susceptibility of the workers to the occupational factors was also revealed.

Based on the Mendelian approach to heredity, modern genetics describes inheritance of characters belonging to the category of intraspecific difference. The other large category of characters, intraspecific similarity, stays out of investigation. In this review, the genome part responsible for intraspecific similarity is considered as invariant and regulatory. An approach to studying the invariant part of the Drosophila melanogaster genome is formulated and the results of examining this genome part are presented. The expression of mutations at genes in the invariant genome part is different from that of Mendelian genes. We conclude that these genes are present in the genome in multiple copies and they are functionally haploid in the diploid genome. Severe abnormalities of development appearing in the progeny of mutant parents suggest that the mutant genes are genes regulating ontogeny. A hypothesis on an elementary ontogenetic event is advanced and the general scheme of ontogeny is presented. A concept on two types of gene allelism (cis- and trans-allelism) is formulated. This approach opens a possibility for studying genetic material responsible for the formation of intraspecific similarity characters at different taxonomic levels on the basis of crossing individuals of the same species.

Different aspects of the presence of multiple copies of ribosomal operons in prokaryotic genomes are reviewed. Structure of prokaryotic ribosomal operons is briefly described. The available data are summarized regarding the copy number of ribosomal genes in various prokaryotic genomes, the degree of polymorphism of their individual copies, physiological and evolutionary aspects of the presence of the multiple copies of ribosomal genes. The review also considers the influence of the presence of multiple copies of ribosomal genes on the results of identification of prokaryotic isolates and of the studies of prokaryotic diversity in environmental samples based on phylogenetic analysis of 16S rRNA gene sequences.

A modified version of quantitating repetitive sequences in genomic DNA was developed to allow comparisons for numerous individual genomes and simultaneous analysis of several sequences in each DNA specimen. The relative genomic content of ribosomal repeats (rDNA) was estimated for 75 individuals, including 33 healthy donors (HD) and 42 schizophrenic patients (SP). The rDNA copy number in HD was 427 +/- 18 (mean SE) per diploid nucleus, ranging 250-600. In SP, the rDNA copy number was 494 +/- 15 and ranged 280-670, being significantly higher than in HD. The two samples did not differ in contents of sequences hybridizing with probes directed to a subfraction of human satellite III or to the histone genes. Cytogenetic analysis (silver staining of metaphase chromosomes) showed that the content of active rRNA genes in nucleolus organizer regions is higher in SP compared with HD. The possible causes of the elevated rRNA gene dosage in SP were considered. The method employed was proposed for studying the polymorphism for genomic content of various repeats in higher organisms, including humans.

Thermotolerance was studied in a wide spectrum of Drosophila species and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilis group exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogaster Oregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters of Drosophila and variation in the numbers of gene copies and hsp70 isoforms in group virilis were found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genus Drosophila is discussed.

A specific index of nucleotide sequence redundancy, the specific restriction length of a finite frequency dictionary, was determined for a complete set of genes in some viral genomes and a genome of a bacterium, Bacillus subtilis. The distribution of the gene number over the specific restriction length was shown to be bimodal for viral genomes and unimodal for the Bac. subtilis genome. These results agree with earlier data.

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

The rearrangement of bacterial chromosomes induced by intragenomic recombination is considered. The role of stochastic and programmed genome rearrangements in bacterial adaptation to the environment and in cell differentiation is discussed.

Polytene chromosomes of salivary glands of various Drosophila melanogaster strains containing two doses of the normal Su(UR)ES allele have a constant set of intercalary heterochromatin (IHC) sites. Their DNA is underreplicated, which leads to breaks and ectopic contacts emerging at a certain rate. Almost no underreplication, breaks, or ectopic conjugation are present in mutants lacking the normal Su(UR)ES gene product. It could be expected that an increase in the number of the Su(UR)ES+ gene doses would, in turn, drastically increase ectopic conjugation and breakage. To test this hypothesis, a strain of D. melanogaster was obtained with two additional doses of Su(UR)ES+ introduced into its genome. The flies with four gene doses exhibited a considerable increase in ectopic conjugation: both the proportion of regions participating in conjugation and the number of chromosomes with numerous contact nodes were increased. As a result, chromosomes that were straight and well-stretched in homozygotes for the mutation in Su(UR)ES became twisted and wound and contained many loops or nodes. Many chromosomes were wound too tightly for cytological analysis. Four doses of Su(UR)ES+ considerably increased the number of weak "points." For example, the 2R chromosome has only 3 weak points in strains with two doses of Su(UR)ES+ and as many as 22 weak points in the strain with four doses. In the transgenic strain, the frequency of breaks in previously known weak points increased, and new breaks appeared in 19 additional sites. All new break points appeared in the regions that were earlier described as regions of late replication in the S phase.

Three sublines of an inbred laboratory line of Drosophila melanogaster with the initial copia transposition rate 2 x 10(-2), 2 x 10(-3), and 5 x 10(-4) per copy per generation were reared for several dozen generations under conditions of low effective population size (by full-sib crosses or in a small mass culture of 10 females x 10 males). All six lines were tested for the transposition rate, location pattern, and copy number of copia in euchromatic genome regions and for fitness inferred from the intraspecific competition index. The copia transposition rate remained constant in both versions of the lines with an initially lower rate and decreased by an order of magnitude in both versions of the line with an initially higher rate. New copia insertions behaved as selectively neutral and were accumulated in the genome. Each new copy decreased fitness by less than 1% on average. Some of the existing unfixed insertions remained segregating after long-term inbreeding and were assumed to provide a selective advantage to heterozygotes.

Mice of mi/+ and +/+ genotypes of the mutant stock microphthalmia (mi) were mated inter se and with those of CC57BR/Mv and CBA/J inbred lines. In all types of crosses, the offsprings of mi/+ genotype had unpigmented fingers and distal tail parts (penetrance 100%). This trait is determined mostly by the mutant mi gene, the expression of which is not affected by modifier genes. However, the degree of mi gene expression, white spotting, on other body parts (wrist, foot, ventral body side, and parietal head part) varied widely in mice obtained from different crosses; penetrance ranged from 0 to 100%. The results obtained indicate that in these cases, the expression of mi gene was strongly affected by modifier genes. The different frequency of the mi gene effects, in heterozygote offspring obtained from reciprocal crosses, can result from the imprinting of the modifier genes in maternal and paternal gametes.

Transformation of Drosophila melanogaster using P-element-based vectors yielded 129 sublines, which carried mini-white gene copies in the different genome regions. Dependence of mini-white gene expression on the location, gene dosage, and sex of the transformed individuals was analyzed. The mutation lzb was shown to suppress mini-white gene expression, the degree of suppression depending on the location and dosage of the mini-white gene.