Ubiquitin, a 76-residue protein, is known to occur in chromatin as a part of an unusual bifurcated protein, ubiquitin-H2A semihistone. Using a new approach, two-dimensional hybridization mapping of nucleosomes (1), we have recently found that transcribed copia and heat-shock D. melanogaster genes are heavily ubiquitinated, while tandemly repeated, non-transcribed, heterochromatic chromosomal regions contain virtually no ubiquitin-H2A semihistone (2). Experiments in this proposal are designed to explore both the immediate and more remote implications of this discovery. The major proposed project are: (1) Detailed two-dimensional hybridization mapping of the distribution and degree of nucleosome ubiquitination in transcribed Drosophila melanogaster genes, such as copia, hsp 70, hsp 83, and several others. We shall ask whether the borders of a heavily ubiquitinated chromosomal region are also the borders of the transcriptional unit, whether ubiquitinated nucleosomes from transcribed genes occur in clusters or are interspersed with ubiquitin-less nucleosomes, and whether the degree of nucleosome ubiquitination in a transcriptional unit is a function of the frequency of transcriptional initiation. (2) We shall test whether ubiquitinated nucleosomes are preferred ligands in vitro for certain chromosomal proteins, such as HMGs, RNA polymerase, DNA topoisomerases and ubiquitin itself. (3) In addition to the already available monospecific anti-ubiquitin serum, we shall prepare monoclonal antibodies against ubiquintin and ubiquitin conjugates such as uH2A, and use them to visualize ubiquitinated chromosomal regions in situ, to detect ubiquitinated proteins other than uH2A and uH2B among electrophoretically resolved chromosomal proteins and to separate ubiquitin-containing oligo-and mononucleosomes from ubiquitin-lacking ones in vitro. (4) We shall study certain enzymological aspects of the metabolism of ubiquitinated nuclear proteins and nucleosomes, the major aim being the understanding of possible relationships between two recently discovered ubiquitin functions, its highly preferential if not exclusive localization in transcribed chromosomal regions (2) and its role as a component of an ATP-dependent cytoplasmic proteolytic system (7-9). The results of these studies will contribute to the understanding of both structure and functioning of eukaryotic chromosomes.
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