FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii

• Published in: Genetics (Austin) 1997-05, Vol.146 (1), p.121-133
• Main Authors: Zhang, D. (Washington State University, St. Louis, MO.), Lefebvre, P.A
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN RECOMBINADO; ADN RECOMBINE; AMMONIA; AMMONIAC; AMONIACO; Animals; ANION; ANIONES; ANIONS; CARTE GENETIQUE; Cell Cycle Proteins; CELL STRUCTURE; CHLAMYDOMONAS REINHARDTII; Chlamydomonas reinhardtii - enzymology; Chlamydomonas reinhardtii - genetics; CHLORATE; CHROMOSOME MAPS; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor Proteins; ENZYMIC ACTIVITY; ESTRUCTURA CELULAR; EXPRESION GENICA; EXPRESSION DES GENES; FAR 1 GENE; FAR 1 LOCUS; FLAGELLA; Freshwater; Fungal Proteins - genetics; Fungi; GENE; GENE EXPRESSION; GENES; Genes, Reporter; Genetic Complementation Test; GENETIC MAPPING; GENETIC MAPS; GENETIC REGULATION; GENETICA; GENETICS; GENETIQUE; INDUCED MUTATION; INSERTIONAL MUTAGENESIS; Investigations; LOCI; LOCUS; MAPAS GENETICOS; MUTACION; MUTACION INDUCIDA; Mutagenesis, Insertional; MUTANT; MUTANTES; MUTANTS; MUTATION; MUTATION PROVOQUEE; NIT1 PROMOTER; NITRATE REDUCTASE; Nitrate Reductases - antagonists & inhibitors; Nitrate Reductases - genetics; NITRATO REDUCTASA; PF14 GENE; Promoter Regions, Genetic; PROMOTERS; PROTEINAS; PROTEINE; PROTEINS; RECOMBINACION; RECOMBINAISON; RECOMBINANT DNA; RECOMBINATION; Recombination, Genetic; REGULATORY GENES; REPORTER GENES; Repressor Proteins; RESTRICTION MAPPING; Saccharomyces cerevisiae Proteins; SPONTAEEOUS MUTANTS; SPONTANEOUS MUTATIONS; STRUCTURE CELLULAIRE
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/146.1.121

In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression. (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NIT1;PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.