Open AccessMolecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.
Author(s) -
John V. Paietta,
Robert A. Akins,
Alan M. Lambowitz,
George A. Marzluf
Publication year - 1987
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.7.7.2506
Subject(s) - biology , neurospora crassa , gene , mutant , microbiology and biotechnology , genetics , molecular cloning , regulator gene , biochemistry , restriction map , cloning (programming) , permease , clone (java method) , crassa , restriction enzyme , gene expression , plasmid , computer science , programming language
The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.