Molecular cloning and characteristics of allele variants (GATA)n, the microsatellite locus Du281 of parthenogenetic caucasian rock lizard (Darevskia unisexualis) genome.

Abstract

Hypervariable mini- and microsatellites represent the most widespread and universal system of molecular markers and are widely used in population and evolutional biology, genomic marking, paternity determination, and medico-biological studies [1, 2]. These repeats are classified with the most instable regions of eukaryotic genomes and are characterized by the highest mutation rates reported for genetic loci [3, 4]. The studies of individual mini- and microsatellite loci showed that changes in them significantly vary depending on the species, repeat type, alleles, age, and sex. However, the genesis of mini- and microsatellite loci, intensively studied in the human [5] and some other bisexual species [6], practically has not been studied in species with clonal type of reproduction. Obligate parthenogenetic reptiles, whose reproduction system implies clonal reproducing of maternal genotype in a series of generations, is a unique model system for monitoring of genetic variability in loci with increased mutation activity [7]. Earlier, multilocus DNA fingerprinting was used to study variability of several types of mini- and microsatellite DNA markers in four parthenogenetic species of the genus Darevskia ( D. dahli, D. armeniaca, D. unisexualis , and D. rostombekovi ) [8‐11]. It was shown that genetic heterogeneity of parthenogenetic species with respect to some microsatellite markers may be detected against the background of species-specific DNA fingerprints. The most variable were the loci that contained (TCC) n , (TCT) n , and (GATA) n microsatellites in D. unisexualis genome [8, 9, 12‐14]. DNA fingerprints of parthenogenetic D. unisexualis families demonstrated high frequency of mutations for these loci already in the firstgeneration progeny [9, 14]. Furthermore, mutant (GATA) n fingerprint phenotypes were also detected in the progeny of population D. unisexualis samples. This finding is indicative of the mutant nature of genotypic diversity of D. unisexualis populations with respect to (GATA) n loci. However, the molecular nature and mechanisms of occurrence of these mutations remain obscure. It is conceivable that certain progress in this field may be reached with the use of genetic engineering studies of variable loci. In this study, we for the first time cloned and sequenced one variable locus of D. unisexualis genome and revealed the nature of structural variations of its allele variants. Total cell DNA was isolated form blood of adult D. unisexualis by standard phenol‐chloroform method using protein kinase K. DNA was hydrolyzed with endonuclease Sau 3A (Amersham) until complete degradation and ligated with the pUC12 vector, treated with Bam H1 restriction endonuclease using standard procedure. The ligase mixture was used to transform XL-1 Blue E. coli cells (Stratagene). The clonotheque obtained was then used for searching for recombinant clones containing (GATA) n microsatellites. The screening of approximately 1000 recombinant clones using