Meiosis is essential for all sexually reproducing organisms and the studies described here will further our understanding of this process not only in maize but in all organisms. The mechanism of meiosis is a topic of major medical interest since inaccurate chromosome segregation (aneuploidy) during meiosis is causal in several congenital malformations, a major cause of premature termination of pregnancy, and of poor gamete production in humans. Our goal is to understand the mechanism of chromosome segregation during meiosis, particularly how homologous chromosomes pair and synapse. Maize is the only organism where there is a large collection of mutants that affect meiosis, and it is possible to do superb cytology. Entrance into meiotic prophase is under control of a cell cycle dependent switch, ameiotic 1 (am1) whose function is required to convert a mitotic to a meiotic cell cycle. The function of aml will be studied at a molecular, biochemical and cytological level. We will use expression microarrays to determine whether meiotic gene expression is altered in aml mutant alleles. Chromosome structure will be studied in absence of first division 1 (afd1) nuclei where RAD51 installation is very severely reduced. For various afdl alleles, we will correlate extent of RAD51 installation with extent of leptotene/zygotene chromosome remodeling. We will make anti-AFD1 antibody to immuno-purify AFD1 interacting proteins, and for localization of FD1 on meiotic chromosomes. We propose that RAD51 complexes are required for both the homology search and recombination. To analyze potential recombination defects in our 20-desynaptic mutants, we will use antibodies against key recombination pathway components such as SPO 11, RAD51, BLM or MSH4 and MLH1 to classify their position in the pairing/recombination pathway based on deficiencies in protein complex distribution. We will make double mutants between members of various classes of RAD51 loci deficient mutants to analyze potential epistatic interactions. We will further cytologically and molecularly characterize the three mutants poor homologous synapsis1 (phs1), desynapticCS (dsyCS), and segregation II (seglI) that are severely deficient in RAD51 foci and determine whether they are deficient in a step required to load RAD51 complexes onto chromosomes. We will clone dsyCS and seglI, using transposon-tagging strategies, characterize their function, and if time permits, clone other genes defective in later stages of the pairing/recombination pathway.
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