title: Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) point mutation by recombination with a truncated nonfunctional ARG7 gene.
abstract: Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.
authors.affiliation: Lehrstuhl für Genetik, Universität Regensburg, Universitätsstr. 31, 93040 Regensburg, Germany. wolfgang.mages@biologie.uni-regensburg.de
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Mages
authors.forename: Oliver
authors.initials: O
authors.lastname: Heinrich
authors.forename: Gerda
authors.initials: G
authors.lastname: Treuner
authors.forename: Daniel
authors.initials: D
authors.lastname: Vlcek
authors.forename: Ivana
authors.initials: I
authors.lastname: Daubnerova
authors.forename: Miroslava
authors.initials: M
authors.lastname: Slaninova
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001123:Argininosuccinate Lyase / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D017354:Point Mutation; D015800:Protozoan Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013489:Suppression, Genetic; D014170:Transformation, Genetic
chemical_list: D015800:Protozoan Proteins; D001123:Argininosuccinate Lyase