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 NAD(+)-specific glutamate dehydrogenase (gdh-1) of Neurospora crassa was disrupted using the process of repeat-induced point mutation (RIP). Plasmids containing incomplete copies of the gene, along with selectable markers, were introduced into germinated conidia by electroporation. The sexual progeny of a transformant containing an ectopically integrated copy of a plasmid, harbouring the 5' flanking region and a part of the coding sequence of gdh-1 DNA, was examined for the occurrence of RIP by (i) Southern blot analysis of the genomic DNA digested with the isoschizomers MboI and Sau3A, (ii) Northern blot analysis of total RNA in cultures subjected to repression and induction conditions for NAD-GDH, (iii) direct assessment of enzymatic activity, and (iv) evaluation of protein levels by Western blot analysis using a polyclonal anti-GDH IgG preparation. Attempts were made at delineating different regions of the gene exhibiting RIP by using 32P-labelled DNA probes, corresponding 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 changes in specific activity under repression and derepression conditions. Mutant progeny, thus recovered, included isolates with altered regulatory features, such as constitutive expression, inability to elicit derepression, higher-than-wildtype GDH levels under derepression and inefficient repression.
Vijayaraghavan
Y
Y
Department of Biological Sciences, University of Calgary, AB, Canada.
Kapoor
M
M
D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003433:Crosses, Genetic; D018274:Electroporation; D004794:Enzyme Repression; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D005800:Genes, Fungal; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics*; D017354:Point Mutation; D014170:Transformation, Genetic / Q000235:genetics
D005969:Glutamate Dehydrogenase