Linked Life Data

2182390

Source:http://linkedlifedata.com/resource/pubmed/id/2182390

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1990-5-16
pubmed:databankReference
pubmed:abstractText
We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
86
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
153-62
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori.
pubmed:affiliation
Genencor, Inc., South San Francisco, CA 94080.
pubmed:publicationType
Journal Article