The proper chromosome segregation during cell division depends on the maintenance and regulation of the association between sister chromatids. To attain a clear understanding of sister-chromatid cohesion a study of the protein. MEI-S332, will be implemented. The objectives of the experiments in this proposal will determine where MEI-S332 binds to on the centromeric region of meiotic chromosomes and to determine how its disappearance at the metaphase II/anaphase II transition is regulated. The fulfillment of these objectives is essential for understanding the regulation of sister- chromatid cohesion. The following questions regarding MEI-S332's function will be tested: 1) Is MEI-S332 bound to the heterochromatin flanking the centromere or to the kinetochore itself? The localization of MEI-S332-GFP on an inverted chromosome and on deleted minichromosomes will be used for the purpose. 2) Is MEI-S332's disappearance from the centromeric region at anaphase II a result of cell cycle regulated proteolysis or post translational modification such as phosphorylation? Western blot analysis will be performed to test which of these mechanisms regulate MEI-S332 localization. 3) What regions of the protein dictate its localization to the centromeric region? A site-directed mutagenesis will be utilized to define essential regions of MEI-S332 that dictate its localization and disappearance. 4) What effects do other mutations have on MEI-S332-GFP behavior will be examined in the genetic background of mutants that are defective in chromosome segregation and cell cycle control during meiosis.
