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

该项目旨在利用细胞黏菌盘基网柄菌分离和分析与受精有关的基因。为此，采用了一种有效的插入突变方法，称为REMI(限制酶介导的整合)。获得了如下三组有趣的突变体：1、KMCs 1、2、3：虽然mat A细胞通常只与mat A细胞交配，但这些突变体与mat A细胞交配。因此，交配型基因座似乎受到影响。2.TMC1，有性和无性发育均有缺陷的突变体：该突变体在细胞融合中是正常的，但由于不能趋化cAMP而在融合后发育中存在缺陷。对TMC1的分析表明，两种不同的发育模式之间的分子机制很大程度上是共同的(cAMP中继系统除外)。3.MCF1，一个完全不能进行有性繁殖的突变体：该突变体不能进行有性细胞融合，这表明膜融合的关键基因(S)被载体插入破坏。然而，不幸的是，在载体的插入部位附近有大约20kb的缺失，克隆相关基因需要基因组行走。作为替代，覆盖基因组相应区域的YAC重叠群可以作为克隆该基因的起始材料。除了上述插入突变之外，我们还对盘藻配子的mRNAs文库中克隆成员的结构和功能进行了分析。鉴定出了三个配子特有的新基因。

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.