Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Allen M. Orville, Committee Member ; Sheldon W. May, Committee Member ; Jung H. Choi, Committee Member ; Mostafa A. El-Sayed, Committee Member ; Donald F. Doyle, Committee Chair.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The gene for bovine prothrombin is 15.6 Kbp in length which encodes a mRNA of 2025 nucleotides plus a poly(A) tail. The prothrombin gene is composed of 14 exons separated by 13 introns, all of which vary in size. The positions of the introns found within the prothrombin gene provides some insight into the evolution of prothrombin and provide evidence on the origin of introns. Within the activation peptide and leader sequence of precursor prothrombin, some of the introns appear to separate structural and functional protein domains. Introns are found to separate certain domains, including the pre-peptide, the propeptide and Gla region, and each of the kringles. This organization of exons may reflect the evolution of the prothrombin gene as the result of the fusion of exon(s) containing protein domains by exon shuffling. The activation peptide appears to be constructed from four domains: a pre-peptide, a pro-peptide and Gla region, and two kringles. On comparison of the exon organization of the serine protease domain of prothrombin and other serine protease genes, it was found that none of the introns of the prothrombin gene are shared with any of the other serine protease genes. This absence of shared introns is in contrast to the shared introns found for the shared domains of the activation peptide and leader. The positions of the introns of the serine protease domain of serine proteases genes does not appear to reflect the evolution of the serine protease from protein domains, but rather the result of intron insertion into the serine protease coding regions. Intron insertion would also explain the origin of the few introns of the activation peptide that do not appear to separate protein domains. In conclusion, the organization of the exons and introns of the gene for prothrombin reflect both the origin of introns by insertion events, and the use of introns in exon shuffling. The insertion of introns, and the subsequent possibility of exon shuffling appear to have been essential for the evolution of the multidomainal proteins, such as prothrombin, which are essential for vertebrate life. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The LexA repressor of Escherichia coli represses a set of genes that are expressed in response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires RecA protein. Under physiological conditions in vitro, RecA-dependent cleavage also occurs. At alkaline pH, however, the specific cleavage reaction occurs spontaneously without RecA, a reaction which is termed autodigestion. The LexA repressor is, therefore, thought to cleave itself with RecA acting to stimulate autodigestion. A set of lexA (Ind⁻) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function were isolated. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction. Many of the mutant proteins were purified in order to further characterize their properties in both autodigestion and RecA-mediated cleavage. All of these mutant proteins are found to be deficient in both cleavage reactions. A mutant protein, replacing Lys-156 to Arg, requires a higher pH condition than the wild-type protein does for both cleavage reactions. The results suggest that deprotonation of Arg-156, and by inference Lys-156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and that in the latter reaction RecA acts to reduce the pKa of Lys-156, allowing efficient cleavage of wild-type repressor under physiological conditions. Finally, several mutant proteins affecting amino acids around the cleavage site and the proposed nucleophile in the cleavage reaction (Ser-119) could not efficiently act as a competitive inhibitor in the RecA-mediated cleavage of wild-type repressor, presumably because they affect RecA binding.

The LexA protein of E. coli is a repressor of at least 20 genes in the SOS regulon, and by this function plays a major role in regulating the SOS response. Two different genetic approaches have been taken to define the DNA binding domain of LexA repressor. First, several mutant repressors which are defective in DNA binding have been isolated. The mutations generating these repressors were dominant to lexA+, indicating that the mutant proteins can act in trans to interfere with binding of normal repressor to an operator sequence. The repressors may be defective due to elimination or disruption of contacts made between side chain(s) within the protein and the DNA helix but dominant because they can still interact with other monomers of LexA protein. In a second experiment to define the DNA binding domain of LexA protein, a novel genetic selection has been used to isolate DNA binding specificity mutants. The recA operator (CTG TATGA.GCATA CAG), a known lexA binding site, has been altered in a symmetric fashion. This choice was based on the assumption that the dyad symmetry of the operator indicates at least two repressor monomers bind to each operator such that each monomer recognizes one half of the operator. A class of mutant repressors which restored binding to this altered operator but had little affinity for the wild-type recA operator was isolated. This type of mutation allowed the identification of amino acids in the repressor which are likely to make specific contacts with base-pair(s) in the DNA binding site. By examining the effects of a series of amino acid substitutions on repressor specificity, it was possible to show that a glutamic acid residue at position 45 (E45) contacts the first and last base-pair of the consensus recA operator (CTG TATGA.GCATA CAG). Both negative dominant and operator recognition mutations were located in a small region that was previously identified to specify a helix-turn-helix motif based on sequence similarity to other repressors. These studies therefore suggest that LexA protein may bind to DNA by a helix-turn-helix motif similar to these repressors.

Starch gel electrophoresis was used to compare 14 proteins encoded by 15 loci for seven species of the family Icteridae. A close genetic relationship among these species was classified into three groups. The Agelaiine group contained Agelaius phoeniceus, Sturnella magna, and S. neglecta. The Quiscaline group contained Euphagus cyanocephalus, Cassidix mexicanus, and Quiscalus quiscula. Molothrus ater, the most divergent, was placed in a separate group. Divergence times for the seven species were compared to the literature. Heterozygosity of the seven populations of the two species of Sturnella were compared to determine factors influencing their divergence. Two factors proposed were heterosis in S. neglecta and possible hybridization between S. neglect and S. magna.

Thesis (M.S.)--Florida State University, 2003. / Advisor: Dr. Thomas E. Joiner, Florida State University, College of Arts and Sciences, Dept. of Psychology. Title and description from dissertation home page (viewed Mar. 02, 2003). Includes bibliographical references.