Germline mutations of tetranucleotide DNA repeats in families with normal children and reproductive pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortuses. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a complex of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortuses with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 x 10(-3) versus 2.32 x 10(-3) per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.     

Isolation and Characterization of Biochemical Properties of DNA Methyltransferase <Emphasis Type="Italic">Fau</Emphasis>IA Modifying the Second Cytosine in the Nonpalindromic Sequence 5′-CCCGC-3′

A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.     

Isolation and investigation of natural yeast strains resistant to heavy metal salts and radionuclides

The collection of yeasts (more than 2000 strains) from extreme natural environment of Kamchatka peninsular and Kuril Islands was created. 448 strains were selected for their resistance to salts of such heavy metals as Cu, Cd, Co and to high temperature (37-52 degrees C). 72% of strains appeared to be resistant to one or more selective factors. We obtained several strains able to grow on medium with 0.1 M/L nonradioactive strontium and (or) cesium. Four of this strains accumulated radioactive isotope 90Sr with 45-80% efficiency. Thus, we demonstrated that yeast strains from nature could be used for bioremediation of industrial waste solutions, polluted by radionuclides and salts of heavy metals.     

Functionalized nanocrystal-tagged fluorescent polymer beads: synthesis, physicochemical characterization, and immunolabeling application

A methodology for incorporating solubilized CdSe/ZnS core/shell nanocrystals (NCs) into functionalized carboxylated polystyrene latexes 0.3-1 microm in diameter via a swelling procedure was developed and used for the production of homogeneous, highly fluorescent polymeric beads (HFPBs), which were found to be comparable in brightness to standard polymeric microspheres doped with organic fluorophores and more photostable than the latter by more than 50 times (Fluoresbrite yellow-orange microspheres were used as an example). The three-dimensional (3D) confocal analysis of individual 1-microm HFPB demonstrated that the beads were doped with the NCs almost homogeneously. HFPBs 0.3 microm in diameter were conjugated with anti-mouse polyvalent immunoglobulins and used for immunofluorescent detection of p-glycoprotein, a mediator of the multidrug resistance phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. The photostability of NCs-tagged HFPBs offers obvious advantages for the reconstruction of 3D confocal fluorescence images of antigen distribution, and their exceptionally high brightness combined with photostability permits the detection of a single antigen molecule using a standard epifluorescence microscope.     

Poly(ADP-ribose) polymerase-1 as a regulator of protein-nucleic acid interactions in the processes responding to genotoxic action

Poly(ADP-ribose) polymerase-1 (PARP-1), nuclear protein of higher eukaryotes, specifically detects strand breaks in DNA. When bound to DNA strand breaks, PARP-1 is activated and catalyzes synthesis of poly(ADP-ribose) covalently attached to the row of nuclear proteins, with the main acceptor being PARP-1 itself. This protein participates in a majority of DNA dependent processes: repair, recombination; replication: cell death: apoptosis and necrosis. Poly(ADP-ribosyl)ation of proteins is considered as mechanism, which signals about DNA damage and modulate protein functioning in response to genotoxic impact. The main emphasis is made on the roles of PARP-1 and poly(ADP-ribosyl)ation in base excision repair (BER), the process, which provides repair of DNA breaks. The main proposed functions of PARP-1 in this process are: factor initiating assemblage of protein complex of BER; temporary protection of DNA ends; modulation of chromatin structure via poly(ADP-ribosyl)ation of histones; signaling function in detection of the levels of DNA damage in cell.     

Detection of aneuploidy in spontaneous abortions using the comparative hybridization method

Comparative genomic hybridization (CGH) technique was used to examine a set of ten spontaneous abortions whose cell cultures were characterized by the lack of proliferation in vitro, and thereby, were not available for the analysis by means of routine cytogenetic methods. Five abortions (50%) had aneuploidy of autosomes, including trisomy 10, 14, 18, and 21, and monosomy 22. The latter variant of unbalanced chromosomal abnormalities is rarely detected in spontaneous abortions by use of conventional cytogenetic methods. The results were validated by using fluorescent in situ hybridization (FISH) analysis with centromere-specific DNA probes. Embryos with trisomy 10 and monosomy 22 displayed mosaicism with the frequencies of abnormal cell clones constituting 68 and 33% respectively. The advantages and limitations of the applying of CGH technique for detection of genomic abnormalities in both nonmosaic and mosaic forms are discussed.     

Effect of inhibitors of two isoprenoid biosynthetic pathways on physiological and biosynthetic characteristics of <Emphasis Type="Italic">Dioscorea deltoidea</Emphasis> cell suspension culture

The effect of phosmidomycin and mevinolin, which inhibit MEP and MVA isoprenoid biosynthetic pathways, respectively, on the growth (biomass accumulation, growth index, specific growth rate), physiological (respiration intensity and ratio between the cytochrome and cyanide-resistant respiration types), and biosynthetic (steroid glycoside biosynthesis) characteristics of the cell suspension culture of Dioscorea deltoidea Wall. has been studied. Both inhibitors decreased the growth index of a cell culture by 20–25%, but their influence on the cell growth dynamics was different. Mevinolin treatment reduced the maximum biomass accumulation by 20% as against the control but did not change the character of a growth curve. Phosmidomycin treatment caused a significant growth delay (a 6-day lag phase) followed by a short active growth period (μ = 0.29 days^-1). Treatment of cells with inhibitors did not significantly influence on their total oxygen uptake rate, whose average value at different growth phases was equal to 100–200 mg О₂/g of dry cell weight per hour, but cardinally changed characteristics of respiratory metabolism. The inhibitors increased the activity of a cyanide-resistant respiration and decreased the intensity of a cytochrome respiration; each inhibitor worked in its specific manner. In the case of mevinolin, the maximum level of cyanide-resistant respiration (70% of the total respiration intensity) was observed at initial growth phases; during the further cell culture growth, this value gradually reduced to 6–8%. Phosmidomycin treatment caused a reverse dynamics: at the initial growth phases, the contribution of cyanide-resistant respiration was 30%, whereas at the stationary and degradation phases it increased to 50–60%. The treatment of cells with phosmidomycin resulted in a double increase in the content of furostanol glycosides at the stationary growth phase, whereas the use of mevinolin-containing medium reduced the content of these compounds as against the control. Inhibitors also influenced on the ratio of individual glycosides, such as protodioscin, deltoside, and their 26-S-isomers. The obtained results confirm the hypothesis of a possible intermediate exchange between the plastid (MEP) and cytosolic (MVA) isoprenoid biosynthetic pathways; this exchange is directed mainly from the plastids to the cytosol.     

About the sex ratio in connection with early embryonic mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortionssensu stricto with a developed embryo without any significant intrauterine delay of development (n=100), nondeveloping pregnancies (n=176), and anembryonic fetuses (n=66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and the 46.XY karyotype. The presence of 46.XY may reflect the specific genetic mechanisms of the prenatal mortality of embryos with the normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with the normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for nondeveloping pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of the prevalence of embryos with the 46.XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio from the control group consisting of medical abortions (1,11). Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46.XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical “chances ratio” index also provided evidence that embryos with the 46.XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.