RESUMO
We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.
Assuntos
Anemia Falciforme/genética , DNA/genética , RNA/genética , Alelos , Anemia Falciforme/diagnóstico , Mapeamento Cromossômico , Eletroforese em Gel de Ágar , Globinas/genética , Humanos , Mutação , Hibridização de Ácido Nucleico , Polimorfismo Genético , Ribonuclease T1RESUMO
Transcription of tRNA genes carried by transducing bacteriophages phi80psu3+ (tRNA1Tyr) and lambdah80T (tRNA2Tyr, tRNA2Glysu36+, tRNA3Thr) was studied in vitro in a system consisting of whole bacteriophage DNA and purified RNA polymerase. In contrast to unusual requirements for tRNA1Tyr gene transcription from DNA fragments, the transcription on whole bacteriophage DNA was found to be relatively not salt sensitive, did not require glycerol and rifampicin-resistant complexes with RNA polymerase were formed in the absence of nucleoside triphosphates. Termination factor rho stimulated the transcription of the tRNA genes as well as that of 4S RNA on lambdah80T DNA template. The stimulatory effect of rho was abolished by rifampicin and seems to be due to the release of RNA polymerase and reinitiation of transcription.
Assuntos
Escherichia coli/genética , RNA de Transferência/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Reguladores , Glicerol/farmacologia , Cloreto de Potássio/farmacologia , Fator Rho/metabolismo , Rifampina/farmacologia , Moldes Genéticos , Transcrição Gênica/efeitos dos fármacosRESUMO
Two Escherichia coli tRNA gene clusters, tRNA1Tyr (su3+ and su3-) and tRNA2Tyr, tRNA2Gly (su+36), tRNA3Thr, were transcribed in a purified in vitro system. Evidence indicates that the adjacent tRNA genes are transcribed together as a common precursor of large size, which, on incubation with crude cell extracts, yields mature tRNA molecules.
Assuntos
Precursores de Ácido Nucleico/biossíntese , RNA de Transferência/biossíntese , Transcrição Gênica , Sequência de Bases , Sistema Livre de Células , Escherichia coli/metabolismo , Peso Molecular , Óperon , RNA de Transferência/análiseRESUMO
A gene specifying tyrosine transfer RNA has been purified and transcribed in vitro. The purification procedure made use of two specialized transducing phages carrying the tRNA(Tyr) gene of Escherichia coli inserted into their DNA in opposite orientations. The separated heavy strands of the two phages were annealed and the single-stranded tails of the resulting hybrid were removed by digestion with Neurospora endonuclease. The size of the purified double-stranded structures was determined by electron microscopy. These isolated duplexes served as template for the in vitro transcription of tRNA(Tyr)-like molecules.