RESUMEN
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.
RESUMEN
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.
Asunto(s)
Metilación de ADN , Neoplasias Pulmonares/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Supresoras de Tumor/genética , HumanosRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales/genética , Metilación de ADN , ADN de Neoplasias/genética , Receptor alfa de Estrógeno/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Supresoras de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , ADN de Neoplasias/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Masculino , Proteínas Supresoras de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Población Blanca/genética , Humanos , Mutación Puntual , Polimorfismo de Longitud del Fragmento de Restricción , SiberiaRESUMEN
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.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Enzimas de Restricción del ADN/química , Arilamina N-Acetiltransferasa/química , Humanos , Mutación Puntual , Polimorfismo GenéticoRESUMEN
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.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Polimorfismo de Longitud del Fragmento de Restricción , Población Blanca/genética , Alelos , Enzimas de Restricción del ADN/metabolismo , Europa (Continente)/etnología , Frecuencia de los Genes , Heterocigoto , Humanos , Isoenzimas , Mutación , SiberiaRESUMEN
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.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleósidos/metabolismo , Temperatura , Thermus thermophilus/enzimología , Autorradiografía , Secuencia de Bases , Catálisis , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Estabilidad de Enzimas , Escherichia coli , Datos de Secuencia Molecular , Mutación/genética , Nucleósidos/química , Alineación de Secuencia , Thermus thermophilus/genéticaRESUMEN
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.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Estabilidad de Enzimas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Thermus thermophilus/enzimología , Cationes Bivalentes/farmacología , Cromatografía , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , ADN Polimerasa Dirigida por ADN/química , Durapatita , Estabilidad de Enzimas , Calor , Cinética , Peso Molecular , TermodinámicaRESUMEN
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.