Meiotic Kinetochores of Maize

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9513556 Dawe The long term goal of this study is to identify the structural and molecular properties of the kinetochore that ensure normal meiotic chromosome segregation. Kinetochores have a variety of critical roles in cell division, such as ensuring that the chromatids do not separate prematurely, and acting as the biological motors that pull the chromosomes along the spindle. In meiosis, the kinetochores have more specific roles that relate to their critical function in ensuring that the genetic information is transmitted accurately from generation to generation. Maize is an excellent organism for studying kinetochores, because of the unusually large size of the chromosomes and of the kinetochores themselves. In prior studies, advanced three dimensional light microscmpy has been used to identify subtle structural features, first in the chromosomes and more recently in the kinetochores using a human antiserum that recognizes maize meiotic kinetochores. The major cell biological advantages of maize are coupled with extensive genetic resources with which to analyze kinetochore function. One such resource is an unusual variant of chromosome 10 that induces facultative centromeres ("neocentromeres") as a part of a more complex meiotic drive system. In the presence of Abnormal chromosome 10, quiescent heterochromatic regions called "knobs" are converted into meiotic kinetochores that stretch the chromosome arms towards the spindle poles. Neocentromeres and true centromeres are probably closely related, because knobs have strong sequence similarity to true centromeres. However, unlike true centromeres, mutations of neocentromere formation can be readily identified. The method for identifying neocentromeres relies on the fact that their formation is closely associated with a preferential segregation, or meiotic drive, of genetic markers that are linked to knobs. If the genetic marker affects kernel pigmentation, meiotic drive is visible as an excess of pigmented kernels. In prior stud ies, a meiotic drive mutation called Ab10-smd1 was identified, which proved to be a defect in neocentromere formation. This proposal contains experiments leading to the cloning and characterization of Ab10-smd1 by virtue of the fact that the mutation was induced by a transposable element. It should be possible to identify the genes that regulate normal meiotic kinetochore behavior by homology to Ab10-smd1 and other genes like it. Additional studies are proposed that relate to how neocentromeres promote meiotic drive, and how normal kinetochores direct meiotic chromosome segregation. It will be possible to verify a long-standing model for how neocentromeres lead to meiotic drive by directly labeling knobs with fluorescent oligonucleotides. Labeling the knobs as well as the kinetochores and spindle using specific antibodies will make it possible to study the interaction of these three components during chromosome segregation. Finally, experiments are proposed to analyze neocentromere behavior using time lapse three dimensional light microscopy. The conspicuous nature of the neocentromeres coupled with a powerful new light microscope workstation will make it possible to study meiotic kinetochores in the living state. Such time lapse microscopy will also provide an added level of resolution to the characterization of neocentromere mutations. ***