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Studies on the sequence-selective nuclease, bovine pancreatic DNase IDoherty, Aidan Joseph January 1992 (has links)
No description available.
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The mechanism of interaction of the linker histone with DNA and nucleosomesEllen, Thomas Patrick 27 June 2003 (has links)
This dissertation examines the interaction of the linker histone with DNA and with
nucleosomes. The first goal of the project was to characterize the interaction of the
linker histone with DNA. Three factors previously reported to influence the linker
histone's interaction with DNA were examined: ratio of linker histone to DNA sites
of binding, monovalent ions in the local environment, and conformation of the DNA
molecules. Evidence obtained through gel mobility shift assays demonstrates the
strong preference by the linker histone for DNA with superhelical torsion, i.e.,
supercoiling, and the negative cooperative mode of binding that the linker histone
exhibits in association with supercoiled DNA.
The second part of the dissertation examines the location of linker histone binding
on the nucleosome, and documents the pronounced tendency of the linker histone to
bind to two DNA duplex strands. A preparation of homogeneous nucleosome core
particles, consisting of a defined 238 base pair DNA fragment and the core histone
octamer positioned precisely on this DNA, was used as a substrate for the UV-induced
crosslinking of the linker histone to the DNA of this nucleosome. By site-specific
labeling of a single site on the DNA of the nucleosome, the linker histone was
observed crosslinked at that labeled site, confirming that the linker histone binds at the
pseudo-dyad axis of the nucleosome. This evidence was used to support a model of
linker histone binding to the nucleosome that invokes the association of the linker
histone with no fewer than two duplex strands of DNA of the nucleosome. / Graduation date: 2004
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Binding and assembly of H5 (and the globular domain of H5) onto DNACarter, George John 07 January 1998 (has links)
In order to better characterize linker histone interactions with DNA, avian erythrocyte-specific linker H5 and the trypsin-resistant globular domain of H5 (GH5) were used in DNA binding studies. To begin, H5 displayed a considerably higher binding
affinity for DNA than the isolated globular domain (GH5), supporting the importance of the terminal tail domains in binding. This conclusion is based upon binding curves conducted in low-salt solution, and on the considerably-higher salt concentration required
to prevent protein-DNA contact. Linker histones also induce DNA-protein aggregation in a process that was found to result in protein insolubility in 2% SDS, and included protein-protein interactions that did not require the terminal tail domains. In addition, DNA supercoiling appeared to impede the aggregation process; this that may be attributable to binding of linker histones in isolated clusters, as gauged by a limit in the number of observed dithiobis (succinimidyl) propionate (DSP)-crosslinkable contacts. In a related study, the property of GH5 to bind, then organize onto DNA was investigated. GH5 crosslinked onto DNA with dithiobis (succinimidyl propionate), then cleaved with chymotrypsin, displayed highly uniform contacts that appeared to involve the C-terminal four amino acids, and suggests protein-protein interactions are important for binding. This finding may be relevant since GH5 (and H5) were observed to self-associate free in solution in an arguably specific manner. Finally, the exposure of Phe 93 to chymotrypsin was used to identify the surface of the globular domain that contacts DNA for the binding
of intact H5. Results suggests that the side of the protein opposite to the recognition helix preferentially binds to DNA, supporting a novel winged-helix protein DNA-binding mechanism.
Furthermore, parallel studies with octamers reconstituted onto a DNA fragment with twelve copies of the 208 b.p. rDNA 5s gene from Lytechinus variegatus, shows that H5 had a high binding affinity with all detectable protein binding to the reconstituted complex. H5 binding conferred protection to a site located near the dyad axis from endonuclease digestion, supporting the contention that H5 binds near or at the nucleosome dyad axis. H5 binding also was observed to condense fibers as observed from agarose gel electrophoresis, although velocity analytical sedimentation studies indicate that H5 in itself was not sufficient to fully compact chromatin fibers; rather H5 and 30 mM NaCl, in combination, were required. Results indicate that the chromatin-reconstituted "208-12 DNA" makes an excellent model for analyzing the effect of linker proteins on chromatin morphology. / Graduation date: 1998
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Protein-DNA interactions molecular modeling and energetics /Plaxco, Kevin W., Goddard, William A., January 1994 (has links)
Thesis (Ph. D.)--California Institute of Technology, 1994. UM #94-06,216. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.
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The topology and geometry of DNA and DNA-protein interactions /Buck, Dorothy E. January 2001 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 110-115). Available also in a digital version from Dissertation Abstracts.
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Interactions of the parA protein in the partition of DNA during bacterial cell division /Zong, Qiuling. January 1997 (has links)
Thesis (M.A.)--Central Connecticut State University, 1997. / Thesis advisor: Kathy A. Martin-Troy, Ph. D. "... in partial fulfillment of the requirements for the degree of Master of Arts in Biology." Includes bibliographical references (leaves 23-24).
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DNA sequence selectivity and kinetic properties of de novo designed metalloprotein dimersWong-Deyrup, Siu Wah. January 2007 (has links)
Thesis (Ph. D.)--University of Iowa, 2007. / Supervisor: Sonya J. Franklin Includes bibliographical references (leaves 161-168).
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DNase I : wild type and mutants studied with a novel fluorescence based assayShipstone, Emma Jane January 1998 (has links)
No description available.
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Identification of a hNP220 splice variant (hNP220e) and its protein-protein interaction with MAPRE1. / Identifications of a hNP220 splice variant (hNP220e) and its protein-protein interaction with MAPRE1January 2003 (has links)
Chan chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-95). / Abstracts in English and Chinese. / Dedication --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Abbreviations --- p.vi / List of Figures --- p.ix / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1. --- Thesis synopsis --- p.1 / Chapter 1.2. --- hNP220 protein --- p.1 / Chapter 1.2.1. --- Domain organization --- p.1 / Chapter 1.2.2. --- Known splice variants --- p.5 / Chapter 1.2.3. --- Subcellular localization --- p.7 / Chapter 1.2.4. --- Proposed roles in transcriptional activation and RNA processing --- p.7 / Chapter 1.2.5. --- Interaction between C-terminal of hNP220 and FHL2 --- p.9 / Chapter 1.3. --- Hypothesis --- p.12 / Chapter 1.4. --- Principles of key methods --- p.14 / Chapter 1.4.1. --- RLM-RACE --- p.14 / Chapter 1.4.2. --- CytoTrap® two-hybrid system --- p.15 / Chapter CHAPTER 2 --- Materials and Methods --- p.18 / Chapter 2.1. --- Cloning protocol --- p.18 / Chapter 2.1.1. --- Amplification of DNA fragment --- p.18 / Chapter 2.1.2. --- Purification of PCR product --- p.19 / Chapter 2.1.3. --- Restriction endonuclease digestion --- p.20 / Chapter 2.1.4. --- Dephosphorylation of cloning vector 5'-termini --- p.20 / Chapter 2.1.5. --- Insert/vector ligation --- p.20 / Chapter 2.1.6. --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.21 / Chapter 2.1.7. --- Transformation of ligation product into chemically competent bacterial cells --- p.22 / Chapter 2.1.8. --- Small-scale preparation of bacterial plasmid DNA --- p.22 / Chapter 2.1.9. --- Screening for recombinant clone --- p.24 / Chapter 2.1.10. --- Dideoxy DNA sequencing --- p.24 / Chapter 2.1.11. --- Midi-scale preparation of recombinant plasmid DNA --- p.25 / Chapter 2.2. --- Determination of the transcription start site (TSS) of hNP220 gene --- p.27 / Chapter 2.2.1. --- RNA ligase-mediated rapid amplification of cDNA 5'-end (5-RLM-RACE) --- p.27 / Chapter 2.3. --- Isolation and identification of the third splice variant of HNP220 (hNP220ε) --- p.29 / Chapter 2.3.1. --- PCR from human heart/testis cDNAs pool --- p.29 / Chapter 2.3.2. --- RT-PCR --- p.29 / Chapter 2.3.3. --- Northern hybridization --- p.30 / Chapter 2.4. --- Human tissue distribution of hNP220 --- p.31 / Chapter 2.4.1. --- RT-PCR --- p.31 / Chapter 2.4.2. --- Northern hybridization --- p.31 / Chapter 2.5. --- Visualization of the subcellular localization patterns of GFP-tagged hNP220ε in HepG2 cell line --- p.32 / Chapter 2.5.1. --- Cloning of hNP220a and hNP220s into vector pEGFP-Cl --- p.32 / Chapter 2.5.2. --- Transfection of GFP fusion constructs into HepG2 cell line --- p.32 / Chapter 2.5.3. --- Epi-fluorescence microscopy --- p.33 / Chapter 2.6. --- Identification of the protein-protein interaction between hNP220ε and MAPRE1 --- p.34 / Chapter 2.6.1. --- CytoTrap® XR HeLa Cell cDNA Library screening --- p.34 / Chapter 2.6.1.1. --- Cloning of hNP220ε into yeast two-hybrid bait vector pSos --- p.34 / Chapter 2.6.1.2. --- Preparation of cdc25Ha & cdc25Hα yeast competent cells --- p.34 / Chapter 2.6.1.3. --- Autonomous activation study of bait fusion construct pSos-hNP220ε --- p.36 / Chapter 2.6.1.4. --- Cotransformation of pSos-hNP220ε and CytoTrap® XR HeLa Cell cDNA Library --- p.36 / Chapter 2.6.1.5. --- Verification of interaction by yeast mating --- p.38 / Chapter 2.6.1.5.1. --- Generation of yeast plasmid segregant for mating --- p.38 / Chapter 2.6.1.5.2. --- Yeast mating in 96-well plate --- p.39 / Chapter 2.6.1.6. --- Identification of putative interaction partner --- p.39 / Chapter CHAPTER 3 --- Results --- p.42 / Chapter 3.1. --- Transcription start site of the HNP220 gene is located 312 nucleotides upstream the initiation codon --- p.42 / Chapter 3.2. --- Third splice variant of hNP220 gene hNP220s) is identified --- p.44 / Chapter 3.3. --- In silico analysis of hNP220ε --- p.54 / Chapter 3.4. --- hNP220a and hNP220s are ubiquitously expressed in human fetal and adult tissues --- p.65 / Chapter 3.5. --- hNP220ε shows a punctate subnuclear localization pattern in HepG2 cell line --- p.67 / Chapter 3.6. --- hNP220ε interacts with MAPRE1 --- p.69 / Chapter CHAPTER 4 --- Discussion --- p.71 / Chapter 4.1. --- "Identification of hNP220s, the third splice variant of hNP220 gene" --- p.71 / Chapter 4.2. --- Biological resemblance between hNP220α (hNP220) and hNP220ε --- p.73 / Chapter 4.3. --- Protein-protein interaction between hNP220ε and MAPRE1 --- p.74 / Chapter 4.3.1. --- MAPRE1 protein --- p.77 / Chapter 4.3.2. --- Wnt signaling pathway --- p.78 / Chapter 4.4. --- Potential roles of hNP220 in the regulation of chromosome stability and oncogenesis --- p.82 / Chapter 4.5. --- Summary --- p.85 / Chapter 4.6. --- Concluding questions --- p.86 / Chapter 4.7. --- Future work --- p.87 / References --- p.89 / Appendix --- p.96
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Biochemical characterization and mutational analysis of human uracil-DNA glycosylaseChen, Cheng-Yao 09 December 2004 (has links)
PCR-based codon-specific random mutagenesis and site-specific mutagenesis
were performed to construct a library of 18 amino acid changes at Arg276 in the
conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase
(UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to
apparent homogeneity by conventional chromatography. All of the R276 mutant
proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein
(Ugi) in vitro, suggesting that the active site structure of the mutant enzymes was not
perturbed. The catalytic activity of all mutant proteins was reduced; the least active
mutant, R276E, exhibited 0.6% of wild-type UNG activity, whereas the most active
mutant, R276H, exhibited 43%. Equilibrium binding measurements utilizing a 2-
aminopurine-deoxypseudouridine DNA substrate showed that all mutant proteins
displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed
cross-linking of the R276 mutants to single-stranded DNA was much less
compromised. Using a concatemeric [³²P]U·A DNA polynucleotide substrate to assess
enzyme processivity, UNG was shown to use a processive search mechanism to locate
successive uracil residues, and Arg276 mutations did not alter this attribute. A
transient kinetics approach was used to study six different amino acid substitutions at
Arg276 (R276C, R276E, R276H, R276L, R276W, and R276Y). When reacted with
double-stranded uracil-DNA, these mutations resulted in a significant reduction in the
rate of base flipping and enzyme conformational change, and in catalytic activity.
However, these mutational effects were not observed when the mutant proteins were
reacted with single-stranded uracil-DNA. Thus, mutations at Arg276 effectively
transformed the enzyme into a single-strand-specific uracil-DNA glycosylase. The
nuclear form of human uracil-DNA glycosylase (LTNG2) was overproduced in E. coli
cells and purified to apparent homogeneity. While UNG2 retained ~9 % of UNG
activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of
NaC1 and MgC1₂ stimulated UNG2 catalytic activity as well as the rate of rapid
fluorescence quenching; however, the rate of uracil flipping was reduced. When
UNG2 bound pseudouracil-containing DNA, conformational change was not detected. / Graduation date: 2005
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