Master of Science (MS)
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.
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Ge, Yu, "Characterization of the Promoter and Insulator Activities of the Scs' Insulator" (2012). LSU Master's Theses. 3574.