BACKGROUND Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors. RESULT We constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 105 transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype. CONCLUSION So far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis.

中文翻译：

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菌株的构建，以鉴定调节着丝粒凝聚保护（CCP）和动粒单向姐妹单向（SKM）的新因素。

背景技术减数分裂-I是染色体分离的独特类型，其中每个染色体均与其同源物对齐并分离。不同真核生物中减数分裂I同源物分离的机制取决于它们的着丝粒和线粒体结构，而后者又主要依赖于两个过程，首先依赖于一种被称为monopolin的特殊的四种蛋白质复合物，其次是着丝粒的内聚保护（CCP）。然而，在哺乳动物中尚未鉴定出复合物。此外，在发芽酵母中，该过程中可能还有其他因素，包括某些减数分裂特异性因子和一些非减数分裂特异性因子。结果我们构建了两个菌株。在第一个菌株中，我们表达了Mam1和Cdc5，这导致了姊妹动粒单向（SKM）；在第二个菌株中，我们表达了即使在有丝分裂中也能增强CCP的Rec8和Spo13。这些蛋白质在有丝分裂分裂的细胞中的表达引起染色体的共取向，从而导致细胞死亡，继而导致染色体的错误分离。然后，我们利用这些菌株从酵母和哺乳动物中筛选cDNA文库，以鉴定参与CCP和SKM的新因子。最后，用pRS316 gal cDNA文库转化表达Spo13和Rec8的SGY4119菌株，并筛选转化体对半乳糖的致死性。我们筛选了〜105个转化子菌落。在这些〜3000个菌落中，能够在半乳糖平板上存活，该平板根据所需的表型缩小到6个。结论到目前为止，仅在两种酵母中鉴定出了减数分裂特异的线粒体蛋白。最近，在哺乳动物中，已经鉴定出减数分裂特异的动粒蛋白（MEIKIN）具有相似的功能。到现在为止，已经确定哺乳动物中的一种蛋白质和发芽酵母中的四种蛋白质monopolin复合物可以使着丝粒协调一致。还必须确定更多新颖的因素。这就是为什么我们希望使用功能基因组学方法来进行遗传筛选的原因。由于在酵母中已经鉴定出的蛋白质列表并非如间接证据所显示的那样详尽，因此我们希望使用相同的酵母菌株来鉴定可能参与减数分裂执行的其他新型酵母蛋白质。这就是为什么我们希望使用功能基因组学方法来进行遗传筛选的原因。由于在酵母中已经鉴定出的蛋白质列表并不像环境证据所显示的那样详尽，因此我们希望使用相同的酵母菌株来鉴定可能参与减数分裂执行的其他新型酵母蛋白质。这就是为什么我们希望使用功能基因组学方法来进行遗传筛选的原因。由于在酵母中已经鉴定出的蛋白质列表并不像环境证据所显示的那样详尽，因此我们希望使用相同的酵母菌株来鉴定可能参与减数分裂执行的其他新型酵母蛋白质。