Thesis (Ph. D.)--University of Washington, 2006. / Includes bibliographical references (leaves 180-197).

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Anatomy and Neurobiology. Bibliography: leaves 151-202. Also available online.

In Cellulomonas fimi the cenB gene encodes a secreted endoglucanase (EngB) involved in the degradation of cellulose. The cenB gene carried on a 5.6 kb C fimi DNA fragment encodes a polypeptide of Mr 110,000 in Escherichia coli. The level of expression of the gene was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. The intact EngB polypeptide is not required for enzymatic activity: active polypeptides of Mr 95,000 and 82,000 also appear in E. coli and a deletion mutant of cenB encodes an active polypeptide of Mr 72,000. The intact and truncated EngB both bind to microcrystalline cellulose. A simple, rapid affinity chromatography procedure on Avicel was developed for the purification of intact EngB and of the 72,000 deletion derivative. Alignment of the amino-terminal amino acid sequence of the purified intact EngB from E. coli with the partial nucleotide sequence of the cloned C. fimi DNA showed that the mature EngB is preceded by a sequence encoding a putative signal polypeptide of 32 amino acids, a translational initiation codon and a sequence resembling an E. coli ribosome binding site 4 nucleotides before the initiation codon. The signal peptide functions and is correctly processed in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of β-galactosidase. The truncation of EngB does not affect its export to the periplasm of E.coli. In the intact EngB, 25% of the residues are hydroxyamino acids. It displays features common to endo-β-1 ,4-glucanases, since it has a high activity on carboxymethylcellulose. The kinetic parameters for carboxymethylcellulose hydrolysis of both intact and truncated EngB are not significantly different. C. fimi protease cleaves intact EngB, in a specific manner, to generate two polypeptides of Mr 65,000 and 43,000; the former has the capacity to bind Avicel. A polyclonal antibody raised against the purified intact EngB recognizes a C. fimi extracellular protein of M 110,000 as well as 5 polypeptides of lower molecular weight. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Leishmania major (L. major) is an intra-macrophage protozoan parasite which differentiates from a promastigote to an amastigote upon transmission from its insect vector at 25°C to its mammalian host at 37°C. This temperature shift occurs in the same range as that used to elicit the heat shock response in prokaryotes and higher eukaryotes in which the induction of genes encoding heat shock proteins is seen. Ubiquitin is a heat inducible protein and one of the most conserved eukaryotic proteins known. Genomic libraries made from major DNA were initially screened with the ubiquitin gene from yeast. DNA sequence analyses of positive clones revealed at least 5 ubiquitin coding elements arranged head to tail without intervening sequences. The predicted protein sequence showed that ubiquitin in Leishmania differs from that of yeast and barley at 5 out of 76 amino acid positions and from that of human at only 2 positions. Further characterization revealed another ubiquitin encoding locus believed to carry only one ubiquitin encoding element. Comparisons of ubiquitin mRNA levels from L. major grown at 26°C, 37°C, and 42°C suggest that ubiquitin gene expression in these particular parasites is constitutive and that prolonged exposure at a non-lethal temperature results in a reduction of ubiquitin-specific mRNA. However, a direct correlation between parasite differentiation and ubiquitin gene expression was not defined as it could not be determined whether the described experimental conditions actually established differentiated states of L. major. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.

Gonadotropin releasing hormone (GnRH) is a key reproductive hormone in vertebrates and exerts its effects via the GnRH receptor (GnRHR) to result in the synthesis and release of the gonadotropin hormones in the pituitary gonadotrope cells. GnRHR expression is likely to be regulated in a tissue- and cell- specific manner. A variety of hormones, including GnRH itself, estrogen, progesterone, inhibin, and testosterone have been shown to regulate GnRHR expression. Steroidogenic Factor-1 (SF-1), a member of the orphan nuclear receptor transcription factor family, regulates the expression of both the gonadotropin hormones in the pituitary and the steroidogenic enzymes in the gonads and adrenal gland, and provides a potential molecular mechanism for coordinate control of reproductive function. SF-1 binds to a gonadotrope-specific element (GSE) in the promoters of the gonadotropin hormones. Our studies involved investigating whether SF- I plays a role in tissue-specific regulation of GnRHR gene expression. A genomic clone of the mouse GnRHR gene contains a putative SF- I site at about -15 relative to the translation start site. We demonstrate the presence of a factor with SF-1-like DNA-binding activity in the gonadotrope cell lines, αT3-1 and αT4, by gel retardation assays. DNasel footprinting reveals that the major DNA-binding activity in αT3-1 cells on the GnRHR promoter occurs at the SF-1-like site. The SF-1-like sequence specificity of the interaction is demonstrated by gel redardation and DNasel footprinting assays using specific and mutated oligonucleotides as competitors. Northern blot analysis suggests that GnRHR expression is not solely dependent on the presence of SF-1, as αT4 cells do not express GnRHR but a SF-1 transcript is seen in these cells. Promoter function was analysed by constructing plasmids containing 563 bp of the GnRHR gene 5' to the ATG translation start site linked to a luciferase reporter gene, followed by transfection of these constructs into different cell lines. In addition, a mutant construct containing a mutated SF-1 site was tested. We demonstrate that this 563 bp of the GnRHR gene contains strong promoter activity in both pituitary gonadotrope (αT3-1) and somatotrope (GH₃) cells, but not in non-pituitary (COS-1) cells. Thus promoter activity appears to be tissue specific but not gonadotrope specific. The presence of a mutated SF-1 site in the 563 bp GnRHR gene fragment did not significantly effect the promoter activity, showing that binding of SF-1 protein to this site is not necessary for high levels of GnRHR expression in the pituitary gonadotropes.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20936号 / 理博第4388号 / 新制||理||1630(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.