Ultradian oscillators of the circadian clock in Saccharomyces cerevisiae

Sara S Dick, Purdue University

Abstract

The cell surface protein ENOX1 has properties of an ultradian oscillator of the cells’ circadian clock in that the period length of the ENOX1 activity in minutes equals the circadian day length in hours. How the ultradian and circadian timing are linked is difficult to study in mammals or plants, or even in cell culture. Because yeast grow by tip growth rather than cell enlargement, ENOX1 activity can be blocked in, for example, Saccharomyces cerevisiae without affecting growth, making yeasts ideal organisms for investigating the effect of mutations on clock function. To enable study of the relationship between circadian day and ENOX1 activity, I have established that the yeast, Saccharomyces cerevisiae, has an ENOX1 activity similar to that of other eukaryotes. In contrast to other eukaryotes, Saccharomyces cerevisiae has a second ENOX1-like activity with a period length of 25 min that is distinguishable from the traditional ENOX1 on the basis of the longer period length along with resistance to an ENOX1 inhibitor, simalikalactone D, and failure to be phased by melatonin. In addition, two periods are apparent in measurements of oxygen consumption indicating that the consumption of oxygen to water occurs independently by homodimers of both of the two forms of ENOX. A heterodimer would produce one period of oxygen consumption. There is a third period of 40 min that corresponds to the metabolic rhythm of oxygen consumption in yeast during continuous culture. The yeast exhibit circadian maxima of GAPDH activity at 24 h, 25 h and 40 h, indicative of three independent clock outputs, corresponding to the observed 24, 25 and 40 min ultradian period lengths. Yeast libraries were screened for increased growth rate and lack of oscillatory behavior in NADH levels. The deletion library screen identified 32 genes lacking periodic fluctuations in NADH levels, including SIR1, a protein involved in histone modifications. The overexpression library screen based on increased growth rate yielded 85 genes, some of which were involved in chromatin remodeling, including members of the COMPASS complex responsible for silencing near telomeres, ADA2 and MAK10, members of acetyltransferase complexes, and HDA2 and UME1, members of histone deacetylase complexes. As the clock genes are regulated in part by silencing and relaxation of chromatin at clock gene promoters in response to NADH or NAD+ levels, these genes have the potential to be involved in clock gene function. As each of the three ultradian oscillators of Saccharomyces cerevisiae result in fluctuations in NADH levels, pyridine nucleotide fluctuations generated by ultradian oscillations emerge as the controlling influence that modulates core clock genes to create the circadian day.

Degree

Ph.D.

Advisors

Morre, Purdue University.

Subject Area

Microbiology|Biochemistry

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