[MOLECULAR GENETIC CHARACTERISTICS OF ALLELE VARIANTS OF MICROSATELLITE LOCI DU281, DU215, AND DU323 IN THE PARTHENOGENETIC LIZARDS DAREVSKIA ROSTOMBEKOVI (FAMILY LACERTIDAE) ].

One of the main questions-in the study of the unisexual (parthenogenetic) species of vertebrates is the determination of their genetic diversity. Nuclear and mitochondrial genetic markers can be used for this purpose. One of the most effective genetic markers is the microsatellite DNA, which mutates at a high rate. Development and characteristics of such markers are necessary in studies of parthenogenetic species. In this work, the analysis of the allele polymorphism of three microsatellite loci was performed for the first time via locus-specific PCR in the populations of parthenogenetic species Darevskia rostombekovi (n = 42) and bisexual parental species D. raddei (n = 6) and D. portschinskii (n = 6). All examined individuals of the parthenogenetic D. rostombekovi were heterozygous. Two to five alleles, depending on the locus, were found in the studied populations of the parthenogenetic species. It was shown that the differences were due to the varying structure of the microsatellite cluster and to single nucleotide substitutions at fixed distances in the DNA regions adjacent to the cluster. The allele structure variations form haplotype markers specific for each allele and inherited from the parental bisexual species. It was determined which alleles of the parthenogenetic species were inherited from the maternal species and which from the paternal species. Characteristics of distribution, frequency of occurrence, and combination of alleles of microsatellite loci, which determine the distinctive features of each D. rostombekovi population were obtained. The data can be used in the future to determine the clonal diversity and possible ways of its formation in the populations of the parthenogenetic species D. rostombekovi.

[Genetic Differentiation among Natural Populations of the Lizard Complex Darevskia raddei as Inferred from Genome Microsatellite Marking].

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the F(st) genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the F(st) genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the F(st) distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.

[Characterization of retrotransposon Bov-B LINE reverse transcriptase gene sequences in parthenogenetic lizards Darevskia unisexualis and bisexual species D. nairensis and D. valentini].

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.

[Comparative Analysis of Polymorphisms of the Serotonin Receptor Genes HTR1A, HTR2A, and HTR1B in Hadza and Datoga Males].

The distribution of the allele and genotype frequencies of polymorphic loci of serotonin receptor genes (HTR1A, rs6295; HTR2A, rs6311; HTR1B, rs6296) in Hadza (n = 197) and Datoga males (n = 230) living in Tanzania was determined. It was shown that the populations significantly differ by the frequencies of alleles and genotypes of the rs6295 locus of the HTR1A gene. The G-allele (0.779) and the genotype G/G (0.590), which are markers of increased risk of suicidal and impulsive behavior, respectively, are revealed in Hadza with high frequency. It was found that the frequency of homozygous G/G of the rs6296 locus of the HTR1B gene, which is a marker of increased risk of outward directed aggression, is higher in Datoga (0.563) than in Hadza (0.457). The allele and genotype frequencies of the rs6311 locus of the HTR2A gene do not differ among the Hadza and Datoga males. The data on the distribution of allele and genotype frequencies of the HTR1A, HTR2A, and HTR1B genes can be used to determine the associations of the identified markers with various forms of human aggressive behavior.

[The 3'-UTR polymorphism of dopamine transporter gene in hadza and datoga males].

The study of VNTR-polymorphism and the molecular struc ture of 3'-UTR of the dopamine transporter gene (DAT1/SLC6A3) was performed in hadza and datoga males. It was shown that hadza and datoga differed in allele and genotype frequencies. Allele with 9 repeats in 3'-UTR is more common in hadza as well as homozygous genotype DAT19/9. Allele with 10 repeats is more common in datoga as well as homozygous genotype DAT1 10/10. The molecular structure of the DAT alleles with 3, 8 and 12 repeats was determined for the first time. In addition it was found that DAT1 allele with 11 repeats in datoga significantly differed from previously described ones in other populations in repeats type and arrangement. We suggest that variations of the repeats num ber and type in the 3'-UTR of allelic variants may affect the dopamine transporter gene function.

[Polymorphism of 5-HTTLPR and Stin2 loci of the serotonin transporter gene in males of African ethnic populations Hadza and Datoga].

A molecular-genetic study of 5-HTTLPR and the Stin2 loci of the serotonin transporter gene (5-HTTL) in males of the African ethnic populations Hadza and Datoga, which differ in the level of culturally acceptable aggression, was carried out. The distribution of allele and genotype frequencies of these two loci was established. It was shown that the frequency distribution of genotypes and alleles among Hadza and Datoga in the examined samples is practically identical by the VNTR-polymorphism of both loci. However, the Hadza populations, as compared to Datoga, showed a significant (p = 0.006) increase in the frequency of the transcriptionally less active allele L(G) of the 5-HTTLPR locus. For the first time, the structure of the allelic variant of locus Stin2 with eight repetitions (Stin2.8) is described and established for African populations. The test for independence of the frequency distribution of the alleles of the studied loci showed highly significant linkage disequilibrium among Hadza (p << 0.001) and Datoga (p = 0.021). In analysis of the genotype combinations of two loci, it was revealed that thestudied populations differed significantly by the L(A)L(G) 10/12 genotype (p << 0.001). When combining the genotypes, no significant differences between the populations based on their expression activity were identified. We assume that the identified combined genotypes reflect the effects of similar behavioral traits for both populations.

[Molecular genetic polymorphism of androgen receptor gene (AR) in African populations of Hadza and Datoga].

The molecular genetic analysis of the polymorphic variants of the CAG repeat-containing locus of the andro- gen receptor (AR) gene was performed in the populations of Hadza and Datoga. Allele frequency distribution patterns were established. Alleles containing 20-25 repeats were the most abundant in both populations were the. The populations studied were compared with Asians (Han), white Americans, and Africans (Ariaal). Sta- tistically significant difference between populations of Hadza and Datoga in the distribution of the AR allelic variants was demonstrated.

[Molecular nature of allelic polymorphism of highly variable microsatellite locus Du161(arm) in unisexual lizard Darevskia armeniaca (Lacertidae)].

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du 161 (arm) was discovered. Analysis of allelic polymorphism was carried out using locus-specific PCR of the lizard DNA specimens from 13 isolated Armenian populations (N = 138). In the sample examined, a total of 12 Du 161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du 161 (arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du 161 (arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from microsatellite cluster, and by single nucleotide insertions, as well as by longer insertions located within and outside of the microsatellite cluster. Moreover, point mutations formed different combinations (haplotypes), typical of certain alleles. These combinations can be used for the analysis of the origin and inheritance of these alleles in D. armeniaca, as well as for investigation of their interspecific variation in the representatives of the genus Darevskia.

[Polymorphism of the dopamine D4 receptor (DRD4) and serotonin transporter (5-HTTL) gene promoter regions in African tribes of Hadza and Datoga].

Molecular genetic analysis of the allelic variants of the DRD4 and 5-HTTL gene promoter regions was performed in African tribes of Hadza and Datoga, characterized by different levels of socially acceptable aggression. It was demonstrated that Hadza and Datoga people differed in the structural organization of one of the 5-HTTL alleles (extra long allele xL). Analysis of the allele length polymorphism of both genes showed that in the Hadza and Datoga samples examined, variation parameters, as well as the genotype and allele frequency distribution pattern were almost the same. At the same time, analysis of the SNP polymorphism at the A/G substitutions of the 5-HTTL locus revealed a substantial decrease of the active allele L(A) frequency in the population of Hadza compared to the population of Datoga (chi2 = 3.77; d.f = 1; P = 0.052).

Isolation of eukaryotic DNA fragments containing structural genes and the adjacent sequences.

In Drosophila melanogaster structural genes are located close to moderately reiterated sequences. One of the clones obtained contains the DNA related to intercalary heterochromatin of D. melanogaster. These are individual differences in the distribution of genetic material in polytenic chromosomes of different stocks of D. melanogaster. The techniques that allow isolation of DNA fragments containing structural genes at the beginning, in the middle, or the end of the coding strand have been elaborated.

[Somatic mosaicism in mice detected using the RAPD-PCR method].

99 DNA samples of organs and tissues of 18 mice were examined using the method of PCR amplification with random primers. Among 27 oligonucleotide primers tested, 4 producing stable, well-reproducible profiles of amplification products were chosen for further analysis. Using 2 of these primers we detected differences in RAPD-profiles in some tissues in several individuals. These differences were associated with the modification of mobility, or with the fragment gain/loss in the RAPD profile, and could be caused either by genomic rearrangements, or mutations involving the regions of the DNA-primer pairing. Different epigenetic factors may also contribute to this process.

[Molecular genetic characteristic of dinucleotide loci in parthenogenetic lizards Darevskia unisexualis].

In the present study, the first molecular genetic investigation of dinucleotide (GT)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis was performed. New polymorphic locus, Du214, (GeneBank ac no. EU252542) was identified and characterized in detail. It was demonstrated that allele of this locus differed in the size and structure of microsatellite locus, as well as in point mutations, the combinations of which enabled the isolation of stabile fixed double nucleotide substitutions A-A (alleles 2 and 4) and G-T (alleles 1, 3, 5, and 6). Double nucleotide substitutions described were also identified in the orthlogous loci of the parental species genomes, D. raddei (G-T) and D. valentine (A-A). Based on the analysis of allele distribution pattern at this locus in all populations of parthenospecies D. unisexualis, mathematic model was elaborated and realized. Using this model, frequencies of allelic variants for all populations of the species of interest were calculated and population genetic structure of D. unisexualis was characterized. Genetic contribution of each population to the species gene pool was determined. The data obtained demonstrated that microsatellite variation was one of the factors of clonal and genetic diversity of a parthenospecies.

[Molecular structure of allelic variants of microsatellite loci Du281 and Du47 in unisexual and bisexual lizard species of the genus Darevskia].

Experimental data on the molecular structure and variability of microsatellite loci in unisexual and bisexual lizard species of the genus Darevskia were analyzed. The allelic variants of Du281 and Du47 were found to differ in the number of monomers, the structure of microsatellite clusters, and point mutations in these clusters and flanking DNA. Interspecific comparison of alleles of these loci revealed both variable regions in the microsatellite clusters and allele-specific evolutionarily conserved nucleotide groups. In general, the results of comparative structural analysis of allelic variants testify to a high genetic similarity of the unisexual and bisexual lizard species studied and reveals the characteristic features of their interspecies variability.

Enhanced heterogeneity of the LR2 segment in the human ribosomal intergenic spacer.

Human ribosomal intergenic spacer (rIGS) contains in its central part two highly homologous 2 kb repeats, LR1 and LR2. In this paper, we investigate heterogeneity of the variable LR2 segment (LR2var) in the human rIGS. More than 500 LR2var copies from ten unrelated human genomes have been cloned and sequenced. Prolonged (G)n (AG)m compound microsatellite clusters with 'n' and 'm' notions fluctuating in random manner span central parts of almost all LR2var variants. Nucleotide sequences flanking the central microsatellite clusters are represented by more than 30 structural groups, with the two major (A and B) and six minor (C-H) ones. The analysis of sequencing data let us propose that the LR2var variability can be derived by various ways, including microsatellite DNA slip-strand mispairing during replication, non-equal crossover and segmental DNA exchange between LR1var and LR2var through the mechanism of gene conversion.

[Molecular and genetic characterization of allelic variants of microsatellite loci in parthenogenetic Caucasian rock lizards Darevskia armeniaca (Lacertidae)].

Using a pair of primers selected for the loci deltau 215, deltau 281, and deltau 323 of Darevskia unisexualis monolocus PCR analysis of orthologous loci was carried out in populations of the related parthenogenetic species D. armeniaca and in populations of bisexual parental species D. valentini and D. mixta. It was shown that the studied loci were polymorphic and represented in populations of D. armeniaca by several allelic variants. We cloned and sequenced PCR amplification products of the allelic variants of deltau 215, deltau 281, and deltau 323 loci. It was found that allelic differences of microsatellite loci were caused by variation in a number of tandem repeats in the microsatellite clusters and point mutations in the flanking regions. Interspecies comparison of the orthologous locus deltau 215 between parthenogenetic species D. armeniaca and parental species showed that two allelic variants of deltau 215 in D. armeniaca were inherited from the parental bisexual species D. mixta and D. valentini. The third allelic variant was not found in parental species and appeared because of mutation processes in genome of parthenospecies. For the first time, the information about the molecular nature of allelic polymorphism of these microsatellite loci of parthenogenetic species D. armeniaca was received in this study.

[Polymorphism of the 12S rRNA gene and phylogeography of the Central Asian tortoises Agrionemys horsfieldii gray, 1844].

Based on intraspecific polymorphism of 12S rRNA gene, genetic variation of isolated populations of the Central Asian tortoise, Agrionemys horsfieldii, was for the first time investigated on a large part of the species distribution range, encompassing Uzbekistan, southern Kazakhstan, and northern and eastern Iran. In 59 tortoises, four haplotypes were discovered, including two (AH1 and AH2), described earlier. Haplotype AH1 was detected in 52 tortoises, inhabiting southern Kazakhstan and Uzbekistan. Haplotype AH2 was found in four tortoises from the border territory between Uzbekistan, Tajikistan, and Afghanistan. Two novel haplotypes, AH3 and AH4, were detected in the three tortoises from Iran. Based on nucleotide substitutions in the 12S rDNA sequence, the possible divergence time between the tortoises from different parts of the range was estimated. Possible pathways of the formation of modern intraspecific groups of A. horsfieldii are discussed.

Molecular characterization of allelic variants of (GATA)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae).

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.

[Nucleotide sequences of the microsatellite locus Du215 (arm) allelic variants in the parthenospecies Darevskia armeniaca (Lacertidae)].

Using monolocus PCR analysis with the pairs of primers designed for the Du215 locus of Darevskia unisexualis, allelic polymorphism at the orthologous locus in the populations of the related parthenospecies D. armeniaca was investigated. It was demonstrated that Du215 (arm) locus was polymorphic and in the populations of parthenospecies D. armeniaca (n = 127) represented by at least three allelic variants, differing from each other by the size and composition of microsatellite cluster, and by single nucleotide substitutions in flanking DNA. Unlike the Du215 locus, Du215 (arm) was shown contain not only GATA, but also (GACA) repeats, which were absent in D. unisexualis. Thus, in this study, the data on the molecular nature of allelic polymorphism at one of the microsatellite loci of the parthenospecies D. armeniaca were reported.

[Evolutionary divergence of the ribosomal internal transcribed spacer 2 (ITS2) in lizards].

During the pre-rRNA cleavage pathway, the excision of ITS2, a eukaryotic specific insertion, remains the most elusive processing step, even in yeast. Comparison of the ITS2 sequences in different organisms permits to reveal conservative, presumably functionally important elements as well as obtain new information about ITS2 divergence in evolution. We have cloned and sequenced the ITS2 of three lizard species, Agama caucasia (Agamidae), Darevskia armeniaca and Lacerta strigata (Lacertidae) and detected in them a set of specific and conservative structural elements employing secondary structure consensus for vertebrate ITS2. Furthermore, we have performed an alignment and comparative analysis of the ITS2 sequences for the two lizards families. It enables us to propose that modern lizard species formation in evolution was accompanied by ITS2 duplication in the rDNA of their common progenitors.

[Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)].

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.