Thesis (M.S.)--Marshall University, 2004. / Title from document title page. Document formatted into pages; contains vii, 123 p. including illustrations. Includes abstract. Includes bibliographical references (p. 122-123).

A new approach in mapping restriction fragments by means of primed extension was proposed but was found to be unfeasible after studying the extent of T4 polymerase mediated DNA synthesis. The maximum length of DNA replication mediated by T4 polymerase was studied using ØX-174 DNA as template primed by a restriction fragment of the same DNA. Both nucleotide incorporation kinetics and alkaline gel electrophoresis were used to study the products of DNA synthesis. Although the incorporation kinetics suggested that the primer was extended by approximately 100 nucleotides, the electrophoretic mobilities of the products suggested much less extension. The effect of T4 gene 32 protein (unwinding protein) was also studied. This protein was purified by DNA cellulose chromatography to near homogeneity and was shown to be nuclease free. The purified protein stimulated nucleotide incorporation three-fold when added to the usual T4 polymerase reaction mixture. Contrary to the kinetic results, however, the gel mobilities of the products again showed only limited extension of the primer. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Experiments with ØW-14-infected, thymidine-requiring mutants of P_. acidovorans strain 29 demonstrated that deoxyuridine but not thymidine was a precursor of thymine in ØW-14 DNA. Deoxyuridine was also a precursor of the a-putrescinylthymine found in ØW-14 DNA. The biosynthesis of a-putrescinylthymine and thymine was mediated by enzyme activities appearing after infection. ØW-14 DNA synthesis and DNA modification was resistant to the antibiotics trimethoprim and 5-fluorodeoxyuridine. This indicated that endogenous thymidine biosynthesis was unlike that observed in the uninfected host or in other biological systems. These observations helped demonstrate that hydroxy-methyluracil-containing nucleotides were precursors of thymine and a-putrescinylthymine-containing nucleotides (Neuhard et al., 1980). The absence of a-putrescinyl thymine and thymine nucleotides in 0W-14-infected cell nucleotide pools suggested that these nucleotides might be synthesized from hydroxymethyluracil at the polynucleotide level. Degradative analysis of nascent ØW-14 DNA demonstrated the presence of hydroxymethyluracil. Enzymatic degradation of pulse-labelled, nascent 0W-14 DNA followed by TLC suggested the presence of three or more novel nucleotides not found in uniformly labelled DNA samples. These observations were consistent with neutral CsCl analysis of pulse-labelled ØW-14 DNA. This DNA contained unusual heavy density components. ØW-14 ts and amber mutants were screened for defects in DNA replication or DNA modification by CsCl gradient and/or degradative analysis. Some DO mutants were identified. In addition, two DNA modification mutants were found. Am 42 made ØW-14 DNA containing lower-than-normal levels of a-putrescinylthymine and increased levels of thymine. Am 37 accumulated intermediates in a-putrescinylthymine biosynthesis. The conditionally lethal nature of the DNA modification lesion was demonstrated. DNA synthesis was adversely affected by this mutation but DNA precursor supplies were not impaired. Two atypical mononucleotides were purified from am 37 DNA. One was identified as hydroxymethyldeoxyuridylate. The second nucleotide was an acid-labile derivative of hydroxymethyldeoxyuridylate. Analysis of [6- ³H]-uracil and ³²PO₄ labelling ratios, chemical and enzymatic degradation and chromatographic analysis of this nucleotide demonstrated that it was the novel compound 5-(hydroxymethyl-0-pyro-phosphoryl)-deoxyuridylate (abbreviated to hmPPdUMP). 5-(hydroxymethyl-O-pyrophosphoryl)-uracil was shown to be a precursor of a-putrescinylthymine by in vitro modification of am 37 DNA with ØW-14 wild-type infected P. acidovorans cell-free extracts. In vitro modification confirmed that a-putrescinylthymine was formed at the polynucleotide level. ØW-14 DNA modification was not necessarily coupled to replication. The presence of hydroxymethyluracil in am 37 DNA agreed with the suggestion that hmPPura was formed by pyrophos-phorylation of hydroxymethyluracil in nascent DNA. HmPPdUMP had chromatographic properties similar to one of the compounds detected in pulse-labelled ØW-14 wild-type DNA. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, December 2002. / Includes bibliographical references. Also available on the Internet.

Aluminium is an element of increasing clinical importance. It not only has uses as a medicinal substance but also in recent years it has been shown to be the cause of considerable toxicity, particularly in the setting of chronic renal failure. Diseases that have been shown to be associated with aluminium, or in which it has been implicated, include dialysis dementia, renal osteodystrophy and Alzheimer's disease. This thesis has studied aspects of the interaction between aluminium and the kidney. The work has addressed two major issues. Firstly, a study is described where Malvin's stop-flow technique was used to determine any excretory/absorptive tubular site for Al in the pig kidney. Al was found to be excreted in the distal nephron of the pig kidney. Secondly, the toxic effects of Al in vitro on the DNA of pig kidney cell line LLC-PKl were investigated, in an attempt to elucidate some of the mechanisms of toxic action. DNA synthesis was measured using ³H-TdR incorporation. Over increases of both time (9-72 h) and Al concentration (0.01-8.0 mM), ³H-TdR incorporation was diminished. Effects were evident at concentrations as low as 0.05 mM Al. The production of DNA strand breaks was assessed by the increase in size of cell nucleoids (ie DNA in supercoiled form). Nucleoid size was analyzed in a Epics 753 Fluorescence Activated Cell Sorter interfaced with an MDADSII data acquisition and analysis system. After 90 min incubation with Al (over the concentration range 0.001-32 mM), an increase in nucleoid size was noted at concentrations above 0.05 mM. The data demonstrate that Al exerts an effect on kidney cells in vitro which is expressed as diminished DNA synthesis and production of DNA strand breaks. These effects on DNA may have important long-term implications on various disease states associated with Al toxicity.

The in vivo synthesis of deoxyribonucleic acid from labeled precursors was studied in the rat intestinal mucosa in an attempt to elucidate the complex process of DNA replication. In one set of experiments, the rats were injected with ³H-thymidine and then starved for 24 hours, in which time the stable DNA became labelled with tritium. (14)C-thymidine was then administered and the animals were sacrificed 5 minutes later. By this procedure the newly synthesized DNA was labelled with (14)C. The DNA, was fractionated by chromatography on a methylated-albumin kieselguhr column. Only one main peak of DNA was eluted with a sodium chloride solution ranging in concentration from 0.5-0.6 M. The thermal denaturation temperature for the DNA in each.fraction from this peak was determined and the G + C content was calculated:, Within the DNA peak obtained from MAK chromatography, the G + C content of the DNA decreased with increasing fraction number. In addition to these differences in base composition, there were differences in metabolic activity between the fractions, which were indicated by their ³H/ (14)C ratios. The ³H/ (14)C ratio of the DNA fractions from MAK chromatography increased with fraction number to a maximum at fraction 4 or 5 and then decreased. It was found that the ³H/O.D. ratio of the fractions was not constant, thus suggesting that the tritium might be unevenly distributed throughout the fractions. If the time interval between the ³H and (14)C-thymidine injections was reduced to 3 ½ hours, the ³H/O.D. ratio became constant while the pattern of the ³H/14C ratios remained unchanged. If (14)C-thymidine was administered 20 minutes before the animals were sacrificed, the ³H/(14)C ratio of the DNA fractions from MAK chromatography increased with increasing fraction number. From these results it was concluded that small molecular weight, newly synthesized DNA, which was highly labelled with (14)C, was being incorporated with time into the high molecular weight, stable DNA fraction, which is labelled with ³H. During these experiments it was observed that the pattern of ³H/(14)C ratio versus fraction number varied according to the treatment given to the DNA sample prior to the preparation for radioactive counting. If the sample was denatured by heating to obtain its T(M) value, and then dialyzed against distilled water, small molecular weight nucleotides passed into the dialysate. The denatured DNA sample also gave different results from the native DNA sample on digestion with snake venom phosphodiesterase. On the denatured sample, the pattern of release of ³H and (14)C labelled material into the acid soluble material, indicated that both these labels were uniformly distributed along the DNA chain. On the other hand, with the native 5 min. DNA samples, the release of (14)C labelled material into the acid soluble fraction was that expected for DNA which had incorporated (14)C-preferentially into the 3’ terminal positions. The separation of the pyrimidine clusters of DNA indicated that those were not uniformly labelled with (14)C and ³H. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound. In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes. An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness. Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.

Lau Lai Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-141). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Research initiative and significance --- p.1 / Chapter 1.2 --- Terminal differentiation --- p.4 / Chapter 1.3 --- Controversial terminal differentiation in cardiomyocytes --- p.5 / Chapter 1.4 --- Molecular switch from hyperplasia to hypertrophy in neonatal myocardial development --- p.7 / Chapter 1.5 --- Interferons --- p.8 / Chapter 1.6 --- Functions induced by interferons --- p.9 / Chapter 1.7 --- Interferons in cardiomyocytes --- p.12 / Chapter 1.8 --- Interferon-inducible transmembrane gene family --- p.13 / Chapter 1.9 --- Our hypothesis and objective --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Sequence analysis --- p.18 / Chapter 2.2 --- Cell culture --- p.18 / Chapter 2.3 --- Induction of differentiation of H9C2 cells --- p.19 / Chapter 2.4 --- In vitro induction of IFITMs by interferon treatments --- p.19 / Chapter 2.5 --- RNA isolation --- p.20 / Chapter 2.5.1 --- Experimental animals and sampling --- p.20 / Chapter 2.5.2 --- Total RNA Isolation --- p.20 / Chapter 2.5.3 --- RNA Quantification and Quality Check --- p.21 / Chapter 2.5.4 --- Purification by Qiagen-RNeasy Column and DNase I Digestion --- p.21 / Chapter 2.6 --- First-strand cDNA synthesis --- p.22 / Chapter 2.7 --- Quantitative real-time polymerase chain reaction --- p.22 / Chapter 2.8 --- Cloning protocol --- p.25 / Chapter 2.8.1 --- "Construction of pEGFP-IFITMl, pEGFP-IFITM2 and pEGFP-IFITM3 fusion proteins" --- p.25 / Chapter 2.8.1.1 --- Amplification of DNA fragments --- p.25 / Chapter 2.8.1.2 --- Purification of PCR product --- p.26 / Chapter 2.8.1.3 --- Restriction endonuclease digestion --- p.26 / Chapter 2.8.1.4 --- Insert/vector ligation --- p.27 / Chapter 2.8.1.5 --- Preparation of chemically competent bacterial cells --- p.27 / Chapter 2.8.1.6 --- Transformation of ligation product into chemically competent bacterial cells DH5a --- p.28 / Chapter 2.8.1.7 --- Recombinant clone screening by PCR --- p.29 / Chapter 2.8.1.8 --- Small-scale preparation of recombinant plasmid DNA --- p.29 / Chapter 2.8.1.9 --- Dideoxy DNA sequencing --- p.30 / Chapter 2.8.1.10 --- Large-scale preparation of recombinant plasmid DNA --- p.30 / Chapter 2.8.2 --- "Construction of IFITMl-pcDNA4, IFITM2-pcDNA4 and IFITM3- pcDNA4 constructs" --- p.33 / Chapter 2.8.2.1 --- Amplification of DNA fragments --- p.33 / Chapter 2.8.2.2 --- Insert/vector ligation --- p.33 / Chapter 2.8.2.3 --- Transformation of ligation product into one shot® TOP1 OF´ة chemically competent E. coli cells --- p.34 / Chapter 2.9 --- Transient transfection --- p.36 / Chapter 2.10 --- Subcellular fractionation --- p.37 / Chapter 2.11 --- Isolation of total protein cell lysate --- p.38 / Chapter 2.12 --- Protein concentration determination --- p.38 / Chapter 2.13 --- Protein gel electrophoresis and western blotting --- p.39 / Chapter 2.13.1 --- Preparation of SDS-polyacrylamide gel --- p.39 / Chapter 2.13.2 --- Preparation of protein samples --- p.39 / Chapter 2.13.3 --- SDS-polyacrylamide gel electrophoresis --- p.40 / Chapter 2.13.4 --- Protein transfer to nylon membrane --- p.40 / Chapter 2.13.5 --- Antibodies and detection --- p.40 / Chapter 2.13.6 --- Stripping membrane --- p.41 / Chapter 2.14 --- Bromodeoxyuridine proliferation assay --- p.42 / Chapter 2.14.1 --- Bromodeoxyuridine labeling and detection --- p.42 / Chapter 2.14.2 --- Cell number determination --- p.42 / Chapter 2.15 --- Fluorescence microscopy --- p.43 / Chapter 2.16 --- Confocal microscopy --- p.43 / Chapter 2.17 --- Statistical analysis --- p.44 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequence analysis --- p.45 / Chapter 3.1.1 --- Primary structure analysis --- p.45 / Chapter 3.1.2 --- Transmembrane he lice prediction --- p.46 / Chapter 3.1.3 --- Conserved domain prediction --- p.51 / Chapter 3.1.4 --- Sequence alignments across different species --- p.52 / Chapter 3.2 --- Differential expression during rat myocardial development --- p.53 / Chapter 3.3 --- Altered mRNA levels during differentiation of H9C2 cells --- p.55 / Chapter 3.4 --- "Cloning of IFITMl, IFITM2 and IFITM3" --- p.60 / Chapter 3.5 --- Subcellular localization --- p.61 / Chapter 3.5.1 --- Fluorescence microscopy --- p.61 / Chapter 3.5.2 --- Subcellular fractionation --- p.70 / Chapter 3.6 --- "In vitro induction by interferons-α, β and γ" --- p.72 / Chapter 3.7 --- "DNA synthesis after in vitro induction of interferons-α, β and γ" --- p.79 / Chapter 3.8 --- "Proliferating cell nuclear antigen expression after in vitro induction of interferons-α, β and γ" --- p.87 / Chapter 3.9 --- "DNA synthesis after overexpression of IFITM1, IFITM2 and IFITM3" --- p.93 / Chapter 3.10 --- "Proliferating cell nuclear antigen expression after overexpression of IFITM1, IFITM2 and IFITM3" --- p.95 / Chapter 3.11 --- "β-catenin and cyclin D1 expression after in vitro induction of interferons-α, β and γ" --- p.97 / Chapter 3.12 --- "β-catenin and cyclin D1 expression after overexpression of IFITMl, IFITM2 and IFITM3" --- p.101 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- "Upregulation of IlFITMl, IFITM2 and IFITM3 during myocardial development" --- p.103 / Chapter 4.2 --- "Subcellular localization of IFITMl, IFITM2 and IFITM3" --- p.105 / Chapter 4.3 --- "Induction by interferons-α, β and γ" --- p.107 / Chapter 4.4 --- Inhibition of DNA synthesis by interferons-α and β and IFITM1 --- p.109 / Chapter 4.5 --- Involvement of IFITM family in canonical Wnt pathway --- p.112 / Chapter 4.6 --- Other possible pathways involved --- p.117 / Chapter CHAPTER 5 --- FUTURE PROSPECTS / Chapter 5.1 --- Production of antibodies --- p.118 / Chapter 5.2 --- Silencing or knockout approach --- p.118 / Chapter 5.3 --- Target genes of Wnt/β-catenin signaling --- p.119 / Chapter 5.4 --- Other signaling pathways involved --- p.119 / Chapter 5.5 --- Use of primary cardiomyocytes --- p.120 / APPENDIX --- p.121 / REFERENCES --- p.124