Generation of new mutants ofnmr, the negative-acting nitrogen regulatory gene ofNeurospora crassa, by repeat induced mutation

✍ Scribed by Gabor Jarai; George A. Marzluf

• Publisher: Springer-Verlag
• Year: 1991
• Tongue: English
• Weight: 641 KB
• Volume: 20
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00318516

✦ Synopsis

The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, many chlorate-sensitive progeny were obtained from crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.