Cloning gamete fusion genes in Chlamydomonas reinhardtii

Munevver Aksoy1,2 and Charlene L. Forest2

1The Graduate Center of CUNY, 365 5th Ave. New York, NY 10016 USA 2Dept. of Biology, Brooklyn College of CUNY, 2900 Bedford Ave. Brooklyn, NY 11210 USA

Our goal is to identify the genes involved in gamete fusion in the green alga Chlamydomonas reinhardtii. We have isolated several independently generated fusion-defective mating type minus insertional mutants which we are using to clone these genes. The plasmid pSP124S, containing the ble gene, was inserted as a random mutagen, using the acid-washed beads transformation technique. All of the fusion-defective mutants we have analyzed from 15 Ð 20 % pairs when mated with the opposite mating type (similar to the pair formation in the previously isolated temperature sensitive fusion-defective gam mutants). This indicates that these mutants are capable of undergoing plasma membrane adhesion between activated mating structures of the mutant and wild type gametes but are unable to undergo the subsequent fusion reaction. Using Tail-PCR to identify the flanking genomic DNA for each of the mutants, we have sequenced the flanking DNA for 2 mutants. We are now using BACs that contain the wild type genes found in these genomic regions to complement the mutants. Preliminary results suggest that the 2 genes we have identified are different from the gam gene, indicating that there may be several genes involved in controlling fusion. We are also attempting to clone additional mutants and have begun insertional mutagenesis using the plasmid pHyg3. In addition we have begun bioinformatics analysis of the proteins we believe are coded for by these genes.

e-mail address of presenting author: CLForest@brooklyn.cuny.edu

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