RESUMO
The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.
Assuntos
Proteínas de Bactérias , Genes Bacterianos/genética , Glicosídeo Hidrolases/genética , Streptomyces/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Mutação/genética , Fases de Leitura Aberta/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Streptomyces/química , Streptomyces/enzimologiaRESUMO
Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Asel fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dral map was obtained by positioning the Dral sites relative to the Asel map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Asel and Dral fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The two terminal Asel fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.
Assuntos
DNA Bacteriano/genética , Sequências Repetitivas de Ácido Nucleico/genética , Mapeamento por Restrição , Streptomyces/genética , Cromossomos Bacterianos/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Biblioteca Gênica , Marcadores Genéticos/genética , Streptomyces/isolamento & purificaçãoRESUMO
The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.
Assuntos
DNA Bacteriano/análise , Genes Bacterianos , Plasmídeos , Streptomyces/genética , Southern Blotting , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/genética , Densitometria , Eletroforese , Homologia de Sequência do Ácido NucleicoRESUMO
DNA analysis of several genetically unstable strains of the fungus Ascobolus immersus revealed the presence of at least seven different plasmids. These plasmids ranged from 2 to 20 kb in size, and showed homology to one of them, pA1. In 18 stocks directly isolated from nature, two-thirds harboured plasmids ranging from 3 to 17 kb. Plasmids with homology to pA1 had similar molecular masses (about 8.5 kb). A possible mechanism of plasmid formation from chromosomal DNA is discussed.
