The research proposed in this application is a genetic investigation of the genes that encode the histones H3 and H4 in the budding yeast Saccharomyces cerevisiae. The experiments are divided into two major project areas: (1) the molecular genetics of histone protein structure and function, and (2) the cell division cycle control of histone gene transcription. The N-terminal protein domains of the histones H3 and H4 are critical for cellular functions, such as nuclear division and transcription, and the lysines subject to reversible acetylation play an important role in these functions. A mutational study of the H3 and H4 acetylation sites will be conducted to test the involvement of positive charge density in N-terminal domain function. A scanning point mutant analysis will be conducted to identify functional peptide sites within the terminal domains. Specific histone H4 N-terminal domain mutants are temperature sensitive for growth. Extragenic suppressors of this lethality will be identified, cloned, and characterized to understand the steps and pathways dependent on N-terminal domain function. Double mutants containing both the histone H3 and H4 N- terminal domain deletions are inviable. A screen will be carried out to identify other mutants that are also synthetic lethals in combination with either of the individual H3 or H4 N-terminal domain deletions. These genes will be cloned and characterized to examine the pathways and hierarchy of interactions involved. In a previous mutational screen for temperature sensitive lethal mutants, a histone H4 mutant was identified in which an early step in chromosome segregation is blocked at the restrictive temperature. Genetic and biochemical experiments will be conducted to determine if this block is caused by centromere disfunction or a global disruption of chromatid structure. A mutational screen will be carried out to identify extragenic suppressors of this temperature sensitive lethality. The new genes will be cloned and characterized to determine what steps in mitotic chromosome segregation depend on histone H4 function. Several other histone H3 and H4 mutants also have a delay during the G2 phase of the division cycle. A genetic screen will be conducted to identify new G2 checkpoint mutants predicted to monitor chromosome integrity. Transcription of the histone H3 and H4 genes is tightly regulated within the cell division cycle with expression restricted to the late G1 and S phases of the cycle. This control is mediated by upstream activation site (UAS) elements and upstream repressor site (URS) elements. Saturation mutagenesis will be used to define the precise DNA sequence requirements for these cis-acting regulatory sites. There are multiple copies of the UAS element arranged as inverted repeats in each of the three histone promoters examined to date. The importance of the orientation and spacing of these elements will be tested. A DNA binding protein specific for the histone UAS element has been identified. To understand the mechanism of cell cycle control at the histone UAS, the gene encoding this binding protein will be cloned, sequenced, and characterized. A mutational analysis of the UAS binding protein will be carried out to determine its molecular function, and define the signal transduction pathway for cell cycle position information.
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