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1.
Vestn Ross Akad Med Nauk ; (2): 35-41, 2012.
Artigo em Russo | MEDLINE | ID: mdl-22642176

RESUMO

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/ACRS/sequencing and allele-specific PCR in the real-life regimen (Russian set "KRAS-7M"). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using "KRAS-7M" set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/ACRS/automatic sequencing. False positive results were absent when detecting with "KRAS-7M" and accounted for 2 cases (5,9%) when using PCR/SSCP/ ACRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using "KRAS-7M".


Assuntos
Neoplasias Colorretais/genética , Análise Mutacional de DNA , DNA de Neoplasias/análise , Genes ras , Técnicas de Diagnóstico Molecular , Mutação Puntual , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Feminino , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Estudos Retrospectivos , Sensibilidade e Especificidade
2.
Mol Biol (Mosk) ; 45(5): 863-70, 2011.
Artigo em Russo | MEDLINE | ID: mdl-22393783

RESUMO

Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows determining most frequent mutations in 12, 13 and 61 codons of the KRAS gene. To increase the sensitivity of the method and to make possible the analysis of minor fractions of tumor cells in clinical samples the method of blocking a wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on biochip. Biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly ductal adenocarcinomas. As reference methods, the RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of whole cell population.


Assuntos
Adenocarcinoma , Análise em Microsséries/métodos , Mutação , Neoplasias Pancreáticas , Patologia Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adulto , Idoso , Códon , Sondas de DNA/genética , Feminino , Genótipo , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Sondas de Oligonucleotídeos/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Polimorfismo de Fragmento de Restrição , Proteínas Proto-Oncogênicas p21(ras)
3.
Genetika ; 46(5): 700-8, 2010 May.
Artigo em Russo | MEDLINE | ID: mdl-20583607

RESUMO

To estimate diagnostic value of K-ras mutations during cancer risk group formation, they were studied in the samples of sporadic carcinomas (n = 33) and malignant (n = 13) polyps of large intestine obtained during surgery or polypectomy. Using PCR analysis, restriction analysis, SSCP analysis and automated sequencing, eight various point mutations were revealed. Six of them were located in codon 12 and two, in codon 13 of the K-ras gene. Mutation frequency in carcinomas, benign and malignant polyps was 43, 49, and 69%, respectively. In the healthy tissue of the large intestine, no changes in codons 12 and 13 in the K-ras gene were observed. Mutations in the groups of Russian patients examined partially overlapped. In patients with colorectal carcinoma the mutation frequency in the K-ras gene was not associated with disease onset age, location, and the extent of tumor differentiation while it was associated with the stage of tumor process. The maximum mutation frequency was revealed in polyps of patients over 70 years of age as well as in the adenomas of villous histology and large size ((1 cm). No correlation between the K-ras mutation frequency and the extent of polyp dysplasia was observed.


Assuntos
Adenocarcinoma/genética , Pólipos do Colo/genética , Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Genes ras/genética , Mutação , Adulto , Fatores Etários , Idoso , Códon , Análise Mutacional de DNA/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Conformacional de Fita Simples , Federação Russa
4.
Mol Biol (Mosk) ; 43(3): 414-21, 2009.
Artigo em Russo | MEDLINE | ID: mdl-19548527

RESUMO

The purpose of this study was to investigate informativety and clinical significance of most frequent somatic alterations in K-ras, TP53, CDKN2A, MADH4 and more uncommon mutations in BRCA1, BRCA2, CHEK2 genes, which arise on preinvasive stage in sporadic pancreatic adenocarcinomas (PA), in Russian patients. We examined surgically resected and manually microdissected primary PA tissue samples and samples of normal pancreatic tissue for 37 individuals. K-ras mutations in codon 12 were found in 24 tumors (0.65) and none of normal tissue samples. No mutations were detected in BRCA1(185delAG, 300T > G, 4153delA, 4158A > G,5382insC), BRCA2 (695insT, 6174delT) and CHEK2 (1100delC) genes. Informativety for allelic loss of three tumor suppressor genes studied had not statistically significant differences: 60% - for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162); and 65.7% - for MADH4 (D18S363 + D18S474) (t = 0.48). Maximal frequency of loss of heterozygosity (LOH) was observed for CDKN2A - 0.95. For TP53 and MADH4 it was 0.62 and 0.70 respectively. The tumors included 80% cases showing LOH on different chromosomal loci. The combination of K-ras mutations (c.12) and LOH at 9p, 17p and 18q resulted in a high informativety of selected molecular markers: 85.7%. Instability of microsatellites was found only in 9% of PA.


Assuntos
Adenocarcinoma/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Genes ras , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Adenocarcinoma/metabolismo , Adulto , Idoso , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais , Quinase do Ponto de Checagem 2 , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 9 , Feminino , Marcadores Genéticos , Humanos , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/genética
5.
Mol Biol (Mosk) ; 41(1): 37-42, 2007.
Artigo em Russo | MEDLINE | ID: mdl-17380889

RESUMO

Ovarian cancer (OC) is one of the leading cause of cancer death in women. Inherited BRCA1 and BRCA2 mutations strikingly increase OC risk (with lifetime risk estimates ranging at 10-60%). Mutation 1100delC in CHEK2 gene was shown to be associated with breast cancer in women carrying this mutation. Knowledge of the nature and frequency of population-specific mutations in these genes is a critical step in the development of simple and inexpensive diagnostic approaches to DNA analysis. The frequencies of 185delAG, 300T>G, 4153delA, 4158A>G, 5382insC mutations in BRCA1 gene, 695insT and 6174delT mutations in BRCA2 gene and 1100delC mutation in CHEK2 gene were analyzed using biochips in Russian OC patients. We studied 68 women who received a diagnosis of epithelial OC and 19 women with primary multiple tumors involving the ovaries. The 185delAG, 300T>G, 4153delA and 5382insC in BRCA1 gene were identified. The most prevailing mutation was 5382insC in BRCA1 gene (87.5% of all BRCA1 mutations OC patients, 50.0% in patients with primary multiple tumors involving the ovaries). No mutations in BRCA2 and CHEK2 genes were detected.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Mutação , Neoplasias Primárias Múltiplas/genética , Neoplasias Ovarianas/genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Idoso , Quinase do Ponto de Checagem 2 , Feminino , Frequência do Gene , Genética Populacional , Humanos , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Ovarianas/patologia , Fatores de Risco , Federação Russa
6.
Genetika ; 39(6): 847-54, 2003 Jun.
Artigo em Russo | MEDLINE | ID: mdl-12884527

RESUMO

The spectrum of mutations of the RET protooncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MET 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.


Assuntos
Carcinoma Medular/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Idoso , Éxons , Feminino , Humanos , Masculino , Metionina/genética , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2b/genética , Linhagem , Feocromocitoma/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Federação Russa , Treonina/genética , Glândula Tireoide/patologia , Glândula Tireoide/cirurgia , Tireoidectomia
7.
J Mol Med (Berl) ; 79(10): 609-12, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11692159

RESUMO

Medullary thyroid carcinoma (MTC) occurs as a sporadic tumor or in connection with inherited cancer syndromes of multiple endocrine neoplasia type 2 and familial MTC. Missense RET proto-oncogene mutations and small in-frame deletions are found in most of the cases. In a significant amount of sporadic MTC cases somatic mutation at codon 918 (exon 16), or at codons 609, 611, 618, 620 (exon 10), or codons 630, 634 (exon 11) appear. We report here on three new somatic cell missense mutations of the RET proto-oncogene associated with sporadic MTC. In one tumor mutation at codon 922 TCC(Ser)-->TTC(Phe) in exon 16 was found. In another tumor two mutations at codons 639 GCA(Ala)-->GGA(Gly) and 641 GCT(Ala)-->CGT(Arg) in the exon 11 were observed. Allele-specific PCR followed by sequencing demonstrated the presence of both mutations at the same allele.


Assuntos
Carcinoma Medular/genética , Proteínas de Drosophila , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Carcinoma Medular/patologia , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Humanos , Mutação , Mutação de Sentido Incorreto , Polimorfismo Conformacional de Fita Simples , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Neoplasias da Glândula Tireoide/patologia
8.
Vestn Ross Akad Med Nauk ; (2): 34-7, 2001.
Artigo em Russo | MEDLINE | ID: mdl-11338505

RESUMO

The paper reviews the data on the molecular structure of the protooncogene RET encoding for receptor-type protein kinase, on the mechanism of transformation of the normal protooncogene RET to a dominant transforming oncogene, and on RET mutations detected in patients with the MEN-2 syndrome. Moreover, it presents the authors' own findings. The familial medullary thyroid carcinoma burdened genealogy shows a new point mutation TCG(Ser)-->GCG(Ala) in codon 891, in the exon 15 of the protooncogene RET. This mutation was not detected in the chromosomes of healthy individuals. Analyzing the linkage with two known and two new polymorphic markers showed that there was a cisaggregation of informative polymorphic markers, phenotypic manifestation of the disease, and mutations in the genealogy in question. In the protooncogene RET, there were two new polymorphisms: G/A at position 24 in intron 14 and C/T in codon 836 (exon 14). The rate of the polymorphism encountered in codon 836 proved to be similar for the Russians and the Germans (0.96%), which was also seen for two earlier described polymorphisms in codon 691 (0.80 and 0.81, respectively) and in codon 904 (0.21 and 0.22). At the same time, there were statistically significant differences in the rates of intron 14 polymorphism (0.87 and 0.77, respectively). In a family having MEN 2, a proband displayed TGC-->CGC mutation in codon 634 of the gene RET in the heterozygous state. The mutation results in substitution of cysteine amino acid residue in the cysteine-rich extracellular domain of protein kinase encoded by the gene RET for arginine. The results of molecular analysis were used to confirm its clinical diagnosis and to indicate that effective care should be delivered in MEN 2a.


Assuntos
Carcinoma Medular/diagnóstico , DNA de Neoplasias/análise , Proteínas de Drosophila , Biologia Molecular/métodos , Neoplasia Endócrina Múltipla Tipo 2a/diagnóstico , Neoplasia Endócrina Múltipla Tipo 2b/diagnóstico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/diagnóstico , Alelos , Carcinoma Medular/genética , Carcinoma Medular/metabolismo , Sondas de DNA/química , Diagnóstico Diferencial , Feminino , Frequência do Gene , Marcadores Genéticos , Humanos , Masculino , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Neoplasia Endócrina Múltipla Tipo 2b/genética , Neoplasia Endócrina Múltipla Tipo 2b/metabolismo , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Genético , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo
9.
Genetika ; 34(8): 1157-9, 1998 Aug.
Artigo em Russo | MEDLINE | ID: mdl-9777362

RESUMO

A new point mutation, TCG(Ser)-->GCG(Ala) in codon 891, exon 15 of the RET protooncogene was revealed in two patients from a pedigree with familial medullary thyroid carcinoma (FMTC), but not in healthy persons. A linkage analysis with two well-known and two new intragene polymorphisms showed that informative polymorphic markers, the phenotypic expression of the disease, and the mutation are cosegregated in the studied pedigree. Two new polymorphisms, G/A at position-24 of intron 14 and C/T in codon 836 of exon 14, were found in the RET protooncogene. The frequencies of allele 1 of the polymorphic site in codon 836 were the same (0.96) in the Russian and German populations. This was also characteristic of two polymorphisms revealed earlier, namely, the sites in codons 691 (0.80 and 0.81, respectively) and 904 (0.21 and 0.22). However, the frequency of allele 1 of the polymorphisms in intron 14 differed significantly (0.87 and 0.77, respectively).


Assuntos
Carcinoma Medular/genética , Proteínas de Drosophila , Mutação Puntual , Polimorfismo Genético , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Códon , Éxons , Feminino , Ligação Genética , Marcadores Genéticos , Humanos , Masculino , Linhagem , Fenótipo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret
10.
Genetika ; 33(2): 257-61, 1997 Feb.
Artigo em Russo | MEDLINE | ID: mdl-9162703

RESUMO

Data on the screening of 266 non-delta F508 chromosomes (42 cystic fibrosis patients, 43 carriers, and 48 healthy donors from the Moscow region) for the presence of structural abnormalities within the tenth exon of the CFTR gene conducted by means of the single-stranded conformation polymorphism (SSCP) technique in nonisotope modification are presented. The method used made it possible to detect three SSCP variants, one of which was present in cystic fibrosis patients (23.8%) and carriers (9.3%), but not in healthy donors. Sequencing of the 5 amplified DNA fragments carrying this SSCP variant revealed an A-->G substitution in the 1525-61 position, which indicated the presence of TUB9 polymorphism with allele 1 in the homozygous state in all cases tested. The three SSCP variants described corresponded to the three allelic variants of TUB9 polymorphism as judged by MnlI restriction analysis of the amplified tenth exon sequence. The modified SSCP technique is also suitable for routine screening for the G542X, G55ID, and W1282X point mutations within the CFTR gene. The frequency distribution of polymorphic TUB9 marker alleles across the non-delta F508 chromosomes in the three studied groups were estimated. Homozygotes for the TUB9 allele 1 were shown to have identical GATT-TUB9-M470V haplotypes.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Testes Genéticos/métodos , Heterozigoto , Polimorfismo Genético , Estudos de Casos e Controles , Marcadores Genéticos , Humanos , Moscou , Polimorfismo Conformacional de Fita Simples , Valores de Referência , Mapeamento por Restrição
11.
Genetika ; 33(9): 1303-7, 1997 Sep.
Artigo em Russo | MEDLINE | ID: mdl-9445824

RESUMO

Allelic frequencies of two intron polymorphisms in the cystic fibrosis transmembrane regulator (CFTR) gene, TUB18 and TUB20, were estimated on chromosomes of 67 cystic fibrosis patients and on that of 37 healthy donors from Moscow and the Moscow oblast. Allele 2 of the TUB 18, and allele 1 of the TUB20 were 2.1 and 1.5 times more frequent on the non-delta F508 chromosomes of the cystic fibrosis patients than on chromosomes of healthy donors, i.e. these alleles were in linkage disequilibrium with the CFTR gene. Allele 1 of the TUB18 marker and allele 2 of the TUB20 marker demonstrated absolute linkage disequilibrium with the delta F508 mutation of the CFTR gene. The degree of association between the TUB18 and TUB20 intron polymorphisms and the GATT and T854T intragenic polymorphisms was analyzed. Of all 62 delta F508 chromosomes tested, 98.3% shared the 2-1-1-2 GATT- T854T-TUB18-TUB20 haplotype. Eight major (more frequent) GATT-T854T-TUB18-TUB20 haplotypes were found in 89.5% of normal, and in 97.9% of non-delta F508 chromosomes of cystic fibrosis patients from the Moscow region. Three of these major haplotypes, 2-1-1-2, 1-2-2-1, and 2-2-1-2, were respectively 2.5, 2, and 1.5 times more frequent on non-delta F508 cystic fibrosis chromosomes than on normal chromosomes. Data on screening for the G542X, N1303K, and 394delTT mutations of the CFTR gene, carried out on 134 chromosomes of cystic fibrosis patients from the Moscow region are presented. The frequencies of the G542X and 394delTT mutations were estimated as 1.5%, while the frequency of the N1303K mutation was 2.2%.


Assuntos
Fibrose Cística/genética , Marcadores Genéticos , Íntrons , Mutação Puntual , Polimorfismo Genético , Alelos , Estudos de Casos e Controles , Frequência do Gene , Haplótipos , Humanos , Moscou
12.
Genetika ; 31(4): 532-5, 1995 Apr.
Artigo em Russo | MEDLINE | ID: mdl-7607440

RESUMO

Allelic frequencies at three polymorphic markers in the CFTR gene were detected on chromosomes derived from cystic fibrosis (CF) patients and healthy donors from Moscow and the Moscow region. These polymorphic markers are tetranucleotide tandem repeats GATT in intron 6B, M470V in exon 10, and T854T in exon 14 (fragment A). Frequencies at allele 1 of the M470V marker, along with allele 2 of GATT and T854T, are two times higher for CF patients without delta F508 mutation than for healthy donors, and there is linkage disequilibrium of these alleles of the polymorphic markers analyzed with the CF gene. Allele 1 of M470V and T854T markers, as well as allele 2 of the GATT marker (six repeats), are absolutely linked to mutation F508 of the CFTR gene. Using the polymorphic markers studied, family analysis of CF was carried out in two families.


Assuntos
Fibrose Cística/genética , Polimorfismo Genético , Alelos , Estudos de Casos e Controles , Fibrose Cística/diagnóstico , Frequência do Gene , Marcadores Genéticos , Humanos , Desequilíbrio de Ligação , Moscou , Valor Preditivo dos Testes , Valores de Referência , Sequências Repetitivas de Ácido Nucleico
14.
Vaccine ; 9(3): 207-9, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1645900

RESUMO

Cell substrate DNA was shown to be an abundant contaminant in the clarified preparations of the Sabin type 1, 2 and 3 poliovaccines produced on a continuous cell line (4647). The size of the DNA, as evaluated for the Sabin type 1 poliovaccine, was highly heterogeneous, ranging from 100 to 20,000 base pairs. In view of potential oncogenicity of this DNA a simple and efficient procedure for its elimination is proposed. The method is based on use of protamine sulphate which at the concentration of 2.0 mg ml-1 precipitated cell DNA almost completely without affecting the virus titres.


Assuntos
DNA , Vacina Antipólio Oral/isolamento & purificação , Poliovirus/crescimento & desenvolvimento , Animais , Linhagem Celular , Protaminas
16.
Vopr Virusol ; 35(3): 219-21, 1990.
Artigo em Russo | MEDLINE | ID: mdl-2219855

RESUMO

According to the WHO requirements, the concentration of cellular DNA in vaccine preparations produced by pooling virus from continuous cell lines is limited to 100 ng/dose. In this study, different methods were used for purification of tick-borne encephalitis virus suspensions grown in continuous cultures of cell line 4647 from cellular DNA. Two approaches are proposed based on treatment with DNAse and promamin sulfate which allow one to reduce cellular DNA concentration in the virus preparation to the acceptable level. Prospects of their use in vaccine production are discussed.


Assuntos
DNA/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração/métodos , Centrifugação Isopícnica/métodos , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , DNA/análise , Desoxirribonuclease I/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Filtros Microporos , Protaminas/farmacologia , Vacinas de Produtos Inativados/análise , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Vacinas Virais/análise , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificação
18.
Genetika ; 20(3): 373-81, 1984 Mar.
Artigo em Russo | MEDLINE | ID: mdl-6370789

RESUMO

Properties of a temperature-sensitive in replication mutant pRP3.1ts12 derived from the broad host range RP1 plasmid have been studied. pRP3.1ts12 is a shortened variant of the temperature-sensitive RP1ts12 mutant carrying a deletion in a region from 2.3 to 7.6 MD. In contrast to RP1ts12, the plasmid pRP3.1ts12 is a leaky ts mutant and is characterized by an elevated frequency of reversions to the temperature-independent phenotype. Temperature-independent derivatives of pRP3.1ts12 were studied. Approx. 15% of these were found to induce mucoid growth of the host cells. As revealed from restriction endonuclease analysis, most of the latter derivatives contain deletions of small DNA segments in the region 0.56 to 2.3 MD of the RP1 map. The possible nature of the gene(s), whose deletions suppress the temperature-sensitive ts12 mutation and results in superproduction of Escherichia coli capsular poly-saccharide is discussed.


Assuntos
Deleção Cromossômica , Escherichia coli/genética , Genes Bacterianos , Mutação , Plasmídeos , Temperatura , Mapeamento Cromossômico , Replicação do DNA , DNA Bacteriano/genética , Fenótipo , Recombinação Genética
19.
Genetika ; 19(10): 1582-92, 1983 Oct.
Artigo em Russo | MEDLINE | ID: mdl-6317516

RESUMO

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.


Assuntos
Cromossomos Bacterianos/ultraestrutura , Escherichia coli/genética , Plasmídeos , Elementos de DNA Transponíveis , Escherichia coli/ultraestrutura , Marcadores Genéticos , Recombinação Genética , Temperatura
20.
Mol Biol (Mosk) ; 16(4): 837-56, 1982.
Artigo em Russo | MEDLINE | ID: mdl-6289085

RESUMO

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.


Assuntos
Elementos de DNA Transponíveis , Escherichia coli/genética , Plasmídeos , Proteínas de Bactérias/genética , Composição de Bases , Cromossomos Bacterianos , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Mutação , Recombinases Rec A , Temperatura
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