MEI-9 FUNCTION IN DROSOPHILA MEIOTIC RECOMBINATION

在减数分裂过程中，基因重组对于同源染色体的准确分离是至关重要的。许多减数分裂重组途径现在都是在分子水平上了解的，这主要是由于对酿酒酵母的研究。然而，该途径中的最终事件仍然知之甚少，很少有蛋白质被确定为执行这些事件的候选蛋白。果蝇梅-9基因在这些后期阶段起作用。Mei-9蛋白是核苷酸切除修复(NER)核酸内切酶的一种成分，它可以切割经历单链到双链转变的DNA结构，并具有特定的极性。人类MEI-9的同源物是XPF，这是一个与遗传性疾病着色性干皮病有关的基因，与皮肤癌有关。Mei-9减数分裂功能的一个吸引人的模型是它切断Holliday连接来分解重组中间产物。这项提议中的实验试图测试这一模型。减数分裂重组事件的半四分体(两个染色单体)将被描述为来自野生型和mei-9突变体的分子，提供关于重组中间体的结构和它被分解的方式的信息。MEI-9蛋白将与抗MEI-9抗体或与表位标签一起免疫定位。美-9的15个突变等位基因将从分子和遗传学上进行表征，以了解蛋白质的结构与其功能之间的关系。Mus210基因编码果蝇的第二亚单位。该基因的突变会破坏NER，但不会破坏减数分裂重组。因此，这些要么是功能分离等位基因，要么是减数分裂中不需要这种蛋白的活性。如果后者为真，则这是两个亚单位之间功能分离的第一种情况。为了测试这一点，将恢复并鉴定新的Mus210突变。如果Mei-9不需要MUS210，可能是MUS210存在并二聚化，但其活性不是必需的，或者蛋白质存在但不与Mei-9二聚化，或者MUS210不存在于减数分裂细胞中。因此，将对蛋白质的表达进行分析。此外，该蛋白的表达将被强制，以确定它是否干扰Mei-9的功能。最后，将确定梅-9的潜在减数分裂特定合作伙伴。

Genetic recombination is essential for the accurate segregation of homologous chromosomes from one another in meiosis. Much of the meiotic recombination pathway is now understood at the molecular level, largely due to studies in S. cerevisiae. The final events in the pathway, however, remain very poorly understood, and few proteins have been identified as candidates for carrying out these events. The Drosophila mei-9 gene acts during these late stages. The MEI-9 protein is a component of a nucleotide excision repair (NER) endonuclease, which cuts DNA structures that undergo single-stranded to double-stranded transitions with a specific polarity. The human homolog of MEI-9 is XPF, a gene implicated in the hereditary disorder xeroderma pigmentosum, which is associate with skin cancer. An attractive model for the meiotic function of MEI-9 is that it cuts Holliday junctions to resolve recombination intermediates. Experiments in this proposal seek to test this model. Half-tetrads (two chromatids) of meiotic recombination events will be characterized molecular from both wild type and mei-9 mutants, providing information about the structure of the recombination intermediate and the manner in which it is resolved. MEI- 9 protein will be immunolocalized with anti-MEI-9 antibodies or with epitope tags. Fifteen mutant alleles of mei-9 will be characterized molecularly and genetically to learn about how the structure of the protein correlates with its function. The mus210 gene encodes the second subunit in Drosophila. Mutations in this gene disrupt NER, but not meiotic recombination. Hence, either these are separation-of-function alleles, or the activity of this protein is not required in meiosis. If the latter is true, this is the first case of separation of function between the two subunits. To test this, new mus210 mutations will be recovered and characterized. If MEI-9 does not require MUS210, it may be that MUS210 is present and dimerizes, but its activity is not required, or that the protein is present but does not dimerize with MEI-9, or that MUS210 is not present in meiotic cells. The expression of the protein will therefore be analyzed. In addition, expression of the protein will be forced, to determine whether it interferes with the MEI-9 function. Finally, potential meiosis-specific partners of MEI-9 will be identified.