The major aim of this research was to investigate the molecular mechanisms of regulation of transcription of CIT1 and CIT2, the genes encoding citrate synthase in yeast. Specifically addressed are the questions of (1) localization of cis-acting sites required for expression or regulation, (2) the roles of HAP1 and HAP2,3,4 in expression of both genes, (3) identification of other trans-acting factors involved in expression of either gene, (4) localization of cis-acting sites involved in up regulation of CIT2 in response to disruption of CIT1 or rho° status (Liao et al., 1991; Liao and Butow, 1993).
I show here that mutations in HAP2, HAP3, or HAP4 specifically prevent derepression of CIT1. Using deletions and base substitutions, derepression of CIT1 is shown to require candidate HAP2,3,4 binding sites at -290 and -310 (distance upstream from the translational start site). Attempts at demonstrating binding of HAP2,3,4 to CIT1 upstream DNA were unsuccessful. HAP1 appeared to play an important role in lactate-derepressed, but not glucose repressed expression of w. No regions were identified as being responsible for negative regulation by glucose plus glutamate. HAP2,3,4 is not required for this regulation. HAP2,3,4 also was shown to regulate CIT2 to a small degree in glucose- repressed expression and to a large degree in lactate-derepressed expression. No regions were identified as responsible for this activation.
To identify additional m-acting factors involved in expression CIT1 or CIT2 (eg. activators of glucose-repressed expression), yeast containing CIT1-lacZ or CIT2-lacZ fusions were mutagenized and screened for altered expression. Seven mutants with reduced expression of a ClT1-lacZ fusion are currently under study.
Expression of CIT2 (peroxisomal citrate synthase) is not regulated by glucose, but is regulated by the rho status of the strain and by the presence or absence of a functional CIT1 gene (Liao et al., 1991; Liao and Butow, 1993). We identified a region critical for expression of w in lactate-grown cells located between -300 and -370, which is the same region Butow’s laboratory has found to be important in regulation by rho.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-6214 |
| Date | 01 January 1993 |
| Creators | Kell, Christine Smith |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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