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.

Instability of (GATA)n microsatellite loci in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae).

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA)n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)4 as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA)n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA)n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA)n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.

Genetic variation in parthenogenetic Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca, L. unisexualis) analyzed by DNA fingerprinting.

Multilocus DNA fingerprinting has been used to study the variability of some mini- and microsatellite sequences in parthenogenetic species of Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca and L. unisexualis). We demonstrate that these clonally reproducing lizards possess species-specific DNA fingerprints with a low degree of intra- and interpopulation variation. Mean indices of similarity obtained using M13 DNA, (GACA)4 and (TCC)50 as probes were 0.962 and 0.966 in L. dahli and L. armeniaca, respectively. The mean index of similarity obtained using M 13 and GATA probes in L. unisexualis was estimated to be 0.95. However, despite the high degree of band-sharing, variable DNA fragments were revealed in all populations with the microsatellite probes. An particularly high level of variability was observed for (TCC)n microsatellites in populations of L. unisexualis. In fact TCC-derived DNA fingerprints were close to being individual-specific, with a mean index of similarity of 0.824. Fingerprint analysis of parthenogenetic families of L. armeniaca showed that all maternal fragments were inherited together by the progeny, and no differences in fingerprint patterns were observed. On the other hand, while identical DNA fingerprints were obtained from L. unisexualis families with M13 and (GATA)4 probes, use of the (TCC)50 probe revealed remarkable intrafamily variation in this species. It is assumed that the genetic heterogeneity observed in parthenogenetic populations may be explained, at least in part, by the existence of genetically unstable microsatellite loci. Our data serve to illustrate processes of spontaneous mutagenesis and the initial stages of clonal differentiation in natural populations of the lizard species studied.

DNA fingerprinting in captive population of the endangered Siberian crane (Grus leucogeranus).

DNA fingerprinting was used to estimate genetic diversity within the endangered Siberian crane (Grus leucogeranus) captive population consisting of several dozens of founders originating from the two wild populations of eastern and western Siberia. Similarity and difference among captive individuals were demonstrated by the unweighted pair-group (UPGMA) clustering procedure. Quantitative characteristics of the eastern and western captive population groups such as average percentage differences (APD) and heterozygosity showed a high extent of genetic variability of 77.9-79.3% and heterozygosity of 0.85-0.72 within each group. Genetic heterogeneity of the captive population structure observed here provides guidelines for management of the species gene pool in captivity. These data also indicate that monitoring of genetic diversity through DNA fingerprinting can facilitate the efforts of Siberian crane management and restoration.

Genetic differentiation in a captive population of the endangered Siberian crane (Grus leucogeranus Pall.).

DNA fingerprinting, followed by multivariate analysis of data, was used to characterize genetic heterogeneity in captive populations of the endangered Siberian and sandhill cranes. The genetic structure revealed reflected the natural population and species distributions. The relevant groups differed not only from each other, but also from interspecies and inter-population hybrids bred in captivity. In this study we have tested an approach to the analysis of population structure based on individual genotypes. Interpretation of fingerprinting data by means of the analytical system applied here is a useful and reliable procedure for the estimation of genetic relationships between individuals.

Major transcripts containing B1 and B2 repetitive sequences in cytoplasmic poly(A)+RNA from mouse tissues.

The cytoplasmic poly(A)+RNAs containing ubiquitous B1 and B2 repeats of the mouse genome in normal tissues and tumors have been studied. Only one strand of the repeats is represented in cytoplasmic RNA in all the cases. Some tumor cells were found to be enriched in 1.4 kb B1+mRNA, 1.6 kb B2+mRNA and small (0.2-04 kb) B1+ and B2+ poly(A)+RNAs. On the other hand, mouse liver and kidney contained high amounts of 2 kb B2+mRNA. Its content increased noticeably in the regenerating liver, but in hepatoma it dropped to a zero level. Thus, the switching on (or off) of B1- and B2-containing mRNAs occurred noncoordinately. At the same time, the activation of the synthesis of small B2+RNA and small B1+RNA was simultaneous.

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.