5 NITRATE ASSIMILATION-RELATED LOCI ARE CLUSTERED IN CHLAMYDOMONAS-REINHARDTII

Three overlapping clones covering a Chlamydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1. These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium. In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked. They have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1. The nar-3 and nar-4 loci are transcribed divergently from nit-1. DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences. Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized. These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster. In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate. Two of the mutants in which expression of all of the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities. Thus the nar loci cannot be required for nitrite assimilation. Mutants F6 and G1 were unable to grow in either nitrite- or nitrate-containing medium, and lacked nitrate reductase, nitrite reductase, nitrate uptake and nitrite uptake activities. The inability to assimilate nitrite co-segregated with nit-1 in crosses between these mutants and wild type. These results indicate that a complex gene cluster responsible for the assimilation of nitrate has been identified in C. reinhardtii, and that, in addition, at least one locus necessary for nitrite assimilation is genetically linked to this cluster.