The regulatory region of the major immediate early gene (MIE) of the human cytomegalovirus (HCMV) has been analyzed for its capability of fulfilling criteria defining nuclear scaffold/matrix attachment regions (S/MARs). These criteria are: 1) binding the nuclear matrix in an in vitro nuclear matrix binding assay; 2) binding the nuclear scaffold in an in situ nuclear scaffold binding assay; and 3) activating transcription of reporter genes in stabile transformed cells.
The MIE gene was cloned, digested with restriction endonucleases, and the resulting DNA fragments were reacted with nuclear matrices prepared in vitro. Relative to coding region and vector fragments, the regulatory region of the MIE gene bound the in vitro nuclear matrix with the greatest affinity.
HCMV infected fibroblast nuclei were extracted with lithium diiodosalycilate and digested with restriction endonucleases at immediate early and early times post infection. Relative to >90% of the total cellular DNA, the MIE regulatory region bound the in situ nuclear scaffold at both times post infection. In addition, other regions of the viral genome bound the in situ nuclear scaffold at these times post infection.
A reporter gene construct driven by the full length MIE regulatory region and a construct from which an upstream portion of regulatory region was deleted, were separately co-transfected with a neomycin resistance gene into human leukemic monocytes. Stabile transformed cellular clones were selected by culturing the cells in the presence of G418. The reporter gene activity from the full length construct was 4-fold greater than the reporter gene activity of the deleted construct.
This is the first study to show that a portion of the 5' regulatory region of the MIE gene fulfills these criteria of S/MARs. It is also the first to show the HCMV genome associates with the nuclear scaffold in the infected cell nucleus.
Doctor of Philosophy (PhD)
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Library of Congress/MESH Subject Headings
DNA Viruses; Genome, Viral; DNA Replication; Nuclear Matrix -- metabolism; Cytomegalovirus -- physiology; Viral Proteins -- biosynthesis.
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This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Arnold, Brent, "The Association of the Human Cytomegalovirus Genome with the Cellular Nuclear Matrix" (1997). Loma Linda University Electronic Theses, Dissertations & Projects. 1373.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives