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An assay for screening cells for mismatch repair proficiency in vivoWu, Shiau-Yin 30 October 2002 (has links)
Graduation date: 2003
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512 |
Modulation of deoxyribonucleoside triphosphate levels, DNA synthesis rates and fidelity in mammalian cellsMartomo, Stella A. 09 April 2002 (has links)
Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not
symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in
cells. In an in vitro replication system involving semiconservative replication from
an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human
cell extracts in reaction mixtures containing "biologically biased" dNTP pools
estimated from HeLa cell nuclei is not significantly different from that seen when
replication is done with equimolar dNTP concentrations. Significant reduction of
dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations
during replication reaction also did not increase mutation frequency. In contrast, in
vitro replication with dNTP concentrations calculated from normal diploid fibroblast
cells, which are three- to four-fold lower in concentrations, showed a marked
reduction of the observed mutation frequency, showing the importance of overall
dNTP levels during replication on mutation frequency in vitro. When whole-cell
dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels
underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells. / Graduation date: 2002
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Uracil-DNA glycosylase inhibitor protein : role of carboxylic acid residues and use for measuring the fidelity of uracil-excision DNA repair synthesis in human cell extractsSanderson, Russell J. 23 September 1998 (has links)
Graduation date: 1999
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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-protein interactions involved in baculovirus DNA replicationEvans, Jay T. 17 February 1998 (has links)
The yeast two-hybrid system was used to examine
interactions between the nine proteins involved in baculovirus DNA
replication. From the six proteins required for DNA replication,
four protein-protein interactions were identified, including an
interaction between LEF-1 and LEF-2, LEF-3 and itself, LEF-3 and
P143 (Helicase), and an interaction between IE-1 and itself.
The replication factors LEF-1 and LEF-2 interacted in both
yeast two-hybrid assays and glutathione S-transferase fusion
affinity assays. Using the yeast two-hybrid system, we mapped
the interaction domain of LEF-2 to amino acids between positions
20 and 60. Deletion analysis of LEF-1 failed to reveal an
interaction domain, suggesting that either multiple interaction
domains exists or the deletions disrupted secondary structures
required for the interaction. All of the deletions which were
unable to interact also failed to support significant levels of
transient DNA replication, suggesting that this interaction plays a
significant role in DNA replication.
The baculovirus single-stranded DNA binding protein, LEF-3, interacts with itself in yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Deletions of LEF-3, which were unable to interact with full length LEF-3, also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to apparent homogeneity and analyzed by analytical ultracentrifugation, native PAGE and MALDI mass spectrometry, identifying the oligomeric structure of LEF-3 as a homotrimer.
In addition to interacting with itself, LEF-3 also interacts with P143 in yeast two-hybrid assays, immunoprecipitation experiments, and co-purification from a single-stranded DNA agarose column. The yeast two-hybrid system was used to map the LEF-3 interaction domain to the N-terminal 165 amino acids of LEF-3. Deletion analysis of P143 failed to reveal a delimited interaction domain. C-terminal deletions of LEF-3 containing amino acids 1 to 165 were unable to interact with full length LEF-3, indicating that the interaction of LEF-3 with itself (trimerization) is not required for the interaction between LEF-3 with P143. / Graduation date: 1998
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Molecular characterization of specificity and activity of the transposable element IS801Richter, Grace Yukiko 11 August 1995 (has links)
1S801 is a transposable element isolated from Pseudomonas syringae pathovar
(pv.) phaseolicola, the causal agent of halo blight of bean. Fragments of the element are
present in multiple copies on an indigenous plasmid, pMMC7105, of strain LR781, and
have been implicated as sites of homologous recombination leading to imprecise
excision of the chromosomally integrated form of the plasmid. The element, which has
been completely sequenced, is 1512 base pairs in length and is unusual among
transposable elements in that it does not have direct or inverted repeats in its termini.
The terminal regions of the element were uncoupled from the two major open reading
frames, and trans-acting activity of the putative transposase was demonstrated in
Escherichia coli (recA). An alignment of the sequences of thirteen insertions defined
the precise borders of the element, and demonstrated that it does not duplicate its targets
upon insertion. The target specificity of IS801 is similar to, but more degenerate than,
the target specificity of two transposable elements to which it is closely related, IS91
and IS1294. The consensus derived from the aligned target sequences is G/C-A/G-A-C/G, and the target tetramer is found immediately adjacent to the right terminus of the
element upon transposition. IS91 was demonstrated to mobilize 1S801, but not with the
specificity characteristic of 1S801. The structure of 1S801 and the characteristics of
IS91-activated transposition of 1S801 are discussed in light of a proposed model for
IS91 transposition, and it is suggested that 1S801 could have been derived from IS91 by
a modification of its left end. Remnants of IS801 are present near avirulence genes of
various P. syringae pathovars, suggesting that the element has been involved in genetic
rearrangements in the vicinity of these loci. / Graduation date: 1996
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Studies on the hydrophobic effect and its contribution to the stability, crystallization, and helix packing of Z-DNAKagawa, Todd F. 01 December 1994 (has links)
The studies presented here use the B- to Z-DNA transition and Z-DNA
crystallization as model systems to determine the contribution of solvent interactions to
macromolecular structures. The substituent groups of naturally occurring and modified
nucleotide bases affect the hydration and thus the stability of right-handed B-DNA and
left-handed Z-DNA. The free energy for alternating pyrimidine-purine sequences in the
B- and Z-conformations are quantitated as solvent free energies (SFE) from their solvent-accessible
surfaces. The effect of methylation of the C5 carbon of pyrimidine bases on
the stability of Z-DNA was analyzed in comparisons of d(TA) versus d(UA)
dinucleotides. In the minor groove, the N2 amino group of purine bases was examined
by comparing the stability of d(CG) versus d(CI), and d(TA) versus d(TA') dinucleotides
as Z-DNA. These comparisons correctly accounted for the observed effects of the major
and minor groove substituent groups on the relative stability of Z-DNA. These
predictions were confirmed by comparing the amount of salt required to crystallize
various hexanucleotides as Z-DNA.
The relative contribution of solvent and steric interactions in DNA assembly were
studied crystallographically using an asymmetric hexanucleotide which assumes two
discrete and discernible orientations in the crystal lattice. How this sequence orients in
the lattice is a direct measure of the DNA-DNA interactions at the surface of the crystal.
These interaction free energies were directly correlated with differences in solvation for 5
sequences where there was effectively no differences in steric interactions for the two orientations. In the singular case where a large steric clash was expected, the orientation was in fact determined by this interaction. The comparisons indicate that both solvent and van der Waals interactions are significant in DNA assembly with van der Waa ls interactions dominating in situations with large steric interactions.
The analyses of the B- to Z-DNA transition and Z-DNA crystallization based on SFEs support the significance of solvent interactions in determining the structure and assembly of macromolecules. / Graduation date: 1995
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Base inclinations for DNA in solutions and films as revealed by linear dichroismKang, Hunseung 22 November 1993 (has links)
Graduation date: 1994
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519 |
Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells /Hau, Pok Man. January 2007 (has links)
Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 97-105). Also available in electronic version.
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A novel probabilistic framework for microarray data analysis from fundamental probability models to experimental validation /Gelmi, Claudio A. January 2007 (has links)
Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Babatunde Ogunnaike and Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.
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