MEI-9 FUNCTION IN DROSOPHILA MEIOTIC RECOMBINATION

MEI-9 在果蝇减数分裂重组中的功能

• 批准号: 6736250
• 负责人: JEFF J. SEKELSKY
• 金额: $ 21.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2005-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736250
• 关键词: Drosophilidae; alleles; confocal scanning microscopy; endonuclease; enzyme activity; gene conversion; gene expression; genetic recombination; genetic regulation; immunofluorescence technique; meiosis; phenotype; polymerase chain reaction; protein structure function; radiation genetics; transposon /insertion element; western blottings; yeast two hybrid system

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.

遗传重组对于同源染色体在减数分裂中的精确分离是必不可少的。由于对S.啤酒。然而，该途径中的最终事件仍然知之甚少，并且很少有蛋白质被确定为进行这些事件的候选者。果蝇mei-9基因在这些晚期阶段起作用。MEI-9蛋白是核苷酸切除修复（NER）内切核酸酶的组分，其切割经历具有特定极性的单链到双链转变的DNA结构。MEI-9的人类同源物是XPF，这是一种与遗传性色素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有吸引力的模型是它切割Holliday连接以解析重组中间体。本提案中的实验旨在测试该模型。减数分裂重组事件的半四分体（两个染色单体）将从野生型和mei-9突变体中进行分子表征，提供有关重组中间体结构及其解析方式的信息。MEI- 9蛋白将用抗MEI-9抗体或表位标签免疫定位。mei-9的15个突变等位基因将在分子和遗传学上进行表征，以了解蛋白质的结构如何与其功能相关。mus 210基因编码果蝇的第二个亚基。该基因的突变破坏NER，但不破坏减数分裂重组。因此，要么这些是功能分离的等位基因，要么这种蛋白质的活性在减数分裂中是不需要的。如果后者是真的，这是两个子单元之间功能分离的第一种情况。为了测试这一点，将回收和表征新的mus 210突变。如果MEI-9不需要MUS 210，则可能MUS 210存在并二聚化，但不需要其活性，或者蛋白质存在但不与MEI-9二聚化，或者MUS 210不存在于减数分裂细胞中。因此将分析蛋白质的表达。此外，蛋白质的表达将被强制，以确定它是否干扰MEI-9功能。最后，MEI-9的潜在减数分裂特异性合作伙伴将被确定。