Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 193-228).

No description available.

The replication of Moloney Murine Leukemia Virus (MoMLV or MLV) is restricted in mouse embryonic stem (ES) and embryonic carcinoma (EC) cells, but not in differentiated cells. The restriction is mediated by the primer binding site (PBS) of proviral DNA of MLV. A restriction complex can bind to the PBS of MLV and block the transcription of viral genes. Two major components of the PBS-mediated silencing complex were identified in our lab, ZFP809 and Trim28. ZFP809 contains two conserved domains, a zinc finger domain responsible for DNA binding and a KRAB box recruiting Trim28, and hence other transcription repressors, such as HP1γ and ESET. A protein called L1td1 was identified during the purification of PBS-mediated restriction complex. L1td1 is a stem cell specific protein but little is known about the function of L1td1. In differentiated cells, the replication of MLV is not restricted. Overexpression of ZFP809 in differentiated cells is sufficient to re-establish the PBS mediated restriction. However, data from various expression libraries shows that the mRNA levels of ZFP809 in stem cells and differentiated cells are approximately the same, which indicating that there is some post-transcriptional mechanism negatively regulating the protein levels of ZFP809 in differentiated cells. To study the post-translational regulation of ZFP809 may help us understand how retroviral restriction is regulated in different cell types. Here we found that the down-regulation of ZFP809 proteins is due to the rapid degradation of protein but not on mRNA. The protein of ZFP809 is degraded rapidly in differentiated cells but not in stem cells. The last 50 amino acids, as well as the lysine residue within the peptide, are important for the turnover of ZFP809 protein in differentiated cells. The drug MG132 can stabilize the ZFP809 protein in differentiated and in vivo ubiquitination assay show that ZFP809 is heavily ubiquinated in differentiated cells, suggesting that ZFP809 is degraded through the ubiquitin-dependent proteasomal pathway. Interestingly, the protein Trim28, which is an essential factor for in the silencing complex, can promote the degradation of ZFP809. Mutations with the lysine residue mutated to alanine or abolished the interaction between Trim28 are less ubiquitinated. A small drug, MLN4924, which is the neddylation inhibitor, stabilizes ZFP809 in differentiated cells. Overall, these observations suggest that, during the differentiation of mouse stem cells, ZFP809 protein is eliminated by the proteasomal system, which leads to the loss of restriction of MLV in differentiated cells. In addition, we studied the role of L1td1 in retroviral silencing. Knockdown or knockout of L1td1 partially relieves the restriction of MLV replication. Immunoprecipitation and pulldown assays show that L1td1 might interact with Trim28 and ZFP809 bridging by Trim28. In summary, L1td1 might interact with the essential factors of silencing complex and help the silencing of MLV in stem cells. Proteins of the nucleosome remodeling deacetylase (NuRD) complex were also identified during purification of the restriction. The NuRD complex is shown to be involved in the transcriptional repression. However, depletion of single subunits of the NuRD complex does not affect the PBS-mediated retroviral restriction in mouse EC cells.

No description available.

No description available.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 108-111).

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 66-68).

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 69-70).

This investigation is concerned with cell surface antigens present on murine AKR/J mice spleen and thymus cells which have been extracted with papain. Isolation of individual proteins was accomplished by granulated gel electrofocusing. Similar patterns recorded by both electrofocusing procedures identified several proteins limited to the AKR/J and C3Heb/FeJ spleen and thymus samples, which represent Murine Leukemia Virus-associated surface proteins.

This research project deals with the characterization of self-replicating Kirsten murine viral DNA. The replication of this viral DNA and tetracycline resistance conferred to bacteria by this viral DNA will be studied. The restriction endonuclease and Southern blot analysis revealed a fragment of pBR322 from the Hind III and Pst I site that is located in the 3' end of the MLV-K:E molecule. Single stranded sequencing of the two terminal ends of this fragment verified that the 3' end of MLV-K:E contains identical sequence homology to pBR322. The presence of this pBR322 fragment explains the unusual properties of the MLV-K:E molecule. However, tetracycline resistance is less in E. Coli containing MLV-K:E than E. coli containing pBR322 as determined by zone of inhibition assay. This may be due to alteration in the promoter region of the tetracycline gene.