Nuclear proteins associated with gene expression in mouse plasmacytoma cell lines and in rat liver nuclei treated with the carcinogen acetylaminofluorene

The subject matter of this thesis is the role of nuclear non histone proteins in the control of gene expression. To understand how these proteins may be able to regulate gene expression we first look at their molecular environment, viz. the structure of chromatin; some of the evidence linking non histone (chromatin) proteins with gene expression is then examined, together with the background of the experimental work reported here. In the first experimental chapter, the binding of a labelled carcinogen ([14C]-acetylaminofluorene) to rat liver nuclear protein fractions was examined. Most of the recovered carcinogen (80%) was bound to the nonhistone protein fraction. After two weeks the label was more evenly distributed; the highest specific activity being in the protein fraction most tightly bound to DNA. Pre-feeding rats with sodium sulphate and phenobarbitone reduced binding to all nuclear fractions. Labelled proteins were further analysed by isoelectric focusing which revealed a complex binding pattern. In the second experimental chapter the nucleoplasmic and non-histone chromatin proteins from four related and two unrelated mouse plasmacytoma cell lines were analysed by biosynthetic labelling with [35S]-methionine followed by one and two dimensional electrophoresis. In this work in clonal cell lines it was hoped that by looking at cells that were "frozen" at one stage of differentiation, viz. the plasma cell, that it would be possible to both limit the differences between the cell lines and correlate particular proteins with phenotypic differences. The majority of proteins are common to all of the cell lines studied as would be expected if the majority of nuclear proteins are concerned with functions common to all plasma cells. There are however both qualitative and quantitative differences in the nuclear protein patterns of mutant and parent cell lines which appear to correlate with differences in gene expression. The turnover of nuclear proteins in two of the cell lines, MOPC 315.40 (IgA) producer and MOPC 315.32 (λ2 chain producer), which exhibited these differences, was studied using pulse-chase techniques and the half lives of both nuclear protein fractions and nuclear proteins which differed between the cell lines were calculated. Finally, the results of these experiments are discussed in the context of findings by other authors working in similar areas and in the light of recent developments concerning the role of these proteins in gene expression

The release of high mobility group protein H6 and protamine gene sequences upon selective DNase I degradation of trout testis chromatin.

Limited digestion of trout testis nuclei with DNase I selectively degrades the protamine genes. Concomitant with the degradation of transcribed DNA sequences a series of chromosomal proteins are released; among these, the major species corresponds to the high mobility group protein H6. The amounts of H6 released from chromatin by limited DNase I action and that in the residual nuclear pellet have been determined. A very high proportion of H6 is associated with DNase I sensitive chromatin regions