ISOLATION OF THE CHLAMYDOMONAS REGULATORY GENE NIT2 BY TRANSPOSON TAGGING

Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhard tii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, tran sposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was fo und to be genetically and phenotypically associated with the nit2 muta tion. Using the Gulliver element as a probe, one of the transposon-ind uced nit2 alleles was isolated, and a sequence adjoining the transposo n was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 muta tions and mutant rescue by genetic transformation. The NIT2 gene encod es a 6-kb transcript that was not detected in cells grown in the prese nce of ammonium. Likewise, NIT2-dependent genes are repressed in ammon ium-grown cells. These results suggest that repression of the NIT2 gen e may mediate metabolite repression of the nitrate assimilation pathwa y in Chlamydomonas.