In vivo complementation analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardtii.
In vivo complementation between different wild and mutant strains defective for nitrate assimilation has been performed by isolating diploid strains from the appropriate crosses. Twenty-two diploids homozygous or heterozygous with respect to nitrate reduction and able to grow on nitrate medium were obtained and their diploid character demonstrated from analyses of mating type, cell volume, nuclear size and progeny of crosses with haploid wild-type. All diploids were assayed for overall- and terminal-nitrate reductase (NR) activity and for the occurrence of the NR-diaphorase subunit. Data on NR activities in heterozygotes carrying mutation(s) in structural gene(s) (nit-1 or nit-1a, nit-1b) agree with the heteromultimeric nature of the enzyme complex previously described (Franco et al. (1984) EMBO J 3: 1403-1407), and indicate that subunits are exchangeable to form hybrid enzymes. In addition, in vitro complementation tests with mutant nit-1 of C. reinhardtii indicate that this mutant has defective NR-diaphorase subunits but intact terminal subunits. Super-repression caused by the mutant allele nit-2 is suppressed by the wild allele in heterozygotes, which suggests a positive control by the nit-2 product on structural gene(s) transcription. Mutant alleles of genes for the biosynthesis of molybdenum-containing cofactor, either nit-4 or nit-5 and nit-6, were recessive in diploids carrying them. The mutant allele of nit-3, from strain 307, was codominant in all heterozygotes suggesting that nit-3 codes for a protein whose activity is limiting for the molybdenum-cofactor biosynthetic pathway.
Fernández
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Departamento de Bioquímica, Facultad de Ciencias, Universidad de Córdoba, Spain.
Matagne
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D020418:Algal Proteins / Q000235:genetics; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics*; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000172:deficiency / Q000235:genetics*; D009586:Nitrogen Fixation / Q000235:genetics
D020418:Algal Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase