Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (atpC).
Chlamydomonas reinhardtii strain atpC1 is a mutant defective in the nuclear gene that encodes the CF1 ATP synthase gamma-subunit polypeptide. Photoautotrophic growth was restored to atpC1 after it was transformed with wild-type DNA. Transformed strains were acetate-independent and arsenate-sensitive, similar in phenotype to the progenitor wild-type strain from which atpC1 was generated. Three transformed strains were examined in detail. Southern blot analyses demonstrated that the transformants were complements and not revertants. The transforming DNA integrated into the nuclear genome in a nonhomologous manner and at a low copy number. Northern blot analyses showed that the gamma-subunit mRNA in the complemented strains was expressed at the same relative level as that of wild-type. Western blots of total protein showed that whereas atpC1 was unable to synthesize any CF1 gamma-subunit, all three complemented strains could. Furthermore, the Western blot analyses demonstrated that the mutation in atpC1 had a pleiotropic effect on the accumulation of the CF1 beta-subunit which was relieved upon complementation. Cell extracts from atpC1 did not have any CF1-dependent catalytic activity, whereas extracts from all of the complemented strains and the wild-type strain had identical activities.
Smart
E J
EJ
Department of Cell Biology, Texas Southwestern Medical Center, Dallas 75235-9053.
Selman
B R
BR
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017343:Genes, Plant; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D006180:Proton-Translocating ATPases / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D014170:Transformation, Genetic
D016054:DNA, Protozoan; D010940:Plant Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D004247:DNA; D006180:Proton-Translocating ATPases