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Promoter Deletion Analysis of Xylem Cysteine Protease 2 (XCP2) in Arabidopsis thalianaPetzold, Herman Earl III 01 June 2007 (has links)
The process of xylem tracheary element differentiation involves the coordination of vascular cambium activity, cell fate determination, cell expansion/elongation, secondary wall synthesis, programmed cell death, and cellular autolysis. The end result of tracheary element differentiation is a cellular corpse lacking a protoplast and consisting of a thickened cell wall composed mostly of lignin and cellulose. Little is known about the genetic mechanisms regulating the process of tracheary element differentiation. XCP2 expression localizes to tracheary elements according to two independent methods of analysis: promoter reporter experiments and immunogold localization by electron microscopy. XCP2 may be involved in catalyzing the degeneration of the protoplast during the final autolytic stages of tracheary element differentiation. To this date XCP2 function has not been directly demonstrated. In principle, any tracheary element-specific markers can be linked to upstream regulatory genes with roles in tracheary element differentiation. To develop the XCP2 promoter as a tool for identification of transacting factors, a promoter deletion analysis was carried out. Utilizing information from 5â and 3â deletion constructs, a 70-bp region upstream of the XCP2 translational start site is both necessary and sufficient for TE-specific expression of the UidA reporter gene. Mutational analysis of the ACTTTA element at position -113-bp strongly suggests it is a cis element required for XCP2 expression. In silico analysis of an 18-bp promoter region located within 200-bp of the translation start site and including the ACTTTA element revealed high indentity shared between xylem-specific XCP2 homologs from Zinnia elegans, Populus trichocarpa, and XCP1 from Arabidopsis thaliana. / Master of Science
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Towards Identifying Cis and Trans Regulators of Expression of Xylem Cysteine Protease 1 (XCP1) in ArabidopsisStroud, William Jefferson 04 June 2009 (has links)
Secondary xylem, commonly known as wood, is a valuable commercial commodity. Among the major components of wood are the elongated, thick-walled water-conducting cells known as tracheary elements. Understanding tracheary element differentiation and maturation is of scientific and commercial importance as it may lead to broad understanding of cellular differentiation processes as well as ways to increase both the quality and quantity of wood produced by economically important tree species. One way to begin to understand the regulation of tracheary element differentiation is to identify elements that control expression of genes associated with tracheary elements. In Arabidopsis thaliana, Xylem Cysteine Protease 1 (XCP1) is specifically expressed in tracheary elements where it catalyzes microautolysis. Thus XCP1 can serve as a useful model for identifying factors that regulate tracheary element-specific gene expression. A deletion analysis of the XCP1 promoter was conducted to identify promoter elements that are necessary and sufficient for tracheary element-restricted gene expression. Two regions required for tracheary element-specific gene expression were identified. One of these was assembled as a multimeric bait construct and used in yeast one-hybrid assays to identify candidate transcription factors that bind to the XCP1 promoter region. Subsequently, a southwestern blot analysis was used to identify transcription factors displaying specific binding to a previously reported cis-element, CTTCAAAGCCA, found in the XCP1 promoter and other tracheary element-associated genes from multiple species. / Master of Science
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INVESTIGATING THE MECHANISM OF PROMOTER-SPECIFIC N-TERMINAL MUTANT HUNTINGTIN-MEDIATED TRANSCRIPTIONAL DYSREGULATIONHogel, Matthew 30 August 2011 (has links)
Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.
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