Control of gene expression mediated through activation of topologically isolated domains of chromatin anchored at their bases to the nuclear matrix is likely to play a critical role in expression of developmentally regulated genes, in transcriptional activation, and in transcription in general. Considerable efforts have been directed toward analysis of the components of the nuclear matrix and sites of attachment of DNA, with much less analysis of functional roles. This is due, at least in part, to difficulties in identifying systems which will permit functional studies to be readily undertaken. Adenovirus presents an excellent model system for such studies. Adenovirus is genetically and biochemically manipulable. The adenovirus chromosome appears to comprise one chromatin domain, bounded by attachment to the nuclear matrix through the virally-encoded terminal protein covalently attached to the 5' end of each DNA strand. When this attachment is weakened due to introduction of mutations within the terminal protein gene, transcriptional activity of the entire genome is reduced. Terminal protein function also appears to be required for regulation of mRNA transport to the cytoplasm during the late phase of the infection. Adenovirus will be used to examine the mechanistic details by which binding of chromatin to the nuclear matrix affects transcription and transcriptional activation by ElA protein. In vivo competition by transcriptionally distinguishable viruses will be used to examine transcription complex formation and its dependence on binding of DNA to the nuclear matrix. The effects of nuclear matrix association on expression of transiently transfected genes will be examined. Terminal protein binding to the nuclear matrix will be examined in detail, using in vitro systems and in vivo systems in which terminal protein binding will be studied in isolation or, after rebuilding mutations into the virus, as part of the infectious cycle. The region(s) of terminal protein directly involved in binding to the nuclear matrix will be examined using genes containing in-frame deletions to direct synthesis of mutated proteins. Two amino acid insertion mutagenesis will be used to generate additional mutations in the terminal protein gene as a tool for analyzing terminal protein function and in an attempt to find a mutation which leads to conditional nuclear matrix binding. In particular, mutations will be introduced into regions of the protein found to be important for binding to the nuclear matrix. Antibodies have been raised against terminal protein and will be used in analysis of its metabolism. Finally. the role of terminal protein in controlling transport of host and viral mRNAs will be examined.
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