Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Cell & Molecular Biology

First Advisor

Steven Irvine

Abstract

The classical definition of genetic cis-regulatory modules such as enhancers identifies these elements as insensitive to changes in position and orientation relative to the transcription start site (TSS). While this theory is well supported, some recent studies have uncovered examples of constraint in the position or orientation of particular enhancers. This study examines the position and orientation sensitivity of two upstream enhancer elements found in the gene Pax6, which regulates development of the eyes and central nervous system in vertebrates. Late embryonic stages of the organism used in this study, Ciona intestinalis, show expression of Pax6 in the nerve cord and sensory vesicle.

For this investigation Pax6 constructs were created in which the enhancers were repositioned or inverted relative to the TSS. These constructs were electroporated into embryos of C. intestinalis, and expression of the Pax6-GFP fusion gene was measured both by total fluorescence and by number of positive embryos. Alteration of either position or orientation of these enhancers was found to cause a strong decrease in measured Pax6 expression. These results support the conclusion that the upstream enhancers must be in the proper location and arrangement to be fully functional, a finding that is consistent with the results of many other studies of cis-regulatory elements.

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