The transcription of the S. cerevisiae HO gene occurs immediately after and is dependent upon the start of the cell cycle. The long term goal of this project is to determine how this cell cycle regulation is exerted upon the HO promoter. A repeated sequence has been identified (CACGA4) that is necessary and sufficient for the start-dependent, and transient activation of HO transcription.
The specific aims of this proposal are to: 1) determine what forms of regulation are exerted upon the CACGA4 sequence, 2) identify and characterize the trans-acting factors that are responsible for that regulation, 30 determine which factor displays transient activity through the cell cycle, and 4) identify the start- specific signal that is responsible for that transient activation. This work should lead to a better understanding of the early events in the cell cycle and the mechanisms of start. The CACGA4 element acts as a cell cycle regulated upstream activation sequence (UAS). Two proteins that are specifically required for CACGA4- driven transcription (SW14 and SW16) have been cloned and sequenced. IN order to investigate their role in the transient activation of CACGA4, deletion mutant and over-producing strains have been made. Footprint and band-shift assays will be carried out with crude extracts from these mutant strains to see if SW14 or SW16 are required for binding to the CACGA4 sequence. Antibodies will be raised to both proteins and use to see if SW14 or SW16 can be detected in the protein-DNA complex at CACGA4. Antibodies will also be used for observing any qualitative or quantitative changes in these proteins through the cell cycle which might affect their activity. To identify other components of the CACGA4-regulatory system, genetic screens have been devised to look for mutations in negative regulators or positive regulators that result in constitutive CACGA4-driven transcription. Particular attention will be paid to those displaying is lethality, or other defects in growth control. these regulators will be characterized as described above for SW14 and SW16. If cell cycle- specific changes in the CACGA4- protein complex or in the physical state of any of these regulators is observed, the molecules responsible for that cyclic change will be purified and characterized. Such a molecule would be expected to be directly involved in or closely associated with the start of the cell cycle.
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