Browsing by Subject "Histones"
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Item Expanding Genetic Code for Protein Lysine and Phenylalanine Modifications(2012-05-30) Wang, Yane-Shih 1977-The naturally occurring pyrrolysine (Pyl) incorporation machinery was discovered in methanogenic archaea and some bacteria. In these organisms, Pyl is cotranslationally inserted into proteins and coded by an in-frame UAG codon. Suppression of this UAG codon is mediated by a suppressor tRNA, (tRNA_CUA)^Pyl , that has a CUA anticodon and is acylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). The PylRS-(tRNA_CUA)^Pyl pair can be directly applied to incorporate Pyl and other lysine derivatives into proteins at amber mutation sites in E. coli and mammalian cells. In the approach of amber codon suppression, evolved PylRSs were selected to synthesize the proteins genetically with lysine and phenylalanine derivatives which contain native or mimic of post-translational modifications (PTMs) or active chemical functional groups for protein labelling and protein folding studies. A photocaged N^6-methyl-L-lysine has been genetically incorporated into proteins at amber codons in Escherichia coli using an evovled PylRS-(tRNA_CUA)^Pyl pair. Its genetic incorporation and following photolysis to recover N?-methyl-L-lysine at phsyiological pH provide a convenient method for the biosythesis of proteins with monomethylated lysines. Using an evolved PylRS-(tRNA_CUA)^Pyl pair, a Se-alkylselenocysteine was genetically incorporated in histone H3. The H3 with mimics of PTMs such as lysine methylation, lysine acetylation, and serine phosphorylation has been synthesized by selective oxidative elimination of Se-alkylselenocysteine and followed Michael addition reactions with different thiol-containing small molecules. Using evolved PylRS -(tRNA_CUA)^Pyl pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli. The drastic change of the substrate specificity of PylRS from an aliphatic amino acid to short aromatic amino acids indicates that the PylRS-(tRNA_CUA)^Pyl pair can be evolved for genetic incorporation of a large variety of NAAs into proteins in E. coli. Inspired by the consistent mutations on N346 position, the mutants on N346 and C348 were constructed and evaluated with different L-phenylalanine derivatives. Using PylRS - N346A/C348A (tRNA_CUA)^Pyl pair, more than 30 L-phenylalanine derivatives have been genetically incorporated into proteins at defined sites with amber mutation in E. coli. These breakthroughs and development greatly expand the inventory of genetically encoded NAAs and our abilities to do protein engineering in these cells.Item Isolation and characterization of DNA sequences bound by a class of nonhistone proteins(Texas Tech University, 1979-08) Jagodzinski, Linda L.All somatic cells of the same organism contain the same complement of genes. During cellular differentiation transcriptional specialization occurs. This process allows the selected expression of genetic information in specialized cells; e.g., only red blood cell precursors synthesize hemoglobin, only hepatocytes synthesize phenylalanine hydroxylase and serum albumin (159), and only estrogen induced oviduct cells synthesize ovalbumin. During differentiation certain genes function only at specific times and in particular tissues. Hence, portions of the eukaryotic genome must be prevented from expressing, in some manner, their genetic information. Evidence indicates that the chromosomal proteins participate in the regulation of gene activity. How this is accomplished and which components are involved are questions which are now being investigated.Item The Role of Class I Histone Deacetylases in Cardiovascular Development and Disease(2008-05-13) Montgomery, Rusty Lee; Olson, EricHistone acetylation/deacetylation is a dynamic process that coordinates proper gene expression through the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibitors continue to show promise in a multitude of pathological settings such as cancer and heart disease, however the role of individual HDACs remains largely unexplored. Genetic studies have shown class II HDACs regulate developmental and physiological processes through the interaction with and repression of myocyte enhancer factor 2, MEF2, however the biological functions of class I HDACs have not been determined. To potentially understand the role of individual class I HDACs in development and disease, we generated conditional knockout alleles for HDAC1, HDAC2, and HDAC3. Through global and tissue specific analyses, we hope to identify specific roles of these enzymes in developmental, physiological, and pathological settings. Here I show that HDAC1 and HDAC2 act redundantly in controlling cardiac growth, morphogenesis, and contractility. Mice with cardiac-specific deletion of either HDAC1 or HDAC2 are viable and lack obvious phenotypes, however cardiac-specific deletion of both HDAC1 and HDAC2 results in lethality by two weeks of age. These mice show cardiac arrhythmias, dilated cardiomyopathy, and increased expression of calcium channels and skeletal muscle-specific contractile proteins. HDAC3 is an independent regulator of cardiac development. Global deletion of HDAC3 results in embryonic lethality, whereas cardiac-specific deletion of HDAC3 results in massive cardiac hypertrophy by 3 months of age and lethality by 16 weeks. These mice show metabolic abnormalities including up-regulation of genes involved in fatty acid uptake and oxidation, down-regulation of the glucose utilization pathway, and ligand induced myocardial lipid accumulation. Additionally, these hearts show mitochondrial dysfunction and decreased cardiac efficiency. These studies have identified HDAC3 as a central regulator of myocardial energy metabolism. In addition to cardiac studies, tissue-specific deletions in multiple cell-types have led to the discovery that functional redundancy of HDAC1 and HDAC2 is not restricted to postnatal cardiomyocytes, but extends to early cardiomyocytes, endothelial cells, smooth muscle cells, chondrocytes, and neurons. Deletion of HDAC1 or HDAC2 individually in these cell types does not evoke a phenotype, however deletion of both HDAC1 and HDAC2 results in embryonic lethality or neonatal lethality. Taken together, these studies identify HDAC1 and HDAC2 as redundant regulators of multiple cell types during development. Collectively, these studies have identified distinct and specific roles for HDAC1, HDAC2, and HDAC3 during development and disease. Furthermore, these genetic studies have provided mechanistic insight into the pathways regulated by each enzyme. Additional analyses on these mice will prove instrumental to the development of more specific inhibitors for the treatment of a wide array of pathological conditions.