The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa (original) (raw)
Summary
The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). _Bal_31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.
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Abbreviations
CMCase:
carboxymethylcellulase
PAGE:
polyacrylamide gel electrophoresis
IPTG:
isopropyl-β-D-thiogalactoside
CAT:
chloramphenicol acetyl transferase
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Authors and Affiliations
- Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, NE1 7RU, Newcastle upon Tyne, UK
Judith Hall & Harry J. Gilbert
Authors
- Judith Hall
- Harry J. Gilbert
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Communicated by H. Hennecke
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Hall, J., Gilbert, H.J. The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa.Molec. Gen. Genet. 213, 112–117 (1988). https://doi.org/10.1007/BF00333406
- Received: 08 December 1987
- Issue date: July 1988
- DOI: https://doi.org/10.1007/BF00333406