Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene

Summary In the further analysis of a cross in which the mis-sense allele, am³, of the Neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the RIP process whereas the am³ allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. The am³ allele had also acquired a new HindIII restriction site. It had no detectable methylation. The mutations responsible respectively for the new restriction site and the modified enzyme properties were separated from each other, and from the original am³ mutation, by selecting for intragenic recombination on either side of the am³ site. In this way two new effectively wild-type alleles were generated, one characterised by its heat-sensitive and kinetically modified enzyme product and the other by a new HindIII site. These results demonstrate that the RIP phenomenon can be a source of new functional alleles.