Failure of meiotic gene function has profound consequences in humans, leading to aneuploidy, spontaneous abortions, birth defects and infertility. The superb cytology of the maize chromosome, its well developed genetic and genomic resources, the large meiotic mutant collection, and the ability to obtain large amounts of highly synchronized meiocytes make it an excellent organism for integrating cytological, molecular, genomics and proteomic approaches to answer key questions in meiosis. The ability to perform meiosis depends on a switch to the meiotic cell cycle, and the formation of unique leptotene chromosome architecture. We hypothesize that in maize this switch is mediated by the protein AMEIOTIC1 (AM1), and that the indicator of a successful switch is the formation of the leptotene chromosome. Secondly, changes in leptotene chromosome architecture regulate downstream meiotic processes. To test these hypotheses, we will elucidate the mechanism of AM1 function using an allelic series to define functional domains, proteomics to identify interacting proteins, and reverse genetics to resolve the functions of interacting proteins. To identify genes that are regulated by AM1, we will use microarrays and quantitative RT-PCR using meiotic RNA from wild type and ami mutants. To determine how leptotene chromosomes regulate downstream processes, we will analyze the architecture of the leptotene chromosome using ultra high resolution structured illumination (SI) light microscopy and relate structure to function. We will develop an integrated model of chromomere and axial element organization using SI and elucidate changes as chromosomes pair and synapse. We will use ChIP on tiling arrays to identify histone modifications and RAD21/REC8 binding sites in a defined chromosome region. Mutants deficient in intragenic recombination or chromosome architecture will be used to determine how spatial constraints in chromomere architecture affect recombination. We will identify the defining features of the chromatin remodeling that occurs at the leptotene zygotene transition in wild type, and in am1-pra1 cells arrested at this stage, and determine whether small RNA metabolism is responsible for these changes. Finally, we will characterize the role of the three other RAD21/REC8 cohesin complex family members in establishing leptotene chromosome structure and sister chromatid cohesion. Since meiosis is an evolutionarily conserved process what we learn about meiosis in maize is relevant to humans. ? ? ?
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