Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging.
Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii 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, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas.
Schnell
R A
RA
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Lefebvre
P A
PA
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D005786:Gene Expression Regulation / Q000187:drug effects; D005809:Genes, Regulator; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016324:Sequence Tagged Sites
D004251:DNA Transposable Elements; D009566:Nitrates; D012333:RNA, Messenger; D000641:Ammonia