Efficiency of the inbreeding coefficient f and other estimators in detecting null alleles, as revealed by empirical data of locus oke3 across 65 populations of chum salmon Oncorhynchus keta.

A survey of 65 populations of chum salmon Oncorhynchus keta across the species range revealed homozygote excess (947 homozygotes in 2954 fish) at a polymerase chain reaction (PCR)-based simple sequence repeat (SSR) locus oke3 with multiple alleles, whereas re-designed PCR primers indicated that 328 of these homozygotes were actually heterozygotes. Statistically significant high positive values of inbreeding coefficients, f, in multiple populations appeared to be a reliable predictor of null alleles. Based on these data, three methods were checked for their ability to estimate null-allele frequencies.

[Identification and classification of bovine leukemia virus isolates in Russia and Ukraine based on the pol viral gene polymorphism].

Bovine leukemia virus (BLV) is a widespread specific pathogen of cattle. Analysis of the pol viral gene polymorphism has been used to characterize the polymorphism of BLV isolates at stock-breeding farms in Russia and Ukraine. The fragments of the pol gene corresponding to the reverse transcriptase and integrase 494 and 233 bp in size, respectively, have been used for analysis. Phylogenetic analysis has revealed several variants of BLV clustered with a high bootstrap support in Russia and Ukraine. A new classification of BLV variants is suggested. Comparison of phylograms based on the polymorphism of the nucleotide sequences of the integrase and reverse transcriptase domains did not show topological conflicts. Therefore, recombination between BLV variants has not been found.

[A search for null alleles at the microsatellite locus of chum salmon (Oncorhynchus keta Walbaum)].

Population studies with the use of microsatellite markers face a problem of null alleles, i.e., the absence of a PCR product, caused by the mutations in the microsatellite flanking regions, which serve as the sites of primer hybridization. In this case, the microsatellite primer associated with such mutation is not amplified, leading to false homozygosity in heterozygous individuals. This, in turn, results in biased population genetic estimates, including the excess of homozygotes at microsatellite loci. Analysis of the population structure of a Pacific salmon species, chum salmon (Oncorhynchus keta Walbaum), revealed the presence of null alleles at the Oke3 microsatellite locus in the population samples, in which an excess of homozygotes was observed. The analysis was performed using different combinations of modified primers chosen to match the Oke3 locus. The use of these primers enabled identification of true heterozygotes among those individuals, which were previously diagnosed as homozygotes with the use of standard primers. Removal of null alleles eliminated the excess homozygotes in the chum salmon samples described. In addition to the exclusion of false homozygosity, the use of modified primers makes it possible to introduce polymorphic primer variants associated with certain microsatellite alleles into population studies.

[Nuclear mitochondrial pseudogenes].

Transfer of genetic material from mitochondria to the nucleus and their integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) in the nuclear genome are incorporated as non-encoded sequences, which are called nuclear mitochondrial pseudogenes (NUMT-pseudogenes). At present, the formation NUMT-pseudogenes in the nuclear genome is shown in many eukaryotes. They are distributed on different chromosomes, form a "library" of mtDNA fragments, migrated into the nuclear genome and provide important information on the history of the evolution of genomes. Escape of mtDNA from the mitochondria most is associated with damage and mitophagy these organelles. The integration of mtDNA fragments into the nuclear genome may occur during repair of double strand breaks of nuclear DNA (nDNA) arising under the action of endogenous and exogenous agents. Reparation of nDNA double strand breaks with "capture" fragments of mtDNA, occurs by non-homologous end joining and a similar mechanism, but with the involvement microhomology, located on the terminal sequences. Analysis of data allows us to suppose that the rate of formation NUMT-pseudogenes will depend on the rate of double strand breaks in nDNA, activity systems, their repair, as well--the number of mtDNA fragments that have emerged from the organelles, with their further migration into the nucleus. Such situations can be expected, most often after exposure to the damaging agents, in the first place--ionizing radiation. The emergence of new NUMT-pseudogenes, obviously, is changing not only the structure of the genome in the areas of their implementation, but may have a significant impact on the realization of genetic information. Integration NUMT-pseudogenes in the nuclear genome de novo may play a role in the development of various pathologies and aging. NUMT-pseudogenes can make serious errors in analyzing free mtDNA of total cellular DNA (using PCR), as a result of their co-amplification.

[The alterations in profiles of RAPD- and of ISSR-markers in progeny of the single gamma-irradiated mice of BALB/c strain].

Molecular-genetic effects in the offspring of BALB/c male mice exposed to single radiation doses of 1, 2 and 3 Gy were studied. Induced genetic variability was studied using such methods as assessment of variation RAPD- and ISSR-profiles. Comparative analysis of genetic radiosensitivity of stem spermatogonia and of spermatids is presented in the work. The frequency of changes in the patterns of the offsprings of irradiated mice was significantly different from the analogous parameters in the offsprings of the control group already at a dose of 1 Gy. Comparative analysis of genetic radiosensitivity at different stages of spermatogenesis revealed the similar sensitivity of spermatogonia and of spermatids at 1 and 3 Gy and a higer sensitivity of spematogonia at 2 Gy.

[Lesions of the mitochondrial genome and ways of its preservation].

The results of studies on damage, repair, mutation induction, and compensatory amplification of mitochondrial DNA (mtDNA) in mammalian cells are analyzed. The analysis have shown that mtDNA is a more susceptible target than nuclear DNA for endogenous and exogenous genotoxic agents, whereas DNA repair systems in mitochondria function less efficiently then in the nucleus. The rate of mutation accumulation is higher in mtDNA than in the nuclear DNA. In contrast to nuclear DNA, replication of damaged mtDNA is not blocked. MtDNA with multiple or complex lesions may be removed from mitochondria. An important mechanism of mitochondrial genome preservation is compensatory induction of new mtDNA copies. The cascade of events leading to the activation of the expression of nuclear genes controlling DNA repair in response to energy crisis is analyzed. As the key regulators of the activation of mtDNA replication and mitochondrial biogenesis, transcription coactivators PGC-1alpha and PGC-1beta are considered. These coactivators induce the expression of genes for nuclear respiratory factors NRF-1 and NRF-2. In their turn, NRF-1 and NRF-2 control the expression of mitochondrial transcription factors mtTFA, mtTFB-1, and mtTFB-2. MtTFA plays a key role not only in the mtDNA transcription regulation, but also in its stabilization and replication initiation. Thus, induction of the expression of many genes, resulting in activating mtDNA replication and increasing the number of its copies, is an important mechanism of preserving the mitochondrial genome upon the action of endogenous and exogenous damaging agents.

[The analysis of mutations at mini- and microsatellite DNA loci in the family members of a group of occupationally exposed to tritium and tritium oxide plant workers].

Mini/microsatellite (MNS/MCS) loci are efficient tools in solving basic and applied problems in different spheres of biology and medicine due to their unique characteristics - a high frequency of tandem repeats in combination with their wide variability. Specifically, they have been found use as potential markers of genetic effects of ionizing radiation on animals and on human. However there is no general agreement as to the influence of irradiation on the frequency of mutations in hypervariable repetitive DNA sequences up to now. The present work is the study of the mutation frequency at MCS/MNS loci in 19 families of workers occupationally exposed to chronic beta-radiation from tritium and tritium oxide (examined group), and in the control group included 23 families. The results have indicated that the average frequency of microsatellite mutations in the examined group made up 4.7% and exceeds about 7-fold the same parameter of the control group (0.7%). This differences is statistically significant (p = 0.004). The average frequency of minisatellite mutation in the examined group made up 3% while in the control group it was 2 time lower (1.5%), but this difference is not statistically significant. Mutations for 4 MCS and 2 MNS loci were revealed in two children from one family (the total reconstructed dose in their father was about 1000 mSv). If we exclude this family from statistical analysis the frequencies of MCS and MNS mutations in the children of nuclear workers do not statistically differ from the control values.

[Ionizing radiation can activate the insertion of mitochondrial DNA fragments in the nuclear genome].

In analytical review is considered the possibility of the insertion of mitochondrial DNA (mtDNA) fragments into the nuclear genome of cells, exposed ionizing radiation (IR). Many studies show that integration fragment mtDNA in nuclear genome, as well as its fastening as NUMT-pseudogenes, proceed at ancient periods of the evolutions not only, but also at more late periods. The number of the investigations shows that under influence endogenous reactive oxygen species, chemical agent, UV-light and IR mtDNA is damaged with greater frequency, than nucleus DNA. Furthermore, the repair systems in mitochondria are low efficiency. In irradiated by IR cells mtDNA fragments can transition from the mitochondria to the cytoplasm. The binding of mtDNA fragment to a complex with proteins provides them the protection from nuclease destroying. Possibly, at such safe condition they and are carried to nucleus. At inductions of DNA double-strand breaks (under the action of IR and activated their reparation) mtDNA fragments may be inserted to nuclear genome. Such integration of mtDNA to nuclear genome, with shaping NUMT-pseudogenes de novo, may be proceed in irradiated cells in the course of the reparations DNA double-strand breaks by the nonhomologous end-joining pathway. These insertions of mtDNA can cardinally change the structure of nuclear genomes in area of their introduction and render the essential influence upon the realization of genetic information. Available information in literature also allows to suppose that integration mtDNA in nuclear genome can proceed and at raised genomic instability observed in cells at post radiation period. It in equal extent pertains and to malignant cells with raised by instability mitochondrial and nuclear genomes. As the most efficient agent, initiating insertion fragment mtDNA in nuclear genome, is considered ionizing radiation.

[The influence of chronic and of acute irradiation on the polymorphism of RAPD-markers in offspring for irradiated mice].

On mice lines BALB/c and CBA/lac was performed the study of molecular-genetics effects in mice progeny after the chronic (dose rate -0.0017 Gy/day, total dose -0.36 Gy) and acute (dose range 1-3 Gy) exposure of y-radiation on the parents. For variability analysis was used technique of amplification DNA with series of random primers (RAPD-assay). Random primers were used as single primer and in mixture of ones. In this work were held the comparative analysis of the genetic radiosensitivity for stem spermatogonia and spermatides. After the acute exposure the dose dependence for levels of polymorphism of RAPD-markers were obtained. After the chronic irradiation, significant differences from control group were obtained only by use primers mixture M1. Comparative analysis of the genetic radiosensitivity of different stages of mice spermatogenesis are display is similar sensitivity of stem spermatogonia and spermatides after doses of irradiation 1 Gy and 3 Gy. Indicated that after irradiation by dose 2 Gy, spermatogonia are more sensitivity than spermatides.

[The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific]. / Tkanespetsificheskii kharakter povysheniia variabel'nosti mikrosatellit-assotsiirovannykh povtorov v genome potomstva gamma-obluchennykh samtsov myshei.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.

Mitochondrial DNA variation in two South Siberian Aboriginal populations: implications for the genetic history of North Asia.

The mtDNAs of 76 individuals representing the aboriginal populations of South Siberia, the Tuvinians and Buryats, were subjected to restriction fragment length polymorphism (RFLP) analysis and control region hypervariable segment I (HVS-I) sequencing, and the resulting data were combined with those available for other Siberian and East Asian populations and subjected to statistical and phylogenetic analysis. This analysis showed that the majority of the Tuvinian and Buryat mtDNAs (94.4% and 92.5%, respectively) belong to haplogroups A, B, C, D, E, F, and M*, which are characteristic of Mongoloid populations. Furthermore, the Tuvinians and Buryats harbor four Asian- and Native American-specific haplogroups (A-D) with frequencies (72.2% and 55%, respectively) exceeding those reported previously for Mongolians, Chinese, and Tibetans. They represent, therefore, the populations that are most closely related to New World indigenous groups. Despite their geographical proximity, the Tuvinians and Buryats shared no HVS-I sequences in common, although individually they shared such sequences with a variety of other Siberian and East Asian populations. In addition, phylogenetic and principal component analyses data of mtDNA sequences show that the Tuvinians clustered more closely with Turkic-speaking Yakuts, whereas the Mongolic-speaking Buryats clustered closer to Korean populations. Furthermore, HVS-I sequences, comprising one-fourth of the Buryat lineages and characterized by the only C-to-T transition at nucleotide position 16223, were identified as different RFLP haplotypes (B, C, D, E, M*, and H). This finding appears to indicate the putative ancestral state of the 16223T HVS-I sequences to Mongoloid macrohaplogroup M, at least. Finally, the results of nucleotide diversity analysis in East Asian and Siberian populations suggest that Central and East Asia were the source areas from which the genetically heterogeneous Tuvinians and Buryats first emerged.

[Polymorphism of GPIIIA platelet glycoprotein gene PIA1/A2 compared to plasma hemostasis in myocardial infarction patients]. / Polimorfism gena PIA1/A2 trombotsitarnogo glikoprotein GPIIIA v sopostavlenii s plazmennym zvenom gemostaza u bol'nykh infarktom miokarda.

AIM: To investigate gene PIA1/A2 polymorphism and some parameters of plasma hemostasis in postmyocardial infarction (PMI) patients with chronic cardiac failure (CCF). MATERIALS AND METHODS: A total of 58 PMI patients with CCF, pulmonary artery thromboembolism (PATE), phlebothrombosis (PT) were examined. The age of the patients ranged from 24 to 84 years. Polymorphism of platelet glycoprotein GPIIIa gene was assessed according to the standard PCR-RFLP. RESULTS: Occurrence of genotypes PIA1/A2, PIA1/A2 was 70.8 and 29.2%, respectively; of allele PIA1 and PIA2 84.5 and 15.5%, respectively. In PMI patients genotype PIA1/A1 occurred in 71.7% of cases, genotype PIA1/A2--in 28.3%. Incidence of alleles was: 84.0% (PIA1), 16.0% (PIA2). PATE patients had genotype PIA1/A1, PT patients had distribution of the genotypes 50.0% and 50.0%, respectively. In patients who had suffered MI at the age under 45 years prevalence of the genotypes was 63.2% PIA1A1, 36.8% PIA1A2, of alleles 83.6% PIA1, 16.4% PIA2. In patients with a history of MI at the age over 50 the incidence of the genotypes and alleles was, respectively, 75.0% PIA1A1, 25.0% PIA1A2, 87.7% PIA1, 12.3% PIA2. Patients with genotype PIA1/A2 had a significantly higher fibrinogen than PIA1A1. Concentration of soluble fibrin monomeric complex was higher in patients with genotype PIA1/A2 reflecting activation of intravascular clotting. AT-III decrease by 5.4% indicated lower anticoagulant activity in patients with genotype PIA1A2. CONCLUSION: In patients with MI at the age under 45 years gene PIA1A2 and allel PIA2 occurred more frequently than in patients who had MI at older age. Allele PIA2 was associated with the risk of MI onset at young age. It is suggested that patients with genotype PIA1/A2 are at higher risk of thrombotic conditions, of coronary artery thrombosis in particular, than patients with genotype PIA1/A1.

[Use of polymerase chain reaction in clinical diagnostic laboratories in viral hepatitis B and C]. / Ispol'zovanie tsepnoi polimeraznoi reaktsii pri virusnykh gepatitakh B i C v praktike kliniko-diagnosticheskikh laboratorii.

HBV and HCV markers were detected in the blood serum of patients with acute and chronic hepatitis B (HCV), chronic hepatis C (HCV), and acute nonA, non-B, non-C hepatitis by enzyme immunoassay and the polymerase chain reaction (PCR) in order to assess the clinical value of PCR for the diagnosis of HBV and HCV and monitor the antiviral roferon A therapy in patients with HCV. HBV DNA was detected by the PCR in 100% of acute HBV patients and in 65.7% of chronic HBV patients. In chronic HCV, the PCR detected HCV RNA in 71% of cases. After roferon A therapy, HCV RNA was no longer detected in 43.2% of PCR-positive patients. PCR is recommended for the diagnosis of acute and chronic HCV, selecting the patient groups, and assessing the efficacy of specific therapy.

[DNA polymorphism of the BoLA-DRB3 gene in cattle in connection with resistance and susceptibility to leukemia]. / DNK-polimorfizm gena BoLa-DRB3 u krupnogo rogatogo skota v sviazi s ustoichivost'iu i vospriimchivost'iu k leikozu.

Polymorphism of exon 2 of the BoLA-DRB3 gene was investigated by the PCR-RFLP method in a sample of healthy and leukemia-afflicted Black Pied cattle. Allele variety was studied and allele frequencies were determined in a total sample and in the two groups. Alleles mediating resistance (BoLA-DRB3.2*11, *23, and *28) and susceptibility to leukemia (DRB3.2*22, *24, *16, and *8) were revealed in Black Pied cattle. The dominant type of inheritance of the disease resistance was confirmed. On the basis of original and published data obtained earlier for Holstein-Friesian cattle, a conclusion was made about the universal character of the spectrum of BoLA-DRB3 alleles providing resistance and susceptibility to leukemia.

[Optimization of DNA blot hybridization conditions for various types of membranes]. / Optimizatsiia uslovii blot-gibridizatsii DNK dlia razlichnykh tipov membran.

A set of experiments has been conducted to choose the optimal conditions for DNA transfer and fixation on two types of the nitrocellulose, three types of nylon membranes and on capron filters. The buffer capillary transfer systems, electroblotting and gel hybridization are analyzed. Two techniques for DNA binding have been tested under different transfer conditions for all the membrane types: a vacuum fixation at 80 degrees C and a UV-exposure. The results indicate the critical dependence of the efficiency of blot-hybridization on the conditions of UV-treatment. The UV-exposure longer or shorter than the optimal one resulted in a loss of the hybridization efficiency. The optimal DNA-transfer and fixation conditions are recommended for all the membranes tested. The dependence of the optimal transfer and binding conditions on the specific characteristics of different membrane types was demonstrated for maximal sensitivity of the blot-hybridization.

[Genotyping the bovine kappa-casein locus using polymerase chain reaction]. / Genotipirovanie lokusa kappa-kazeina u krupnogo rogatogo skota s pomoshch'iu polimeraznoi tsepnoi reaktsii.

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.