Studies on genetic control of cell recognition and signal transduction mechanisms during fertilization

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This project intended to isolate and analyze genes involved in fertilization using the cellular slime mold, Dictyostelium discoideum. For this purpose, an effective method for insertional mutagenesis called REMI (Restriction Enzyme Mediated Integration) was employed. Three groups of interesting mutants as below were obtained.1.KMCs 1,2,3 with altered partner choice : Although mat A cells normally mate only with mat a cells, these mutants do with mat A cells. Therefore, mating type loci seem to be affected.2.TMC1, a mutant defective in both sexual and asexual development : This mutant is normal in cell fusion but is defective in post-fusion development due to failure in chemotaxis to cAMP.Analysis of TMC1 revealed that molecular mechanisms are largely common (except for the cAMP relay system) between two alternative developmental modes.3.MCF1, a mutant completely impotent for sexual reproduction : This mutant is unable to undergo sexual cell fusion, suggesting that a critical gene (s) for membrane fusion is destroyed by vector insertion. Unfortunately, however, there is a deletion of about 20 Kb in size adjacent to the insertion site of the vector, and genomic walking is necessary to clone the relevant gene. As an alternative, YAC contigs covering the corresponding region of the genome can be used as starting materials for cloning the gene.In addition to the insertional mutagenesis described above, structural and functional analyzes of clone members in a cDNA library made from mRNAs prepared from D.discoideum gametes were performed. Three novel genes specific to gametes were identified.