RESUMO
We have constructed plasmid pDN1050, a new small cloning vector for Bacillus subtilis. pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.
Assuntos
Bacillus subtilis/genética , Clonagem Molecular/métodos , Vetores Genéticos , Plasmídeos , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Dados de Sequência Molecular , Plasmídeos/metabolismo , Recombinação Genética , Mapeamento por RestriçãoRESUMO
The Brassica napus genome has been investigated by DNA fingerprinting with six synthetic oligonucleotide probes complementary to simple repetitive sequences, namely (GATA)4, (GACA)4, (GGAT)4, (CA)8, (CT)8 and (GTG)5. While all sequence motifs were found to be present in the B. napus genome, their organization and abundance varied considerably. Among the investigated probes, (GATA)4 revealed the highest level of intraspecific polymorphism and distinguishes not only between cultivars but even between different individuals belonging to the same cultivar. In contrast, (GTG)5, (GACA)4 and (GGAT)4 produced relatively homogeneous fingerprint patterns throughout different cultivars, while hybridization to (CT)8 and (CA)8 resulted in only a few weak bands superimposed on a smear. The isoschizomeric pair Hpa II and Msp I revealed the presence of methylated cytosines in the vicinity of (GATA)m repeats. The applicability of simple repetitive sequence polymorphisms as molecular markers for Brassica species is discussed.
RESUMO
We have exploited the transient expression of foreign genes introduced into plant protoplasts to investigate the effect of the pea seedborne mosaic potyvirus (PSbMV) 5' untranslated region (5'UTR) on the level of gene expression in pea and tobacco protoplasts. The plant viral 5'UTRs were found to increase translation significantly in comparison to a plasmid containing no 5'UTR of viral origin. The enhancement effect of the 5'UTRs of PSbMV and tobacco etch potyvirus (TEV) was found to be similar in pea and tobacco protoplasts, indicating a host-independent role of the potyviral 5'UTRs in enhancing gene expression. Translational enhancement of the two potyviral 5'UTRs was similar to that of the 5'UTR of tobacco mosaic virus (TMV). This observation makes it attractive to use potyviral 5'UTRs as general translational enhancers in future genetic transformations of plants.
Assuntos
DNA Viral/fisiologia , Elementos Facilitadores Genéticos , Fabaceae/microbiologia , Íntrons , Vírus do Mosaico/genética , Nicotiana/microbiologia , Plantas Medicinais , Plantas Tóxicas , Biossíntese de Proteínas , Sequência de Bases , Clonagem Molecular , Fabaceae/genética , Glucuronidase/genética , Dados de Sequência Molecular , Vírus do Mosaico/fisiologia , Protoplastos , RNA Viral/genética , Nicotiana/genéticaRESUMO
In the industrial process of liquefying starch to make glucose or high fructose syrups, it is crucial that the amylase used is stable and active at about 105 degrees C at pH 6.5 or preferentially at a lower pH. The amylase from Bacillus licheniformis is well suited for this purpose but it is possible that other amylases might perform even better. Therefore, we cloned and characterized amyS encoding a heat-stable alpha-amylase from Bacillus stearothermophilus. Using a newly developed method for creating exact gene fusions by in vivo recombination, we attempted to increase expression of amyS in Bacillus subtilis. However, only by introducing the amyS gene into B. licheniformis, we obtained significantly better yields.
Assuntos
Geobacillus stearothermophilus/genética , alfa-Amilases/genética , Bacillus/enzimologia , Bacillus/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Engenharia Genética/métodos , Geobacillus stearothermophilus/enzimologia , Técnicas In Vitro , Plasmídeos/genética , Recombinação Genética/genéticaRESUMO
We have cloned and sequenced a gene for a heat-stable alpha-amylase from a natural isolate of Bacillus stearothermophilus. Previously, it had been shown that B. stearothermophilus amylase genes may be harboured on indigenous plasmids. We have found that our isolate harbours the amylase gene only on the chromosome and not on its indigenous plasmid.
Assuntos
Cromossomos Bacterianos , Genes Bacterianos , Geobacillus stearothermophilus/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Bacillus subtilis/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Geobacillus stearothermophilus/enzimologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Mapeamento por RestriçãoRESUMO
This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.
