The basic issue to be examined is the mechanism of tissue specific gene expression and DNA replication using mouse polyomavirus. We will focus on the issue of cell specific DNA replication and its relationship to viral and cellular cell specific gene control. There are five specific aims as follows: 1. To continue the genetic analysis of tissue specific polyoma replication in vivo. We will construct further mutants to examine how point deletions within two domains of polyoma (Py) regulatory DNA (A and B enhancer) apparently broaden tissue specificity replication. In addition we will use genetic methods to examine the ability of various sub-enhancer multimers to also replicate in broader organ types. The apparent ability of the B enhancer to restrict viral replication in some but not other cell types will also be investigated. We will begin a genetic analysis of enhancer requirements for persistent infections. The in vivo analysis will be expanded to include in situ hybridization of all infected organs in order to establish the exact cell specificity within organs. 2. We will further examine the effect of Adenovirus E1A on Polyomavirus DNA replication. We will determine if E1A is inducing a stable or possibly cell cycle control of polyoma DNA replication in permissive cells. In addition, we will examine the viral and cellular proteins involved in viral DNA replication to determine if they have undergone some detectable modification. We will also attempt to establish if their is an enhancer sequence that is the target of E1a suppression or if this suppression is not via cis acting regulatory DNA. Various other E1A proteins (from murine adenovirus, mutants in conserved regions) will also be examined to determine if they affect the repression of Py DNA replication we see. 3. The relationship of terminal differentiation to amplified Py DNA replication be further studied. Various primary and established cell lines which can be induced to terminally differentiate will be examined for their ability to replicate wt and enhancer mutant polyomavirus and the generality of the relationship of terminal differentiation to DNA formation and episomal persistence in undifferentiated cells. If a general relationship is confirmed (ie all terminal lines are more permissive for Py DNA replication), a genetic analysis will be done to determine if common regulatory sequences are involved and if distinct but common cellular replication or chromatin proteins are also involved. 4. We will do a more systematic genetic analysis of the sequence requirements for cell specific DNA replication relative to transcription. We will examine the spacing and heterodimer issue of transcriptional elements and better define the elements required for if replication relative to transcription in specific cell types. We will also examine if cell type affects the spacing relationship of enhancer to the origin of replication. 5. Finally, we will attempt to establish an in vitro system for the analysis of cell specific polyoma DNA replication. Initially we will conduct footprinting experiments to determine relative quantities and binding affinities of cellular factors which appear to participate in cis regulated initiation of viral DNA replication.
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