In higher organisms, selective gene expression is a fundamental mechanism used to accomplish the complex processes of differentiation and development. Maize is an attractive developmental system because extensive genetic studies have uncovered mutations defective in normal developmental programs. For example, more than twenty different loci have been identified as affecting pollen development (microsporogenesis). The defects associated with the known male sterile (ms) mutants occur at different points in the pollen developmental pathway. The ability to maintain such mutants in maize proves a distinct advantage over most animal systems in the study of male gamete development. In addition to the nuclear ms genes, there are cytoplasmic male sterile (cms) traits associated with the mitochondrial genome; nuclear genes known as Restorers of fertility (Rf) overcome the cms defect, suggesting that the Rf gene products interact with mitochondria during pollen development. The Rf/cms system in maize offers an exceptional opportunity to study the interaction of the nuclear and mitochondrial genomes in a developmental system. The goal of this proposal is the molecular analysis of the maize pollen developmental pathway. In order to accomplish this goal, techniques to purify microspores in specific stages of development will be refined. Total, in vivo labelled, and in vitro translated proteins will be analyzed for each developmental stage. Messenger RNA (mRNA) will be isolated from staged microspores and used to produce cDNA libraries; these will be screened for sequences that are specifically expressed during each stage of microsporogenesis, and correlated with specific ms mutations and Restorer genes. In addition, the maize transposable element Robertson's Mutator (Mu) will be utilized to obtain an insertion mutation of a particular restorer gene, Rf3. Cloned Mu sequences will then be used as a molecular probe to isolate the Rf3 gene.
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