Chromatin structure of the integrated viral sequences in adenovirus-transformed cells

• Main Author: Aird, Fraser
• Other Authors: Memorial University of Newfoundland. Faculty of Medicine
• Format: Thesis
• Language: English
• Published: 1989
• Subjects: Chromatin; DNA; Adenoviruses; Ela; Newfoundland studies; University of Newfoundland
• Online Access: http://collections.mun.ca/cdm/ref/collection/theses3/id/30152

Thesis (Ph.D.)--Memorial University of Newfoundland, 1989. Medicine Bibliography: leaves 261-305 In the eukaryotic cell nucleus, chromatin is organized into higher-order structures through hierarchical levels of folding and coiling. Active genes are contained in chromatin domains with a more "open" conformation that is preferentially sensitive to DNase 1 digestion compared to inactive chromatin. The spatial organization of chromatin is maintained by the nuclear matrix, the major structural component of the nucleus. Chromosomal DNA is organized into supercoiled loops anchored at their bases to the nuclear matrix, and in most cases examined, active genes are located at or near the base of the loops. -- I have examined the DNase I sensitivity of the integrated viral sequences of four adenovirus type 5-transformed cell lines, and the organization of these sequences relative to the nuclear matrix. DNase I sensitivity was analysed by digesting nuclei with DNase I and monitoring the disappearance of virus-specific restriction fragments by Southern blotting and hybridization. In each cell line, the integrated viral sequences were in a conformation typical of active chromatin, i.e. they were preferentially sensitive to DNase I compared to inactive chromatin. The DNase I sensitive region included not only the active transforming (El) genes, but extended into the adjacent inactive viral sequences. Thus, the integrated viral sequences were contained within active chromatin domains. -- In addition to these extended domains, DNase I hypersensitive sites were detected in the ElA 5'-flanking sequences. These sites were mapped to sequences previously shown to contain the ElA transcriptional enhancers and binding sites for cellular transcription factors. Therefore, the DNase I hypersensitive sites likely reflect alterations in local chromatin structure associated with regulation of transcription of the integrated ElA genes. -- The organization of the integrated viral sequences relative to the nuclear matrix was analysed by assessing the matrix associated and non-associated DNA fractions for their content of viral sequences by Southern blotting and hybridization. There was no enrichment or depletion of the viral sequences in either of these fractions relative to total unfraotionated DNA, consistent with a random organization relative to the nuclear matrix. However, control studies indicated that these results may be due to the conditions used to isolate the nuclear matrix DNA fractions. -- (Keywords: adenovirus type 5; chromatin domain; DNase I sensitivity; hypersensitive sites; nuclear matrix.)