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Identification and regulation of a circadian clock protein, FRQ, and a clock-controlled protein, CCG-1, in Neurospora crassa.

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Call No.	QP84.6 .G37 1996
Author	Garceau, Norman Y.
Title	Identification and regulation of a circadian clock protein, FRQ, and a clock-controlled protein, CCG-1, in Neurospora crassa.
Date	1996
Abstract	To understand how the circadian clock can temporally organize a cell, the biochemical characterization of a clock protein, FRQ, was initiated in Neurospora crassa using immunochemical techniques. FRQ accumulated in constant light to maximum levels by four hours and remained elevated for at least eight hours. Furthermore, the mobility of FRQ by SDS-PAGE decreased with increasing time in light, probably by posttranslational modification. The abundance of FRQ also oscillated with circadian periodicity in constant dark. The peak in protein abundance occurred at mid subjective day and phase-lagged the peak in frq mRNA accumulation by four hours. Using the baculovirus expression system, FRQ was observed by immunofluorescence to accumulate in nuclei of the Sf9 insect cell line. These results are consistent with a role for FRQ as a transcription factor in the negative regulation of its own expression. To study the role of translational regulation in FRQ expression, a 597 bp deletion was made in the 5$\sp\prime$ UTR of the frq mRNA. This construct restored circadian rhythmicity in a frq-null strain, although the period was shortened by one hour. The abundance of frq mRNA and protein were reduced by approximately two fold relative to a strain that was transformed with wild type frq DNA. These results suggest that the period-shortening effects of the deletion are mediated at the level of transcript abundance. To learn about the nature of clock-controlled biochemical processes, the characterization of GCG-1 was initiated using immunochemical techniques. A circadian oscillation in CCG-1 abundance was observed, with maximum accumulation at approximately the same time as the elaboration of conidiospores begins. Furthermore, CCG-1 abundance increased during macroconidiation and this accumulation was blocked in a mutant strain, aconidial-2, that is deficient in the production of macroconidia. Finally, CCG-l was observed by indirect immunofluorescence to accumulate at elevated levels in macroconidia relative to hyphae. These results are consistent with a role for CCG-1 in hyphae and macroconidia.
Description	xv, 212 leaves : ill.
Format	application/pdf
Advisor	Lienhard, Gus
Subject	Circadian rhythms. Proteins -- Synthesis -- Effect of light on. Genetic regulation. Neurospora crassa -- Reproduction -- Effect of light on. Neurospora crassa -- Genetics.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.1502

Title	Page 1
Full Text	Identification and Regulation of a Circadian Clock Protein, FRQ, and a Clock-Controlled Protein, CCG-1, in Neurospora crassa. A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Norman Y. Garceau Dartmouth College Hanover, NH 03755 May 17, 1996

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Identification and regulation of a circadian clock protein, FRQ, and a clock-controlled protein, CCG-1, in Neurospora crassa.

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