Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library.
We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3 x 10(9) treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.
Purton
S
S
Department of Molecular Biology, University of Geneva, Switzerland.
Rochaix
J D
JD
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D003360:Cosmids / Q000235:genetics*; D004247:DNA / Q000032:analysis; D015723:Gene Library; D017343:Genes, Plant; D005816:Genetic Complementation Test; D009154:Mutation; D015183:Restriction Mapping
D004247:DNA; D001120:Arginine; D001123:Argininosuccinate Lyase