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Initial characterization and intracellular localization of two suppressors of position effect variegation in drosophila melanogaster, S2214 and puckered Toub, Omid

Abstract

Emergence of the higher eukaryotic organisms from their prokaryotic ancestors has been closely associated with an increase of the genetic material. This progression has been dependant on machineries that can package the DNA to various extents, from the levels seen in the 30 nm fibers of interphase nuclei to that of metaphase chromosomes. These evolutionary changes in genome organization have correlated with advancements in regulation of gene expression during development. In eukaryotes, cellular differentiation is partly dependent on the mechanisms that would silence the correct genes in a particular tissue and maintain this silenced state throughout subsequent stages of development. To understand the factors involved in such mechanisms many labs, including ours, have used position effect variegation (PEV) to identify proteins that form or remodel the chromatin fiber. Genetic screens have identified S2214, and puckered as genes coding for putative modifiers of PEV. The aim of this thesis, is to characterize S2214, and puckered by addressing two main questions: i) do the mutations in each of these genes modify the phenotype observed in PEV? And ii) do their products localize to the nucleus, and if so to the chromatin? Results show that P element mutations in these genes cause dominant and strong suppression of PEV in wm⁴ and SbV. Moreover, the observed Su(var) activity is reverted upon mobilization of the P elements. I developed and purified an antibody for each gene. Puc, the product of puckered, localized to the nucleus of S2 and KC1cells (which are late embryonic Drosophila cell lines), as well as the nuclei of salivary gland cells of Drosophila melanogaster, but could not be detected on the polytene chromosomes. In addition, S2214, the product of S2214, was found in the nuclear fraction of S2 cells, and could be observed within the nuclei of S2 and KC1 cells as well as those of the salivary glands of Drosophila melanogaster. Furthermore, S2214 was found at several interbands of the polytene chromosomes of these salivary glands. It is our conclusion that gene products of both S2214 and puckered are involved in mechanisms that affect chromatin structure.

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