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Characterization of the Cellular and Organellar Dynamics that Occur with a Partial Depletion of Mitochondrial DNA when Arabidopsis Organellar DNA Polymerase IB is MutatedCupp, John D. 07 August 2012 (has links)
Plant mitochondrial genomes are large and complex, and the mechanisms for maintaining mitochondrial DNA (mtDNA) remain unclear. Arabidopsis thaliana has two DNA polymerase genes, polIA and polIB, that have been shown to be dual localized to mitochondria and chloroplasts but are unequally expressed within primary plant tissues involved in cell division or cell expansion. PolIB expression is observed at higher levels in both shoot and root apexes, suggesting a possible role in organelle DNA replication in rapidly dividing or expanding cells. It is proposed that both polIA and polIB are required for mtDNA replication under wild type conditions. An Arabidopsis T-DNA polIB mutant has a 30% reduction in mtDNA levels but also a 70% induction in polIA gene expression. The polIB mutant shows an increase relative to wild type plants in the number of mitochondria that are significantly smaller in relative size, observed within hypocotyl epidermis cells that have a reduced rate of cell expansion. These mutants exhibit a significant increase in gene expression for components of mitorespiration and photosynthesis, and there is evidence for an increase in both light to dark (transitional) and light respiration levels. There is not a significant difference in dark adjusted total respiration between mutant and wild type plants. Chloroplast numbers are not significantly different in isolated mesophyll protoplasts, but mesophyll cells from the mutant are significantly smaller than wild type. PolIB mutants exhibit a three-day delay in chloroplast development but after 7dpi (days post-imbibition) there is no difference in relative plastid DNA levels between the mutant and wild type. Overall, the polIB mutant exhibits an adjustment in cell homeostasis, which enables the maintenance of functional mitochondria but at the cost of normal cell expansion rates.
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Molecular studies of homologous chromosome pairing in Triticum aestivumThomas, Stephen W. (Stephen William) January 1997 (has links) (PDF)
Errata pasted on front fly-leaf. Bibliography: leaves 139-173. This thesis identifies DNA structures and genes involved in the process of homologous chromosome pairing in allohexaploid bread wheat (Triticum aestivum). In addition to studying late replicating DNA, a speculative model on the action of the pairing genes in allohexaploid wheat and the putative function of the AWWM5 gene is discussed.
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Exploring the Cell Cycle of ArchaeaLundgren, Magnus January 2007 (has links)
<p>Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation.</p><p>For this thesis, a comprehensive study of the cell cycle in the model archaeon <i>Sulfolobus acidocaldarius</i> was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries.</p><p><i>Sulfolobus</i> species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes.</p><p>A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon <i>Methanothermobacter thermautotrophicus</i> was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.</p>
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Cell cycle regulation of DNA precursor accumulation in the yeast Saccharomyces cerevisiaeKoc, Ahmet 11 June 2002 (has links)
In budding yeast, many of the genes that encode enzymes required for DNA
precursor synthesis (MCB genes) are expressed under cell cycle control in late
G1/S. The relationship between MCB gene expression, dNTP synthesis and DNA
synthesis was investigated by using �� factor-synchronized Saccharomyces
cerevisiae. The levels of all four dNTPs increased several-fold when cells crossed
the G1/S boundary. An even larger increase in the dNTP pools occurred at G1/S
when replication initiation was blocked by incubating synchronized dbf4 mutants at
the nonpermissive temperature. Thus, dNTP accumulation at G1/S was not
dependent on replication initiation. Similarly, MCB gene induction at G1/S was
also independent of replication initiation. The accumulation of dNTPs at G1/S was
dependent on Swi6, a protein known to be required for normal MCB gene
regulation during the cell cycle. Treatment with hydroxyurea, an inhibitor of
ribonucleotide reductase, blocked DNA synthesis and prevented the increase in
dNTP levels that normally occurred at G1/S, however, it did not exhaust the basal
levels of any of the four dNTPs. The mechanism responsible for replication arrest
despite the persistence of dNTPs was not dependent on the checkpoint protein
Rad53, as rad53 mutants also failed to exhaust basal dNTPs when incubated in HU.
The inhibitory effect of HU on DNA synthesis was bypassed when dbf4 cells were
allowed to pre-accumulate dNTPs at 37��C before being released to the permissive
temperature in the presence of HU. Accumulation of dNTPs at G1/S was not a
prerequisite for replication initiation, as dbf4 cells incubated in HU at 25��C were
able to initiate replication when cells were switched to the nonpermissive
temperature and HU was removed. The results indicate that DNA chain elongation
in yeast requires a critical dNTP threshold, below which replication forks are
completely arrested. Cells lacking a functional thioredoxin system were deficient
in dNTP synthesis. The rate of accumulation was significantly lower in ��trr1
mutants lacking thioredoxin reductase, and dNTP accumulation at G1/S did not
occur at all in ��trxl ��trx2 double mutants lacking thioredoxin. The results suggest
that thioredoxin serves as the electron donor for ribonucleotide reductase during
DNA precursor synthesis in yeast. / Graduation date: 2003
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Rapid Replication of High Aspect Ratio Molds for UV EmbossingYan, Yehai, Chan-Park, Mary Bee-Eng, Yue, Chee Yoon 01 1900 (has links)
This paper describes a promising fabrication technique for rapid replication of high aspect ratio microstructured molds for UV embossing. The process involves casting silicone rubber on a microstructured master, replicating an epoxy mold using the PDMS rubber mold and finally, metallizing the surfaces of the epoxy mold by electroless plating nickel (EN). The preliminary study suggests that this technique is feasible for rapid replication of high aspect ratio molds for UV embossing. Uniform molds can be replicated rapidly through this technique making the process economical and accessible. / Singapore-MIT Alliance (SMA)
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Exploring the Cell Cycle of ArchaeaLundgren, Magnus January 2007 (has links)
Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation. For this thesis, a comprehensive study of the cell cycle in the model archaeon Sulfolobus acidocaldarius was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries. Sulfolobus species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes. A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon Methanothermobacter thermautotrophicus was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.
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A Quantitative Model of the Initiation of DNA Replication in Saccharomyces cerevisiaeGidvani, Rohan January 2012 (has links)
A crucial step in eukaryotic cell proliferation is the initiation of DNA replication, a tightly regulated process mediated by a multitude of protein factors. In Saccharomyces cerevisiae, this occurs as a result of the concerted action of an assembly of proteins acting at origins of replication, known as the pre-replicative complex (pre-RC). While many of the mechanisms pertaining to the functions of these proteins and the associations amongst them have been explored experimentally, mathematical models are needed to effectively explore the network’s dynamic behaviour.
An ordinary differential equation (ODE)-based model of the protein-protein interaction network describing DNA replication initiation was constructed. The model was validated against quantified levels of protein factors determined in vivo and from the literature over a range of cell cycle timepoints. The model behaviour conforms to perturbation trials previously reported in the literature and accurately predicts the results of knockdown experiments performed herein. Furthermore, the DNA replication model was successfully incorporated into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes.
A screen for novel DNA damage response proteins was investigated using a unique proteomics approach that uses chromatin fractionation samples to enrich for factors bound to the DNA. This form of sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to detect and quantify low abundance chromatin proteins in the budding yeast proteome. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress, pointing to a role for this nuclear import-associated protein in DNA damage response.
The model presented in this thesis provides a tool for exploring the biochemical network of DNA replication. This is germane to the exploration of new cancer therapeutics considering the link between this disease (and others) and errors in proper cell cycle regulation. The high functional conservation between cell cycle mechanisms in humans and yeast allows predictive analyses of the model to be extrapolated towards understanding aberrant human cell proliferation. Importantly, the model is useful in identifying potential targets for cancer treatment and provides insights into developing highly specific anti-cancer drugs. Finally, the characterization of factors in the proteomic screen opens the door to further investigation of the roles of potential DNA damage response proteins.
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Investigation of the Polyprimidine Tract-Binding Protein-Associated Splicing Factor (PSF) Domains Required for the Hepatitis Delta Virus (HDV) ReplicationAl-Ali, Youser 14 October 2011 (has links)
The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.
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Vaccinia virus ribonucleotide reductase : gene sequencing, intracellular localization, and interaction with a DNA-binding proteinDavis, Ralph Eugene, 1957- 07 May 1992 (has links)
Vaccinia virus infected monkey kidney cells had been previously shown
to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA
from mutant virus resistant to hydroxyurea were digested with restriction
endonucleases and were shown to have substoichiometric amounts of the Hind
III F fragment. Additional information from Southern blotting experiments
localized the putative small subunit (R2) gene to the left end of the Hind III F
fragment of the vaccinia virus genome. The entire open reading frame of the R2
gene and the flanking regions was sequenced and the translated sequence
found to be 80% homologous to the mouse R2 polypeptide.
A combination of in situ and in vitro experiments addressed the question
of macromolecular interactions involving vaccinia ribonucleotide reductase
(FIR). Replication of double stranded viral DNA occurs in very discrete loci in
infected cells and these DNA factories can be isolated from gently lysed cell in
sucrose gradients. RR was detected at low levels (less than 5% of the total R2)
with the rapidly sedimenting DNA by using antibodies against FIR. In situ crosslinking
experiments were attempted with no specific interaction determined at
this time. Immunolocalization experiments have given evidence for localization
of large subunit (R1) polypeptide to the viral inclusion bodies.
The most conclusive results utilized anti-idiotypic antibodies against the
antibodies to R2 protein. lmmunolocalization experiments have shown the
putative R2 binding protein to be localized at the sites of viral DNA synthesis.
lmmunoprecipitations show a single predominant viral polypeptide which also
has proven to be a DNA binding (phospho)protein. Screening a lambda phale
expression library of vaccinia with the anti-idiotypic antibody localized the
binding site to the carboxy terminal 81 amino acids in open reading frame 1-3 of
the vaccinia genome. The open reading frame was cloned into a pET11c
expression vector and the partially purified recombinant protein was shown to
have specificity for single-stranded DNA as well as stimulate vaccinia RR
activity. / Graduation date: 1993
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Analysis of the Cellular Proteins, TIA-1 and TIAR, and their Interaction with the West Nile Virus (WNV) 3' SL Minus-Strand RNAEmara, Mohamed Maged 23 April 2007 (has links)
The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.
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