The specific aims of this proposal are to genetically identify and characterize genes required for the assembly and function of centromeres in meiosis. These studies will be carried out in male Drosophila melanogaster, a model genetic organism whose centromeres appear functionally and structurally similar to human centromeres. Mutants have been induced using transposon mutagenesis, and were screened for their effects on the transmission of a meiotically unstable dicentric chromosome (a chromosome with two centromeres). Mutations that either increased or preferentially decreased meiotic transmission of this chromosome were selected. These will be characterized genetically and cytologically to identify candidate genes for centromere structural and/or regulatory proteins. In addition, a collection of deficiency-bearing chromosomes that together delete 80 percent of the autosomal euchromatin will be tested in trans for effects on the segregation of this dicentric chromosome. This deficiency screen will identify regions of the genome that contain genes important for the stability of the dicentric. Selected mutants will mapped by genetic and molecular methods, and cloned either by transposon-based methods or by chromosomal waLking. Cloned cDNAs will be expressed in bacteria or baculovirus to produce antigen for the production of polyclonal antibodies. Additionally, the nature of the dicentric chromosome will be further studied genetically. The two centromeres of the dicentric will be detached and recovered on separate chromosomes to allow tests of the properties of each centromere individually. New dicentric chromosomes will be generated as derivatives of the original dicentric. These will be characterized genetically and cytologically to identify properties that influence the mitotic and meiotic behavior of centromeres of dicentric chromosomes.
