Complementing fusion-defective mutants with Chlamydomonas BACs modified using the Red/ET system
Munevver Aksoy, Sandra Ospina, Arvind Badhey, Shoshana Berkovic, Louana Cheung, Michael Fong, Arwa Gabr, Ozod Isametdinov, Ting-Yi Lai, Natasha Lamont, Bertholin Pierre, Rina Shrestha, and Charlene Forest
Department of Biology, Brooklyn College of CUNY, Brooklyn, NY 11230, USA
We have produced a collection of mutants defective in gamete fusion; UV irradiation-induced, temperature sensitive that can signal and adhere via their activated mating structures but cannot fuse at the restrictive temperature and non-conditional insertional mutants produced to clone the gene(s) causing this phenotype. When it was reported that the Chlamydomonas mutant fusM (described as having a similar phenotype) was defective in a homolog of the Arabidopsis Hap2 and lily GCS1 genes, we decided to determine if this gene was defective in our mutants. To do this, we obtained a plasmid from Dr. W. Snell, containing the gene as well as a BAC from the Chlamydomonas BAC library to complement our mutants. Because Chlamydomonas BACs do not contain a gene to allow direct selection of transformants in complementation analysis, we applied Red/ET recombination to modify the BACs by inserting a selectable marker gene that can be expressed in Chlamydomonas. Cells can then be transformed with only the modified BAC and transformants can be selected on antibiotic without the need to do co-transformations. The insert we created has two selectable markers; a kanamycin cassette for expression in E. coli and a hygromycin cassette for expression in Chlamydomonas. Our insert can be used to replace any of the genes on a BAC, producing modified BACs that can be used for complementation. We have used a BAC we have created with this construct to complement approximately half of our fusion-defective mutants and find the method to be much more effective than co-transformation. This method will decrease the time needed for use of BACs in functional genomics analysis in Chlamydomonas. Funding: The Eunice Kennedy Shriver NICHHD.
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