Cloning a gamete fusion gene using insertional fusion-defective mutants

Munevver Aksoy and Charlene L. Forest

Department of Biology, Brooklyn College, Brooklyn, NY

Our goal is to isolate and analyze the gene responsible for gamete fusion in Chlamydomonas reinhardtii, a unicellular, eukaryotic green algae. To this end, we have generated fusion-defective insertional mutants and are using these mutants to clone the gene. The plasmid pSP124S, containing the ble gene, was inserted as a random mutagen using the acid-washed beads transformation technique. Mutants were selected using a streptomycin selection procedure. Approximately 700 non-mating colonies were isolated and analyzed by phase-contrast microscopy to determine their mating deficiencies. Among the mutants isolated were non-agglutinating mutants, non-motile mutants with low fusion capacity and one motile, fusion-defective mutant, clone 9-5 (cl 5). cl 5 forms 15 % pairs when mated with the opposite mating type (similar to the pair formation in gam mutants). Southern Blot analysis showed 1 insertion for clone-45 (a previously isolated fusion-defective mutant), and 2 insertions for cl 5. While we continue to screen for new mutants, we are now using LMS-PCR to identify the genomic sequences flanking the insertion in the genome of clone-45. After PCR, we will sequence this flanking DNA and compare it to the gamete/zygote EST library available from the Chlamydomonas Genome Center as well as to the full genomic sequence that is now available as a blast database. We then will isolate the wild type gene by screening a Chlamydomonas BAC library.