Studies on structure and function of molecules involved in gamete interactions

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This project intended to reveal the genetic control of gamete interactions using the macrocyst formation of the cellular slime mold, Dtctyostelium discoideum as a model system. For this purpose, immunochemical, forward genetic, and reverse genetic approaches have been taken.1. Results of immunochemical approach : GP138 multigene family, which had been shown to encode gp138, a target molecule for gamete-fusion blocking antibodies, was analyzed. Two new members, GP138C and GP138D, have been isolated as well as two truncated pseudogenes Analysis of total tetra knockout mutants, however, indicated that proteins encoded by GP138 multigenes are distinct from gP138. The possibility that GP138 multigenes are involved in asexual development has been suggested.2. Results of forward genetics : The effective method of insertional mutagenesis called REMI (Restriction Enzyme Mediated In tegration) was used to isolate sexually defective mutants and relevant genes. One of the obtained mutants, MCF1 is unable to undergo sexual cell fusion. Analysis of genome structure around insertion sites revealed an ORF of 6.3 Kb long including two short introns. The relevant gene was named macA. The actual function of macA in cell fusion is being analyzed.3. Results of reverse genetics : Random analysis of genes expressed in the gametes was attempted. First, oligo-(dT) primed directional cDNA library (FC) was constructed and randomly chosen 1,000 clones were sequenced to obtain gene expression profile in the gamete. Since the genes contained in the FC library were predominantly of housekeeping functions, a gamete-specific subtraction library (FC-IC) was constructed. All 901 clones in the FC-IC library were sequenced and characterized. Expression analysis of randomly chosen 100 clones indicated that more than 60 % of the clones are actually gamete specific and FC-IC library is an useful source for analysis of genes involved in gamete interactions.