Non-canonical ribosomal DNA segments in the human genome, and nucleoli functioning.

Ribosomal DNA (rDNA) in the human genome is represented by tandem repeats of 43 kb nucleotide sequences that form nucleoli organizers (NORs) on each of five pairs of acrocentric chromosomes. RDNA-similar segments of different lengths are also present on (NOR)(-) chromosomes. Many of these segments contain nucleotide substitutions, supplementary microsatellite clusters, and extended deletions. Recently, it was shown that, in addition to ribosome biogenesis, nucleoli exhibit additional functions, such as cell-cycle regulation and response to stresses. In particular, several stress-inducible loci located in the ribosomal intergenic spacer (rIGS) produce stimuli-specific noncoding nucleolus RNAs. By mapping the 5'/3' ends of the rIGS segments scattered throughout (NOR)(-) chromosomes, we discovered that the bonds in the rIGS that were most often susceptible to disruption in the rIGS were adjacent to, or overlapped with stimuli-specific inducible loci. This suggests the interconnection of the two phenomena - nucleoli functioning and the scattering of rDNA-like sequences on (NOR)(-) chromosomes.

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2.

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop-stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia+Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.

Genetically unstable microsatellite-containing loci and genome diversity in clonally reproduced unisexual vertebrates.

There are more than 70 known unisexual species of fishes, amphibians, and reptiles. They are all-female populations of interspecific hybrid origin that reproduce without sex via altered gametogenetic mechanisms. They are either sperm independent as in parthenogenesis or sperm dependent as in gynogenesis or hybridogenesis, which causes clonal (or hemiclonal) inheritance. The first two modes of reproduction produce species composed of genetically isolated clones. In many previous papers, origin and ancestry, clonal diversity based on allozyme or mitochondrial DNA variation, ecology and evolution of unisexual vertebrates were discussed. This chapter reviews the role of mutations in genome diversity of some unisexual vertebrates revealed by DNA fingerprinting and/or by locus-specific PCR. It also describes recent data on molecular structure of unstable microsatellite loci and their allelic variants in parthenogenetic lizard species. The available data demonstrate that microsatellite mutations as well as point mutations in flanking regions make significant contribution in genome diversity of, at least some, clonaly reproduced vertebrates.

Genetic variation and de novo mutations in the parthenogenetic Caucasian rock lizard Darevskia unisexualis.

Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containing loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers. No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)(n) containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards.

Genomic variation in parthenogenetic lizard Darevskia armeniaca: evidence from DNA fingerprinting data.

Microsatellites, or short tandem repeats, are abundant across genomes of most organisms. It is evident that the most straightforward and conclusive way of studying mutations in microsatellite-containing loci is to use clonally transmitted genomes or DNA sequences inherited in multigeneration pedigrees. At present, little is known about the origin of genetic variation in species that lack effective genetic recombination. DNA fingerprinting in 43 families of the parthenogenetic lizard species Darevskia armeniaca (131 siblings), using (GACA)(4), (GGCA)(4), (GATA)(4), and (CAC)(5) probes, revealed mutant fingerprints in siblings that differed from their mothers in several restriction DNA fragments. In some cases, the mutant fingerprints detected in siblings were also found in population samples. The mutation rate for new restriction fragment length estimated by using multilocus probes varied from 0.8 x 10(-2) to 4.9 x 10(-2) per band/per sibling. Probably, the most variations detected as restriction fragment length polymorphism have germ-line origin, but somatic changes of (CAC)(n) fingerprints in adult lizards were also observed. These results provide new evidence of existing unstable regions in genomes of parthenogenetic vertebrate animals, which provide genetic variation in unisexual populations.

Genetic differentiation in eastern European and western Asian populations of the liver fluke, Fasciola hepatica, as revealed by mitochondrial nad1 and cox1 genes.

Partial sequences of mitochondrial genes nad1 (316 bp) and cox1 (429 bp) were analyzed to estimate the variability of the liver fluke samples collected in 20 localities in Russia, Belarus, Ukraine, Bulgaria, Armenia, Azerbaijan, Georgia, Turkey, Turkmenistan, and China. The sequences had 4.1% (nad1) and 2.3% (cox1) of variable sites, and 13 and 10 haplotypes were identified among nad1 and cox1 genes, respectively. Spatial analysis of genetic and nucleotide diversity indicated little or no structuring of genetic variation between hosts or regions. The analysis of distribution of both separate and combined (nad1 + cox1) haplotypes revealed the existence of 2 well-defined lineages with 2 main haplotypes and a number of shared divergent haplotypes. Our study showed that the first lineage included the main N1-C1 haplotype, which was found in Australia, China, Georgia, Turkey, Armenia, Azerbaijan, and in all European populations (from Russia, Belarus, Ukraine, Bulgaria). The second lineage was found in all European populations and in populations from Armenia and Azerbaijan. It was suggested that one of the lineages (I) has an Asian origin. The possible source of mtDNA variability and associations between lineage divergence of parasite and its definitive hosts (cattle and sheep) are discussed.

Evolutionary divergence of the pre-promotor region of ribosomal DNA in the great apes.

The human ribosomal intergenic spacer (rIGS) differs considerably on nucleotide sequence and regulatory elements positioning from their counterparts in the mouse, rat and Xenopus laevis. In the present study, we have PCR amplified, cloned and sequenced the rIGS fragments of about 4.5 kb length, located approximately 2 kb upstream of the rRNA transcription start point for the great apes, Pan paniscus, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus. Alignment of the primates' orthologic nucleotide sequences reveals high extent of similarity, with the exception of highly repetitious region between the two Alu repeats, nearest to the onset of transcription. Data obtained have been analyzed for further understanding of the evolution of repetitive sequences. We have also shown, that MARs/SARs distribution patterns in the pre-promoter rIGSs of the great apes and the mouse are surprisingly similar in spite of an absence of similarity in the primary structure and regulatory elements organization in the region under study.

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy.

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.