Molecular cloning of wild-type and mutant osterix and its post-translational modifications in 293 cells

Abstract

Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2014 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 42-43.Bone is one ofthe most important tissues in the human body, it gives both fom and function and any abnormalities can have significant and detrimental effects. This is why it is important to understand how the body triggers and regulates bone growth, SO that we may understand where these abnomalities might occur, and possibly even treat them. One ofthe most important regulatory controIs for bone fomation is the Osterix/SP7 (Osx) gene, a Zinc finger motif containing transcription factor found in osteoblasts. It has been found that in Osterix null mice, they are limited to creating ca皿aginous tissue but camot create bone. The aims ofthis study are to identify ways in which ce11s post-tranSlationally modfty the Osterix protein to further regulate its activity’ specifica11y by phosphorylation and ubiquitination. Mutant foms ofwild type (WT) Osx were created by PCR that deleted each ofthe three zinc fingers (ZF3, ZF2, and ZFl) were inserted into pcDNA3-FLAG tagged vectors. Another mutant form (K41R) was created replacing the lysine-41 with arginine to prevent ubiquitination from occurring to see if this apparent molecular weight gain could be prevented. Transfection and overexpression ... [TRUNCATED