Abstract
BackgroundMeiosis-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.ResultWe 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.ConclusionSo 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.
Highlights
Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog
The homologous chromosome separation mainly depends on the mono orientation of the sister kinetochore because of which the paired homolog separates to the opposite pole
Sister kinetochore co-orientation in meiotic division (MI) depends mainly on two processes, first on a specialized four protein complex known as monopolin and second the centromeric cohesion protection (CCP) by Spo13, shugoshin (SGO) and protein phosphatase 2A (PP2A) [4,5,6,7]
Summary
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). The homologous chromosome separation basically depends on their centromere and kinetochore architecture [2]. On the other side in the MII the sister kinetochore are biorient because of which the sister chromatids separates to the opposite pole In these organisms, sister kinetochore co-orientation in MI depends mainly on two processes, first on a specialized four protein complex known as monopolin and second the CCP by Spo, shugoshin (SGO) and protein phosphatase 2A (PP2A) [4,5,6,7]. Equal cell from each sample were taken and different dilution was made from 1 to 10− 5 and spotting was done on the synthetic complete medium or dropout medium agar plates having galactose or dextrose as a carbon source separately
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