Cloning and preliminary characterization of a molybdenum cofactor gene ofNeurospora crassa

✍ Scribed by Nigel Stuart Dunn-Coleman

• Publisher: Springer-Verlag
• Year: 1984
• Tongue: English
• Weight: 668 KB
• Volume: 8
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00395704

✦ Synopsis

A Neurospora crassa library, constructed in a derivative of the plasmid pBR322 (pRK9), was used to transform two E. coli chlD molybdenum cofactor mutants (chlD, chlD: :Mu). Subsequently, one transformant from each of three independent transformation experiments was restriction mapped. All three transformants had an identical N. crassa DNA insert (4.2 kb). Southern Blot analysis with one of the plasmids (pMoCo, 1:4) showed hybridization to a single band ofN. crassa genomic DNA. When pMoCo plasmid (1:4) was used to transform various E. coli nitrate reductase mutants (chlA, chlB, chlC, chlD, chlE, chlG and chlM), the pMoCo plasmid was capable of restoring E. coli nitrate reductase activity to only the chlD mutant. In vitro reconstitution experiments using wild-type, chlD and chlD; pMoCo cell-free extracts as a source of molybdenum cofactor (MoCo) were performed with the N. erassa MoCo mutants nit-l, nit-7 and nit-8. MoCo from wild-type E. coli cell-free extracts was capable of reconstituting NADPH : nitrate reductase activity to all three N. crassa mutants. MoCo from chlD; pMoCo cell-free extracts was capable of reconstituting more NADPH : nitrate reductase activity to the N. crassa mutants than cell-free extracts from the original chlD mutant.