Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Use of the polymerase chain reaction for typing allelic variants of the human HLA-DQA1 by hybridization with oligonucleotide probes, specific for specific alleles]

Class II HLA molecules are the most useful markers for susceptibility to different autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM) and rheumatoid arthritis (RA). Polymerase chain reaction and hybridization with a set of allele-specific oligonucleotide have been used for analysis of allelic sequence variation. The analysis of frequencies of HLA-DQA1 alleles among 10 patients of the russian population revealed a uneven distribution. We have developed a method for preparing non-radioactive oligonucleotide probes with terminal deoxynucleotidyl transferase and Bio-11-dUTP. Comparison of biotinylated and 32P-labeled hybridization probes gave the same sensitivity for HLA-DQA1 typing of amplified DNA. Amplification of the HLA-DQA1 gene has been successful on 10 pg of total DNA. This amount of DNA is close to the amount of DNA in a single cell. Alternatively, HLA-DQA1 typing could be based on the analysis of buccal cells of saliva that would avoid the problem of individuals who object to giving blood samples.     

Mapping of the major early endonuclease cleavage site of the rat precursor to rRNA within the internal transcribed spacer sequence of rDNA

The endonuclease cleavage of 41 S pre-rRNA to yield 32 S and 21 S pre-rRNA constitutes a major early step in the processing of pre-rRNA in rat liver. The 5'-terminus of 32 S pre-rRNA and the 3'-terminus of 21 S pre-rRNA were precisely located within the rDNA sequence by S1 nuclease protection mapping and use of appropriate rDNA restriction fragments. The 5'-terminus of 12 S pre-rRNA, an initial product of 32 S pre-rRNA processing, was also mapped within the rDNA sequence. The 5'-termini of 32 S and 12 S pre-rRNA coincide and map within a 14-residue T-tract (non-coding strand) at 161-163 bp upstream from the 5'-end of the 5.8 S rRNA gene. The 3'-terminus of 21 S pre-rRNA maps within the same T-tract. These results show that the endonuclease cleavage occurs within a U-tract in the internal transcribed spacer 1 sequence of 41 S pre-rRNA. The homogeneity of the 5'- or 3'-termini of 32 S, 12 S and 21 S pre-rRNA indicates also that the terminal processing of these molecules, if any, is markedly slower. The coincidence in the location of 32 S and 12 S pre-rRNA 5'-termini shows further that the endonuclease cleavage of 32 S pre-rRNA precedes the removal of its 5'-terminal segment to yield 5.8 S rRNA. The absence in the whole pre-rRNA internal transcribed spacer of sequences complementary to the target U-tract suggests that the endonuclease cleavage, generating 32 S and 21 S pre-rRNA, occurs in a single-stranded loop of U-residues.     

Primary and secondary structure of rat 28 S ribosomal RNA.

The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments. The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6). Several regions of high sequence homology with S. cerevisiae 25 S rRNA are present. These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E. coli. The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology. The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent. All G+C-rich segments appear to form strongly base-paired structures. The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule. These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules.     

Protection of particular cleavage sites of restriction endonucleases by distamycin A and actinomycin D.

It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment     

Association of Polymorphic Marker A1/A2 of Gene ITGB3 with Coronary Artery Disease and Myocardial Infarction

The distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3 encoding the 3 subunit of integrin IIb3 in groups of patients with coronary artery disease (CAD), including those who have had myocardial infarction (MI), and in the control group have been compared. Analysis using Fisher's test has not revealed significant differences between these groups with respect to the distributions of the alleles and genotypes of polymorphic marker A1/A2 of gene ITGB3. This indicates that this polymorphic marker is not associated with either CAD or MI in the Moscow population.