Doctor of Philosophy (PhD)
Insulators are DNA elements that have been shown to restrict the communication between enhancers and promoters in transgenic assays. In my PhD work I have attempted to highlight the function and importance of insulators by using the Boundary Element Associated Factors- BEAF -32A and BEAF-32B as model insulator binding proteins. We generated a null BEAFAB-KO allele and BEAFA-KO allele by using homologous recombination method. BEAF-32B was found to be sufficient to obtain viable flies. Our results show that BEAF is required for both oogenesis and development. Position independent expression and enhancer-blocking assays showed that BEAF confers insulator function to scs’ sequence. The role of BEAF in chromatin structure and dynamics was shown by the fact that absence of BEAF caused the male X-chromosome morphology to be disrupted and also through position-effect variegation assay. The above process of gene targeting by homologous recombination to obtain BEAF knock-out resulted in the generation of second-site mutations. We showed through several experimental and control crosses that second-site mutations are generated to a significant extent during the process of homologous recombination. Next we showed that BEAF has a role in maintaining patterns of gene expression. We expressed a dominant negative form of BEAF (BID) under GAL4 UAS control by an eye driver which resulted in a rough eye phenotype and this effect could be rescued by introducing an extra copy of a BEAF transgene. Using this assay as a tool we screened for dominant mutations that modified this eye phenotype. The genes identified in this assay mostly belonged to transcription factors involved in head development, or general transcription factors and insulator binding proteins. We then validated the results obtained in the above genetic screen by showing how several genes which positively interacted with BEAF in the above assay show aberrant levels and patterns of gene expression in BEAF knock-out flies. We also looked at accessibility of certain proteins to DNA in a BEAF knock-out background and found that DNA binding is only subtly affected in the absence of BEAF. The work done here established BEAF as an essential protein. It also showed how BEAF might be involved in the regulation of several important genes. Knowledge gained from these studies would certainly help us to understand the importance of insulators in Drosophila and also in other higher forms of organisms.
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Release the entire work immediately for access worldwide.
Roy, Swarnava, "Genetic Analysis of the Boundary Element Associated Factors-BEAF32A and BEAF-32B" (2008). LSU Doctoral Dissertations. 2902.
Craig M. Hart