MEIOTIC CHROMOSOME SEGREGATION IN C ELEGANS

描述：维伦纽夫博士正在研究其潜在机制 线虫中正常的染色体配对、交叉和分离。 她已经鉴定出至少 10 个由突变定义的新位点 减数分裂染色体不分离。 这些突变被分离于 筛选产生异常高频率的雌雄同体 男性是由于性染色体不分离的结果 表示他有突变。 两个位点的突变仅影响分离 X 染色体，而其他则影响所有染色体的分离 染色体。 维伦纽夫博士提出，him 突变体中的不分离可以 是由于配对、重组、交叉维持或 分离过程。 可以评估交叉的存在 从细胞学角度来看，由于秀丽隐杆线虫卵母细胞通常在晚期停滞 减数分裂前期，附有六对同源物（二价体） 由交叉。 一些 hem 突变体表现出多达 12 个单价， 表明交叉有缺陷或缺失。 大多数突变体都导致无法形成 交叉。 分析的所有七个位点均显示出全球 重组频率。 me14 是这些基因之一 精母细胞分离缺陷。 另一种突变，me8，顺式作用 定义 X 染色体上配对所需的区域；删除 该轨迹导致沿整个交叉的显着减少 X 染色体的长度。 维伦纽夫博士提议阐明 he 基因的功能并 尤其将重点关注 he-14 和 me9。 第一个具体目标将 确定他是表现出配对缺陷的突变体。 生殖细胞 将在减数分裂前期到中期检查细胞核以检测 配对异常。 检测联会中的配对缺陷 将制备和分析复杂的电磁样本。 此外， 荧光原位杂交 (FISH) 将用于确定是否 同源染色体的标记区域正确配对。 另外，鱼 将用于确定配对进行前期的时间点 首先在正常生殖细胞核中观察到。 第二个具体目标是跟进初步调查结果 me9 突变体表现出交叉干扰减少。 STS标记 分布在 X 染色体上将用于评估重组 两个菌株之间的六个标记各不相同，以提供更多 对减少的交叉干扰的完整评估。 更多等位基因 将分离me9以协助me9基因的克隆。 博士。 维伦纽夫提议克隆 me9 和 Him-14 基因。 将采用定位克隆和转化拯救的方法 克隆并确认这些基因的身份。 一旦基因 克隆后，将针对细菌表达的融合物制备抗体 蛋白质和抗血清将用于免疫定位 14 和 me9 蛋白质。 此外，HA 标记的 Him-14 和 me9 基因将被 构建并转化为蠕虫以提供额外的方法 用于免疫定位。 标签的不同位置将用于 获得能够互补的标记基因。 的 这些蛋白质的定位将在各种他身上进行研究 突变体。 最终的具体目标是确定功能上的其他组件 与他互动-14。 Him-14 突变体产生的幼虫主要由 无法存活的非整倍体胚胎，只有少数整倍体幸存者。 将选择 EMS 诱导的 Him-14 抑制剂。 这些 将分析抑制突变体的减数分裂缺陷。

DESCRIPTION: Dr. Villeneuve is investigating the mechanisms underlying normal chromosome pairing, cross over, and segregation in C. elegans. She has identified at least 10 new loci defined by mutations causing meiotic chromosome non-disjunction. These mutations were isolated in screens for hermaphrodites that produced an abnormally high frequency of males as a result of non-disjunction of the sex chromosomes, and are denoted him mutations. Mutations in two loci only affect the segregation of the X chromosomes, whereas the others affect the segregation of all chromosomes. Dr. Villeneuve proposes that the non-disjunction in the him mutants could be due to a defect in pairing, recombination, chiasma maintenance, or the disjunctional process. The presence of chiasmata can be assessed cytologically, since C. elegans oocytes normally arrest at a late stage of meiotic prophase, with six pairs of homologues (bivalents) attached by chiasmata. Some of the him mutants exhibit up to 12 univalents, indicating defective or absent chiasmata. The majority of him mutants characterized result in a failure to form chiasmata. All seven him loci analyzed exhibit a reduction in global recombination frequencies. One of these genes, me14 is specifically defective in spermatocyte disjunction. Another mutation, me8, cis-acting defines a region on the X chromosome required for pairing; deletion of the locus results in a marked decrease in crossing over along the entire length of the X-chromosome. Dr. Villeneuve proposes to elucidate the function of the him genes and in particular will focus on him-14 and me9. The first specific aim will be to identify him mutants that exhibit pairing defects. Germ cell nuclei will be examined during early to mid meiotic prophase to detect anomalies in pairing. To detect pairing defects in the synaptonemal complex, EM specimens will be prepared and analyzed. In addition, fluorescence in situ hybridization (FISH) will be used to determine if tagged regions of homologous chromosomes are properly paired. Also, FISH will be used to determine the time point during prophase when pairing is first observed in normal germ cell nuclei. The second specific aim will be to follow-up on a preliminary finding that the me9 mutant exhibits reduced crossover interference. STS markers distributed over the X chromosome will be used to assess recombination between two strains differing for each of six markers to provide a more complete assessment of the reduced crossover interference. More alleles of me9 will be isolated to assist in the cloning of the me9 gene. Dr. Villeneuve proposes to clone the me9 and him-14 genes. The method of positional cloning and transformation rescue will be used to clone and confirm the identity of these genes. Once the genes are cloned, antibodies will be prepared against bacterially-expressed fusion proteins and the antisera will be used for immunolocalization of the him- 14 and me9 proteins. In addition HA-tagged him-14 and me9 genes will be constructed and transformed into worms to provide an additional method for immunolocalization. Different positions of the tag will be used to obtain tagged genes that are capable of complementation. The localization of these proteins will be studied in a variety of him mutants. The final specific aim is to identify other components that functionally interact with him-14. Him-14 mutants produce broods consisting mainly of inviable aneuploid embryos with only a few euploid survivors. Selection for EMS induced suppressors of him-14 will be performed. These suppressor mutants will be analyzed for defects in meiosis.