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.
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
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RA
Schnell
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PA
Lefebvre
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