Student Theses and Dissertations

Date of Award

2001

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Young Laboratory

Abstract

Although the basic neural mechanism that regulates circadian behavior in Drosophila is known in quite some detail, several aspects of this biological process still remain to be elucidated. In this study a variety of genetic and molecular approaches was used to identify and characterize genes that are involved in circadian rhythms and that may shed light on the gaps in the model. In an F1 screen for altered period length of the circadian locomotor activity cycle the new mutation 2ob9 was isolated. Cloning and sequencing identified the affected gene as a Drosophila homolog of the yeast RNA helicase and splicing factor Prp43. Consistently, splicing efficiency of the timeless gene is reduced in the presence of a dominant negative form of DPRP43. The period-lengthening phenotype is most likely a reflection of the daily cycle of transcription of several clock genes that is delayed in the 2ob9 mutant. The results suggest that splicing may be a rate determining factor in the circadian cycle. Levels of expression as well as temporal and spatial expression patterns are in many cases an integral part of gene function. Thus, over-expression of a particular gene often interferes with its wild type function. Based on this notion, a screening method has previously been developed that allows for tissue specific over-expression of random genes throughout the Drosophila genome. Using this approach, the segment polarity gene shaggy has been identified as an integral regulator of period length of the circadian oscillator in Drosophila; overexpression of shaggy shortens the circadian cycle whereas reduction of shaggy function appears to lengthen to locomotor activity cycle. These behavioral phenotypes coincide with increased and decreased TIMELESS phosphorylation respectively, suggesting that SHAGGY controls period length through posttranslational modification. Furthermore, gain of shaggy function concurs with an advanced nuclear entry of the PERIOD/TIMELESS heterodimer. A model is proposed, whereby SHAGGY determines the rate of PERIOD/TIMELESS nuclear translocation through its effect on the phosphorylation pattern of TIMELESS. This screen also resulted in the identification of the transcription factor ADF-1 as a potential circadian regulator. Flies over-expressing Adf-1 fail to display any circadian rhythmicity of locomotor activity. Increased Adf-1 function dampens the amplitude of PERIOD and TIMELESS oscillation and reduces PDF levels. Thus, molecular oscillator as well as output mechanism of the clock are possible targets of ADF-1. However, mostly due to the lack of loss of function data, a circadian function of Adf-1 could not be ascertained. In an effort to interfere with gene function in a tissue specific manner, inverted repeat sequences specific to the period gene were expressed in neurons responsible for circadian locomotor activity. A consistent lengthening of the circadian period was found. In accordance with a double stranded RNA mediated mechanism, the inverted repeats caused a decrease of endogenous period RNA levels. The spatial selectivity of this approach may provide a tool to investigate behavioral functions of genes that are essential for viability. Using a molecular approach, a correlation between MAP kinase activation and phase responses to photic stimuli was found. Furthermore, light stimulated MAP kinase activation appeared to be dependent on proper functioning of the phosphodiesterase DUNCE. Since dunce had previously been implicated in the entrainment pathway, c A M P metabolism and M A P kinase activity m a y act together in an input pathway of the circadian clock in Drosophila. The implications of each of these findings for the general model of the circadian clock in Drosophila are discussed at the end of the respective chapters.

Comments

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy At The Rockefeller University

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