Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation.
Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.
Kovar
Joy L
JL
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
Zhang
Jun
J
Funke
Roel P
RP
Weeks
Donald P
DP
D000095:Acetolactate Synthase / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics; D005819:Genetic Markers; D006540:Herbicides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D030781:Organisms, Genetically Modified; D017386:Sequence Homology, Amino Acid; D013453:Sulfonylurea Compounds / Q000494:pharmacology; D014170:Transformation, Genetic / Q000187:drug effects
D018076:DNA, Complementary; D005819:Genetic Markers; D006540:Herbicides; D013453:Sulfonylurea Compounds; D000095:Acetolactate Synthase; C042031:sulfometuron methyl