Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction.
The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.
Department of Biochemistry, Wright State University, Dayton, OH 45435.
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Kumar
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Leffak
D000260:Adenoviruses, Human / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D002843:Chromatin / Q000502:physiology*; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D016259:Genes, myc; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014161:Transduction, Genetic
D002843:Chromatin; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease