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In vivo analysis of interactions between trans-acting factors and their target genes

Citation

Mueller, Paul R. (1990) In vivo analysis of interactions between trans-acting factors and their target genes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/yv7h-9335. https://resolver.caltech.edu/CaltechTHESIS:07282014-134702796

Abstract

The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.

The second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Wold, Barbara J.
Thesis Committee:
  • Anderson, David J.
  • Attardi, Giuseppe
  • Simon, Melvin I.
  • Davidson, Norman R.
  • Zinn, Kai George
Defense Date:1 March 1990
Record Number:CaltechTHESIS:07282014-134702796
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:07282014-134702796
DOI:10.7907/yv7h-9335
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8615
Collection:CaltechTHESIS
Deposited By: Bianca Rios
Deposited On:28 Jul 2014 21:22
Last Modified:16 Apr 2021 22:19

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