This application """"""""Genetic and Molecular Analysis of Chromosome Structure"""""""" proposes research to begin to gain an understanding of the genetic basis and the dynamics of chromosome structure, including folding patterns which may change during the cell cycle. The directionality of yeast mating type switching provides the model system for this work. Mating type switching occurs when the information located at the distal silent HMR or HML locus is used in a gene conversion event that results in its insertion at the MAT locus. The switch is directional in that in a given case the HMR locus will be strongly favored, whereas in others the information in HML locus is used. It appears probable that the switch involves a pairing of the MAT locus region with either the HMR or HML locus, depending upon the directionality of the event. This may occur at a specific stage of the cell cycle when these regions of chromosome III could be paired in preparation for switching. Two related, efficient screens for mutants has been designed that will allow identification of genes required for the directionality of mating type switch. For example, the insertion of the ADE2 gene into the HMR allows identification of mutational changes in the directionality of switches since it allow detection by red/white sectoring of yeast colonies. The hypothesis to be tested is that a set of yet unidentified directionality genes encode trans-acting proteins which are required for the precise directionality of mating type switching. Once mutations are identified which result in obvious changes in the red/white sectoring, a number of screens will be employed to eliminate from further consideration trivial mutants which do not appear to be directly relevant to directionality. Mutants that pass these tests will then be sorted into complementation groups, and the genes so identified will be cloned and analyzed. The putative directionality genes will be sequenced, disrupted, mapped, are characterized, including looking for homologies or motifs by comparison with protein data bases. The second goal is to employ a technique known as fluorescent in situ hybridization (FISH) to detect predicted intrachromosomal pairing of the MAT locus with HMR (or HML), and to determine whether such specific pairing of regions of chromsome III occurs during particular stages in the cell cycle. Fluorescent probes which hybridize to segments closely linked to HML, HMR and MAT will be used. This technique has already been successfully employed to examine interchromosomal pairing and thus it is anticipated that it can readily be applied to intrachromosomal pairing of regions of chromosomes. The results of this study will test the hypothesis that switching (and the directionality of switching) depends upon specific pairing of the relevant regions.
