Expression of a foreign gene in Chlamydomonas reinhardtii.
Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens. These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C. reinhardtii. Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin. Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate. The highly biased codon usage in Chlamydomonas does not preclude expression.
Department of Biochemistry, University of Alabama, Birmingham 35294.
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Hall
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Taylor
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Jones
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014170:Transformation, Genetic
D003062:Codon; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D010770:Phosphotransferases; D019868:Kanamycin Kinase