DISRUPTION OF THE NAD(-SPECIFIC GLUTAMATE-DEHYDROGENASE GENE OF NEUROSPORA-CRASSA BY MEANS OF THE RIP (REPEAT-INDUCED POINT MUTATIONS) PROCESS())

The structural gene for the catabolite-repressed, substrate-induced NA D(+)-specific glutamate dehydrogenase (gdh-1) of Neurospora crassa was disrupted using the process of repeat-induced point mutation (RIP). P lasmids containing incomplete copies of the gene, along with selectabl e markers, were introduced into germinated conidia by electroporation. The sexual progeny of a transformant containing an ectopically integr ated copy of a plasmid, harbouring the 5' flanking region and a part o f the coding sequence of gdh-l DNA, was examined for the occurrence of RIP by (i) Southern blot analysis of the genomic DNA digested with th e isoschizomers MboI and Sau3A, (ii) Northern blot analysis of total R NA in cultures subjected to repression and induction conditions for NA D-GDH, (iii) direct assessment of enzymatic activity, and (iv) evaluat ion of protein levels by Western blot analysis using a polyclonal anti -GDH Ige preparation. Attempts were made at delineating different regi ons of the gene exhibiting RIP by using P-32-labelled DNA probes, corr esponding to (i) the complete gene, (ii) a fragment containing the 5' flanking region plus two-thirds of the coding sequence, and (iii) the 5' flanking segment alone. The extent and relative location of RIP, as revealed by these hybridization probes, appeared to correlate with ch anges in specific activity under repression and derepression condition s. Mutant progeny, thus recovered, included isolates with altered regu latory features, such as constitutive expression, inability to elicit derepression, higher-than-wildtype GDH levels under derepression and i nefficient repression.