RESUMO
We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.
Assuntos
Alquil e Aril Transferases/genética , Ácido Mevalônico/metabolismo , Proteínas de Plantas/genética , Streptomyces/enzimologia , Streptomyces/genética , Antibacterianos/biossíntese , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Bacteriano/genética , Desoxirribonuclease BamHI/biossíntese , Desoxirribonuclease BamHI/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Família Multigênica/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The bamHIC gene, controlling the BamHI restriction-modification (R-M) system can functionally be replaced by providing pvuIIC or smaIC in trans. C.BamHI, the protein product encoded by bamHIC, has been purified and shown to bind a 345-bp DNA fragment within the BamHI R-M system.
Assuntos
Desoxirribonuclease BamHI/biossíntese , Desoxirribonuclease BamHI/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Bacillus/enzimologia , Bacillus/genética , Desoxirribonuclease BamHI/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Genes BacterianosRESUMO
A fusion protein produced by a cDNA for rat choline acetyltransferase (ChAT) inserted into a translation vector was used for immunization of rabbits. An antiserum was obtained that could recognize a single protein band in immunoblot analysis of a partially purified enzyme preparation of the rat brain. The antiserum revealed ChAT immunoreactivity in the motoneurons and terminal-like structures in the neuropil of the ventral horn in cryostat sections of the cervical spinal cord of the rat. This antiserum may be of particular use to study the development of the cholinergic neuron.
Assuntos
Colina O-Acetiltransferase/biossíntese , DNA/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Animais , Colina O-Acetiltransferase/genética , Desoxirribonuclease BamHI/biossíntese , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Masculino , Coelhos , Ratos , Ratos Endogâmicos , Proteínas Recombinantes de Fusão/genética , Medula Espinal/imunologia , Medula Espinal/metabolismo , Sistema Nervoso Simpático/imunologia , Sistema Nervoso Simpático/metabolismoRESUMO
The type II restriction endonuclease BamHI has been expressed in E. coli, producing 100-fold more enzyme than the wild type Bacillus amyloliquefaciens H strain. This high yield has facilitated purification to homogeneity of large amounts of the enzyme, along with its crystallization in a form which diffracts to at least 1.9 A in X-ray analysis.
