GLAD-PCR Assay of R(5mC)GY Sites in the Regulatory Region of Tumor-Suppressor Genes Associated with Gastric Cancer.

At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.

[Application of GLAD-PCR Assay for Study on DNA Methylation in Regulatory Regions of Some Tumor-Suppressor Genes in Lung Cancer].

Hypermethylation of the gene regulatory regions are common for many cancer diseases. In this work we applied GLAD-PCR assay for identificating of the aberrantly methylated RCGY sites in the regulatory regions of some downregulated genes in tissue samples of lung cancer (LC). This list includes EFEMP1, EPHA5, HOXA5, HOXA9, LHX1, MYF6, NID2, OTX1, PAX9, RARB, RASSF1A, RXRG, SIX6, SKOR1 and TERT genes. The results of DNA samples from 40 cancer and 25 normal lung tissues showed a good diagnostic potential of selected RCGY sites in regulatory regions of MYF6, SIX6, RXRG, LHX1, RASSF1A and TERT genes with relatively high sensitivity (80.0 %) and specificity (88.0 %) of LC detection in tumor DNA.

[Application of GLAD-PCR analysis for the methylation sites detection in the regulatory areas of tumor-suppressor genes ELMo1 and EsR1 in colorectal cancer].

Aberrant methylation of regulation regions of tumorsuppressor genes is showed for many cancer diseases. In course of this modification an enzyme DNMT3 methylates RCGY sites in CpG-islands of regulation regions producing R(5mC)GY sites. Earlier we developed GLAD-PCR assay to determine R(5mC)GY site in a definite position of human genome. In this work we have applied GLAD-PCR assay to determine R(5mC)GY sites in regulation regions of ESR1 and ELMO1 tumor-suppressor genes. We have studied a fragment of first exon of ELMO1 gene and a part of ESR1 promoter region in DNA preparations from malignant cell line SW837 and colorectal tumor samples. We have checked four sites in each region and found two highly methylated sites: GCGC in first exon of ELMO1 gene and GCGT in promoter region of ESR1 gene. Site GCGT is weakly methylated in healthy tissues and more methylated in the most of colorectal samples. Site GCGC is not methylated in healthy tissues and significantly methylated in 60% of colorectal samples. A possibility to use GLAD-PCR assay for cancer diagnostics is discussed.

[Restriction analysis of N-acetyltransferase 2 gene in Caucasian population of West Siberia]. / Restriktsionnyi analiz gena N-atsetiltransferazy (NAT2) u evropeoidov zapadnoi Sibiri.

Restriction analysis of the NAT2 gene was carried out in inhabitants of Novosibirsk. Polymorphism of this gene for nine known point mutations was studied in a sample of Novosibirsk residents consisting of 109 healthy Caucasians. The frequencies of these mutations did not significantly differ from the frequencies reported for Caucasian populations of other countries. In 79 patients with lung cancer, a region of the NAT2 gene that includes 29.7% of the coding sequence was analyzed for the new mutations by the RFLP analysis. No new mutations were found in this group.

[Use of restriction endonucleases Bst2U I and Acc65 I to detect Kpn I polymorphism of NAT2 gene]. / Primenenie éndonukleaz restriktsii Bst2U I i Acc65 I dlia obnaruzheniia Kpn I-polimorfizma gena NAT2.

RFLP-analysis was made for 30 human DNA samples. The ability of application of restriction endonucleases Acc65I and Bst2UI to find point mutation C481T in NAT2 gene has been demonstrated for the first time. Variants of these enzymes application are discussed.

Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP.

The hybrid protein consisting of Tte DNA polymerase fragment and mutant Taq DNA polymerase (F667Y) fragment in the ratio 20 : 1 was constructed. Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme. The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68%. In the polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA polymerase.

Restriction fragments length polymorphism in the NAT1 gene of Europeans in West Siberia.

Restriction fragment length polymorphism in the NAT1 gene was assayed to reveal 7 mutations (97C>T; 190C>T; 350,351G>C; 402T>C; 752A>T; D(1105); D(1025)) in 74 Europeans from West Siberia. New methods for detecting mutations 350,351G>C, 402T>C, 752A>T, D(1105), and D(1025) were proposed.

Thermostable DNA polymerase from Thermus thermophilus B35: cloning, sequence analysis, and gene expression.

The nucleotide sequences of three thermostable DNA polymerase (Taq, Tth, and Tfl) genes were analyzed and high conserved regions typical for this polymerase family were identified. Using primers for one of the conserved regions, the genomic DNA fragment of T. thermophilus B35 strain was amplified. The resulting fragment was cloned into a plasmid and used as a hybridization probe with digests of T. thermophilus B35 DNA cleaved by different restriction endonucleases. A restriction DNA fragment carrying the full-length Tte polymerase gene was found, cloned, and sequenced. The primary structures of the Tte and Tth DNA polymerase genes were analyzed. The Tte-pol gene was recloned into an expression vector and recombinant protein was purified to homogeneity. The properties of Tte-pol in the polymerase chain reaction were investigated.

Thermostable DNA-polymerase from Thermus thermophilus B35: isolation and characterization of some properties.

Thermostable Tte DNA-polymerase was isolated from the strain Thermus thermophilus B35 which was found in hot spring water. The enzyme with molecular mass 87 kD was isolated using sequential chromatography on DEAE-Sepharose, hydroxylapatite, hexyl-agarose, and heparin-Sepharose. Biochemical properties of Tte DNA-polymerase are similar to those of Tth DNA-polymerase isolated from Thermus thermophilus HB8; however, practical application of Tte-Pol seems to be more favorable due to higher temperature optimum of this enzyme and lack of restriction endonucleases in the initial strain.

[Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus]. / Izuchenie nekotorykh svoistv immobilizovannoi terminal'noi dezoksinukleotidiltransferazy iz timusa krupnogo rogatogo ckota.

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.