A note on the primary structure and expression of an Erwinia carotovora polygalacturonase-encoding gene (peh1) in Escherichia coli and Saccharomyces cerevisiae.
A 1209-base pair (bp) DNA fragment containing the endopolygalacturonase-encoding gene (peh1) from Erwinia carotovora subsp. carotovora was amplified by the polymerase chain reaction (PCR) technique and expressed in Escherichia coli. The nucleotide sequence of the PCR product was determined and found to be highly homologous to the primary structures of other polygalacturonase-encoding genes. The peh1 DNA fragment encoding the mature polygalacturonase was inserted between two different yeast expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS10 and pAMS11. These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae. Transcription initiation signals present in these expression-secretion cassettes were derived from the yeast alcohol dehydrogenase (ADC1P) or mating pheromone alpha-factor (MF alpha 1P) gene promoters. The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of polygalacturonase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). Northern blot analysis revealed the presence of peh1 mRNA in the yeast transformants and a polypectate agarose test was used to monitor polygalacturonase production.
Laing
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Department of Microbiology, University of Stellenbosch, South Africa.
Pretorius
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016973:Pectobacterium carotovorum / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D011096:Polygalacturonase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae; D013728:Terminator Regions, Genetic
D004269:DNA, Bacterial; D011096:Polygalacturonase