MEIOTIC CHROMOSOME SEGREGATION IN C ELEGANS

描述：维伦纽夫博士正在研究 线虫中正常的染色体配对、交叉和分离。 她已经确定了至少10个由突变定义的新基因座 减数分裂染色体不分离。这些突变是在 产生异常高频率的雌雄同体的筛查 雄性是性染色体不分离的结果，并且是 表明他发生了突变。两个基因座的突变只影响分离 X染色体的分离，而其他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能 是由于配对、重组、交叉维持方面的缺陷，或 分离过程。交叉症状的存在可以被评估 细胞学上，由于线虫卵母细胞通常在后期停滞 减数分裂前期，附六对同系物(二价体) 由Chiasmata提供。一些HIM突变体表现出多达12个单价体， 表示有缺陷的或缺少交叉的。 他所描述的大多数突变体都会导致不能形成 十字交叉。所有分析的7个HIM基因座都显示出全球 复合频率。其中一个基因，ME14，是特定的 精母细胞分离有缺陷。另一个突变，me8，顺式作用 定义配对所需的X染色体上的区域；删除 该轨迹导致沿整个 X染色体的长度。 维伦纽夫博士建议阐明HIM基因和 尤其将重点放在他-14岁和我9岁。第一个具体目标是 就是找出有配对缺陷的变种。生殖细胞 核将在减数分裂早期到中期进行检查，以检测 配对中的反常现象。检测联会膜上的配对缺陷 复杂的EM样品将被制备和分析。此外, 将使用荧光原位杂交(FISH)来确定 同源染色体的标记区域是正确配对的。还有，鱼 将用于确定配对的前期时间点 最先在正常生殖细胞核中观察到。 第二个具体目标是对初步调查结果采取后续行动 ME9突变体表现出减少的交叉干扰。STS标记 分布在X染色体上的基因将被用于评估重组 在六个标记中的每一个都不同的两个菌株之间提供更多 完成对减少的交叉干扰的评估。更多等位基因 将分离出一株ME9基因，以协助ME9基因的克隆。Dr。 维伦纽夫提议克隆ME9和HIM-14基因。 将采用位置克隆和转化抢救的方法 克隆并确认这些基因的身份。一旦这些基因 克隆，将制备针对细菌表达的融合的抗体 蛋白质和抗血清将用于免疫定位His- 14和ME9蛋白。此外，HA标记的HIM-14和ME9基因将 构造并转换为蠕虫，以提供另一种方法 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。这个 这些蛋白质的定位将在各种HIS中进行研究 变种人。 最终的具体目标是确定其他功能组件 与他互动--14.HIM-14突变株产生的仔猪主要由 不能存活的非整倍体胚胎只有几个整倍体幸存者。 将进行对EMS诱导的HIM-14抑制物的选择。这些 将分析抑制子突变体是否存在减数分裂缺陷。

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.