Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain.
In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.
Blankenship
J E
JE
Plant Science Center, Cornell University, Ithaca, New York 14853.
Kindle
K L
KL
D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D005799:Genes, Dominant; D005819:Genetic Markers; D005966:Glucuronidase / Q000235:genetics; D008027:Light; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014170:Transformation, Genetic
D005819:Genetic Markers; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D000641:Ammonia; D004247:DNA; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D005966:Glucuronidase