Cell Biology & Molecular Genetics Theses and Dissertations
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Item DIFFERENTIAL ABILITIES OF THE CHICKEN PIT1 ISOFORMS TO REGULATE THE CHICKEN GROWTH HORMONE PROMOTER(2011) Mukherjee, Malini; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pit1, a pituitary-specific transcription factor, regulates differentiation of cells of the PIT1 lineage in the anterior pituitary. PIT1 also regulates the synthesis of peptide hormones from these cell types, including growth hormone (GH). A founding member of the POU-homeodomain family of transcription factors, PIT1 is characterized by a serine-threonine rich N-terminal transactivation domain and a C-terminal POU-domain. Alternative forms of PIT1, differing from each other in the N-terminal domain have been reported in several species, but the functional implication of having multiple isoforms is not known. Several Pit1 isoform mRNAs exist in chickens which have not been characterized. The main aim of this study was to determine which, if any, of the chicken PIT1 isoforms regulated the chicken Gh (cGh) promoter. PIT1β2, a novel isoform of chicken PIT1 was discovered, and known and novel isoforms (PIT1α, PIT1β1, PIT1β2 and PIT1γ) were characterized. A luciferase reporter construct containing 1775bp of the cGh promoter driving expression of firefly luciferase was used to determine the ability of the isoforms to regulate the target gene promoter activity in chicken LMH cells. We showed that three of the isoforms, PIT1α, PIT1β1 and PIT1β2, expressed from recombinant plasmids, regulated the cGh promoter, while PIT1γ did not. All the isoforms localized to the nucleus in both non-pituitary and pituitary cells. Results from gel-shift assays show that PIT1γ did not bind the proximal PIT1-binding site of the cGh promoter as well as the other isoforms, suggesting a possible mechanism behind the inactivity. Our result did not suggest a negative regulatory role for this isoform. In contrast, we found a functional advantage for having multiple isoforms. PIT1β1, the isoform that activated the promoter most strongly, when co-transfected with other activating isoforms, such as PIT1α and PIT1β2, induced significantly higher level of activation than one isoform alone. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Nevertheless, identification of isoforms with specific functions will facilitate identification of their respective interacting partners, which are essential for GH gene expression.Item Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor(2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.