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Title: Dinucleosome reconstitution and transcription
Author: Johnson, Keith Jack
Awarding Body: University of London
Current Institution: Imperial College London
Date of Award: 1986
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DNA is packaged into eukaryotic cell nuclei in a highly condensed form, the chromosomes, the basic repeating unit of which is the nucleosome. It has been postulated that the way in which specific genes are packaged into these structures influences the regulation of expression of the genes and may therefore play a central role in differentiation. Studies todate have shown that the structure of individual genes in cell types in which they are expressed, differ from that in cell types where the gene is quiescent. However, there have been no studies in which the detailed relationship between the DNA sequence and the nucleosomes for an individual gene (transcribed by RNA polymerase II) have been ident ified. In this project I have attempted to reconstitute dinucleosomes on restriction fragments of the human beta-globin gene in order to determine the effects of nucleosomes on promoter usage iji vitro. Using a range of fragments prepared from sonicated calf thymus DNA the optimum size of DNA fragment required to reconstitute dinucleosomes was determined as 350+10 bp. Several different reconstitution procedures were employed, none of them were found to be entirely suitable for recovering purified dinucleosomes. A hybrid of these methods was finally chosen. The published sequence data for the human betaglobin gene was searched for restriction fragments of 350+10 bp containing the promoter region of the gene using a computer programme. These fragments were subcloned and purified in large amounts (> 100 ug ). During the course of these experiments several sub-cloning artefacts were discovered, the problems of sub-cloning are discussed. The transcription of these fragments in vitro was investigated using whole cell lysates prepared from HeLa cells to generate specific run-off transcripts.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available