Identifier

etd-07102012-113238

Degree

Master of Science (MS)

Department

Biological Sciences

Document Type

Thesis

Abstract

Eukaryotic gene expression is regulated by regulatory elements. Insulators are one class of regulatory sequences. They can prevent heterochromatin from spreading into euchromatin, block distal enhancer activity, or both. Insulators function by recruiting several associated proteins. The scs’ insulator, which is located at one end of the 87A7 hsp 70 locus, is bound by BEAF. In the Drosophila genome, more than 85 % of BEAF binding sites are found within 300 bp of transcription start sites, suggesting BEAF may be involved in promoter function. Based on previous insulator position effect assays, two sequences were found to be necessary for full scs’ insulator function. One is the high affinity BEAF binding site called “D”, and the other is a 20 bp sequence called “LS4”. In order to identify the minimal scs’ insulator sequence, several scs’ derivative sequences containing the LS4 region and D site will be tested using a site specific integration system. In this system, ΦC31 integrase mediates specific attB site integration into transgenic attP sites in the Drosophila genome. Two attP fly lines with strong position effects are needed to test scs’ derivative sequences. Currently, 4 out of 12 transgenic attP fly lines show slight position effects. Making new attP landing site flies is in progress to find at least two lines that have strong position effects. Promoter activity assays in S2 cells demonstrated that the scs’ M fragment possesses promoter activity in addition to insulator activity. Electrophoretic mobility shift assays (EMSAs) detected proteins that bind to the LS4 region, and BEAF binding facilitates this binding. DNA affinity chromatography was performed to purify LS4 binding proteins. After two rounds of purification, several candidate protein bands were identified. Further characterization needs to be done to confirm these proteins bind to the LS4 region. If confirmed, the proteins will be identified by mass spectrometry.

Date

2012

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Hart, Craig

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