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Clonagem de genes do operao da L-arabinose de Bacillus subtilis

By Isabel Maria Godinho de Sa Nogueira

Abstract

The aim os this work was to study the genes involved in L-arabinose utilization of Bacillus subtilis. In a previous work (Paveia, 1986) the structural genes for L-arabinose utilization araA, araB and araD - coding for L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase, respectively and mutations in a presumptive regulatory gene araC were mapped. A first approach in this study, the isolation of constitutive mutants for L-arabinose utilization (Ara"c) and mapping of ara"c mutations, showed that they are located in the locus araC between the cys B and his A markers on the B.subtilis chromosome. The characterization of Ara"C mutants by L-arabinose isomerase activity revealed that this activity was reduced in the presence of L-arabinose and repressed by glucose. We have subsequently screened a B.subtilis gene bank for the presence of ara genes. Two plasmids that were able to complement mutations in the structural genes were isolated. Complementation experiments carried out with defined restriction fragments from the cloned DNAs have established their relative order as araA-araB-araD. Such an arrangement contrast with the one found in the Escherichia coli and Salmonella thyphimurium ara operons: araB-A-D. The complete nucleotide sequence of the structural genes was determined showing that the three are adjacent and organized in an operon transcribed from a #sigma# A - like promotors upstream from araA gene. DNA sequencing analysis and Northern hybridization results suggested the presence of two additional open reading frames (ORF4 and ORF5) belonging to the same operon. The products of the structural genes exhibited similarity in aminoacid composition and protein sizes to the enzymes L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase of E.coli and S.thyphimurium. Primer extension analysis and transcriptional fusions showed that the synthesis of ara mRNA is induced by L-arabinose and repressed by glucose. This operon thus appears to be regulated mainly at the transcriptional levelAvailable from Fundacao para a Ciencia e a Tecnologia, Servico de Informacao e Documentacao, Av. D. Carlos I, 126, 1249-074 Lisboa, Portugal / FCT - Fundação para o Ciência e a TecnologiaSIGLEPTPortuga

Topics: 06B - Bioengineering, biomedical engineering, biotechnology, biochemical engineering, 06Y - Biophysics
Year: 1991
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Provided by: OpenGrey Repository
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