The proposal focuses on chromosome replication in the yeast Saccharomyces cerevisiae. Of particular interest is the mechanisms by which replication related events are integrated within the cell cycle, and how processes taking place in the S phase are temporally regulated. Major experimental tools are culture density transfers, equilibrium density banding to separate old and new DNAs, one and two dimensional gel electrophoresis to resolve replication intermediate structures from completely replicated ones, DNA-DNA and RNA-DNA hybridizations using cloned probes for chromosomal sequences, and the construction of novel plasmids. The project will examine: the temporal pattern of replication along extended stretches of chromosomes to obtain evidence whether ARS (autonomous replication sequence) elements are origins of replication; the effect of intermixing early and late replicating chromosome sequences, as an approach to defining cis-acting elements that determine replication time; the time and nature of centromere and telomere replication in the cell cycle; whether """"""""leakage"""""""" replication of specific sequences in cdc7 mutant cells is a property of specific ARS elements; the effect on plasmid replication of different combinations of replication origins and transcription units, as an approach to understanding how the topological conflicts are resolved; possible mechanisms involved in the segregation and amplification of the native 2 Mum plasmid; and, the possibility of identifying chromosomal genes which can suppress defective ARS elements, as an approach to understanding the nature of ARS function. The results of these experiments should, at the very least, yield testable ideas about related mechanisms in mammalian cells and ultimately contribute to an understanding of some human pathologies.
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