RESUMO
A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK- mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.
Assuntos
Escherichia coli/genética , Galactoquinase/genética , Genes Bacterianos , Genes , Regiões Promotoras Genéticas , Streptomyces/genética , Sequência de Bases , Escherichia coli/enzimologia , Galactoquinase/biossíntese , Mutação , PlasmídeosRESUMO
Clinical experience suggests that drugs that interact with and damage DNA are useful in cancer chemotherapy (H. Umezawa , p. 43-72, in V. T. DeVita , Jr., and H. Busch [ed.], Methods in Cancer Research; Cancer Drug Development, vol. XVI, 1979). Prescreening systems for antitumor agents in natural products require assays that are exquisitely sensitive, since the active components are often produced in quantities of micrograms per milliliter or less. One assay used to identify agents that interact with DNA is the biochemical induction assay, utilizing Escherichia coli BR 513 (R. K. Elespuru and R. J. White, Cancer Res. 43:2819-2830, 1983). In this paper we describe a genetic modification of strain BR 513 that displays an expanded spectrum of activity. This strain may provide an improved prescreen for detecting natural products that interact with DNA.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Escherichia coli/genética , Plasmídeos , Ampicilina/farmacologia , Bleomicina/farmacologia , DNA Bacteriano/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/farmacologia , Plasmídeos/efeitos dos fármacos , Transformação BacterianaRESUMO
We have identified lambda transducing bacteriophages carrying deoxyribonucleic acid repair or recombination genes of Escherichia coli K-12 by their ability to infect and express their bacterial genes in mutant cells in an agar overlay. This technique has been used to recognize transducing phages carrying uvrC+, ssb+, and other genes and to isolate phages carrying mutant alleles unable to complement ssb or uvrC cells. Several uvrC mutations were obtained which were suppressor sensitive.
