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Yeast Upf1 Associates With Ribosomestranslating Mrna Coding Sequences Upstream Of Normal Termination Codons: A Dissertation, Ei Ei Min Apr 2015

Yeast Upf1 Associates With Ribosomestranslating Mrna Coding Sequences Upstream Of Normal Termination Codons: A Dissertation, Ei Ei Min

GSBS Dissertations and Theses

Nonsense-mediated mRNA decay (NMD) specifically targets mRNAs with premature translation termination codons for rapid degradation. NMD is a highly conserved translation-dependent mRNA decay pathway, and its core Upf factors are thought to be recruited to prematurely terminating mRNP complexes, possibly through the release factors that orchestrate translation termination. Upf1 is the central regulator of NMD and recent studies have challenged the notion that this protein is specifically targeted to aberrant, nonsense-containing mRNAs. Rather, it has been proposed that Upf1 binds to most mRNAs in a translation-independent manner. In this thesis, I investigated the nature of Upf1 association with its substrates ...


Insights Into Er Translocation Channel Gating. Structural Regulation Of The Transition Between The Closed And Open Channel Conformations: A Dissertation, Steven F. Trueman Oct 2011

Insights Into Er Translocation Channel Gating. Structural Regulation Of The Transition Between The Closed And Open Channel Conformations: A Dissertation, Steven F. Trueman

GSBS Dissertations and Theses

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7 and TM8 in Sec61p) to expose the signal sequence-binding (SSB) site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay ...


Generating Nucleosomal Asymmetry In Saccharomyces Cerevisiae: A Masters Thesis, Yuanyuan Chen Oct 2010

Generating Nucleosomal Asymmetry In Saccharomyces Cerevisiae: A Masters Thesis, Yuanyuan Chen

GSBS Dissertations and Theses

There are two copies of each core histone in a nucleosome, however, it is unclear whether post-translational modifications on each molecule function redundantly or if symmetrical modifications are required to properly regulate gene expression. We tried to address this question by breaking nucleosomal symmetry and measuring its impact on gene expression. Our strategy includes re-engineering specific residues at the H3-H3 interface, generating pairs of mutant proteins, which were predicted by computational methods to form obligate heterodimers. Using S. cerevisiae as a model system, we tested the viability of strains with mutant histones, and analyzed the interaction between by co-immunoprecipitation from ...


Nuclear Dynamics Of A Broken Chromosome: A Dissertation, Pranav O. Oza May 2009

Nuclear Dynamics Of A Broken Chromosome: A Dissertation, Pranav O. Oza

GSBS Dissertations and Theses

In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there ...


Analysis Of Long-Range Chromosomal Interactions In Saccharomyces Cerevisiae: A Dissertation, Adriana Miele Apr 2009

Analysis Of Long-Range Chromosomal Interactions In Saccharomyces Cerevisiae: A Dissertation, Adriana Miele

GSBS Dissertations and Theses

Long-range chromosomal interactions have been discovered in a number of organisms, suggesting that gene regulation through direct physical association with regulatory elements and/or other genes is a common and conserved phenomenon. This thesis investigates the relationship between direct physical contact of genomic loci and how these interactions may play a role in gene regulation. Analysis of such levels of chromosomal organization has been made possible in part by the emergence of Chromosome Conformation Capture (3C). This technique makes use of formaldehyde crosslinking to trap interacting chromosomal fragments, which can be detected after a number of manipulations. By adapting the ...


Chromatin Remodeling And Transcriptional Memory: A Dissertation, Sharmistha Kundu Dec 2008

Chromatin Remodeling And Transcriptional Memory: A Dissertation, Sharmistha Kundu

GSBS Dissertations and Theses

Transcriptional regulation of gene expression is critical for all unicellular and multicellular organisms. The ability to selectively induce or repress expression of only a few genes from the entire genome gives cells the ability to respond to changing environmental conditions, grow and proliferate. Multicellular organisms begin life as a single totipotent cell, which undergoes many cell divisions during embryonic and later postnatal development. During this process, the dividing cells of the embryo progressively lose their pluripotency and adopt restricted cell fates. Cell fate restriction leads different cell types to gain unique transcriptional profiles. This transcriptional profile or gene expression pattern ...


Roles For Histones H4 Serine 1 Phosphorylation In Dna Double Strand Break Repair And Chromatin Compaction: A Dissertation, Melissa Anne Foley Aug 2008

Roles For Histones H4 Serine 1 Phosphorylation In Dna Double Strand Break Repair And Chromatin Compaction: A Dissertation, Melissa Anne Foley

GSBS Dissertations and Theses

The study of DNA templated events is not complete without considering the chromatin environment. Histone modifications help to regulate gene expression, chromatin compaction and DNA replication. Because DNA damage repair must occur within the context of chromatin, many remodeling enzymes and histone modifications work in concert to enable access to the DNA and aid in restoration of chromatin after repair is complete. CK2 has recently been identified as a histone modifying enzyme. In this study we identify CK2 as a histone H3 tail kinase in vitro, identify the phospho-acceptor site in vitro, and characterize the modification in vivo in S ...


Plasma Membrane Localization Of Signaling Proteins In Yeast: A Dissertation, Satoe Takahashi May 2008

Plasma Membrane Localization Of Signaling Proteins In Yeast: A Dissertation, Satoe Takahashi

GSBS Dissertations and Theses

In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway.

Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the ...


Transcript-Specific Cytoplasmic Degradation Of Yra1 Pre-Mrna Mediated By The Yeast Edc3 Protein: A Dissertation, Shuyun Dong Dec 2007

Transcript-Specific Cytoplasmic Degradation Of Yra1 Pre-Mrna Mediated By The Yeast Edc3 Protein: A Dissertation, Shuyun Dong

GSBS Dissertations and Theses

mRNA degradation is a fundamental process that controls both the level and the fidelity of gene expression. Using a combination of bioinformatic, genomic, genetic, and molecular biology approaches, we have shown that Edc3p, a yeast mRNA decay factor, controls the stability of the intron-containing YRA1 pre-mRNA. We found that Edc3p-mediated degradation of YRA1 pre-mRNA: 1) is a component of a negative feedback loop involved in the autoregulation of YRA1, 2) takes place in the cytoplasm, 3) is independent of translation, 4) occurs through a deadenylation-independent decapping and 5΄ to 3΄ exonucleotic decay mechanism, and 5) is controlled by specific cis ...


Regulation Of Cell Polarization And Map Kinase Signaling In The Saccharomyces Cerevisiae Pheromone Response Pathway: A Dissertation, Shelly Catherine Strickfaden Mar 2007

Regulation Of Cell Polarization And Map Kinase Signaling In The Saccharomyces Cerevisiae Pheromone Response Pathway: A Dissertation, Shelly Catherine Strickfaden

GSBS Dissertations and Theses

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and ...


Prevention Of Oxidative Damage By Yeast And Human Oxr1: A Dissertation, Nathan Andrew Elliott Sep 2004

Prevention Of Oxidative Damage By Yeast And Human Oxr1: A Dissertation, Nathan Andrew Elliott

GSBS Dissertations and Theses

Author did not provide abstract.


Functional And Structural Analysis Of The Yeast Swi/Snf Complex: A Dissertation, Corey Lewis Smith Jul 2004

Functional And Structural Analysis Of The Yeast Swi/Snf Complex: A Dissertation, Corey Lewis Smith

GSBS Dissertations and Theses

Modulating chromatin structure is an important step in maintaining control over the eukaryotic genome. SWI/SNF, one of the complexes belonging to the growing family of ATP-dependent chromatin remodeling enzymes, is involved in controlling the expression of a number of inducible genes whose proper regulation is vital for metabolism and progression through mitosis. The mechanism by which SWI/SNF modulates chromatin structure at the nucleosome level is an important aspect of this regulation. The work in this dissertation focuses on how the Saccharomyces cerevisiae SWI/SNF complex uses the energy of ATP-hydrolysis to alter DNA-histone contacts in nucleosomes. This has ...


The Yeast Swi/Snf Complex Structure And Function: A Dissertation, Joan Frances Flanagan Jan 2001

The Yeast Swi/Snf Complex Structure And Function: A Dissertation, Joan Frances Flanagan

GSBS Dissertations and Theses

DNA is packaged within the cells' nucleus as a highly compact chromatin structure ranging between 100-400 nm fibers. The organization and alteration of this structure is mandatory in order to arbitrate DNA-mediated processes of the cell, including transcription, DNA replication, recombination and repair. Many different kinds of enzymes modify chromatin components and, in turn, regulate the accessibility of DNA. These multi-subunited enzymes have emerged as key regulators for several processes of the cell. Central to understanding how DNA-mediated processes are regulated is to comprehend the consequences of these modifications of chromatin, which lead to altered states of either activation or ...


A Study On The Cellular Localization Of Factors Involved In Yeast Nonsense-Mediated Mrna Decay And Their Mechanisms Of Control On Nonsense Mrna Translation: A Dissertation, Alan Baer Maderazo Dec 2000

A Study On The Cellular Localization Of Factors Involved In Yeast Nonsense-Mediated Mrna Decay And Their Mechanisms Of Control On Nonsense Mrna Translation: A Dissertation, Alan Baer Maderazo

GSBS Dissertations and Theses

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance mechanism conserved in eukaryotes. This thesis explores several interesting aspects of the NMD pathway. One important aspect of NMD which is presently the subject of intense controversy is the subcellular localization of NMD. In one set of experiments, the decay kinetics of the ade2-1 and pgk1 nonsense mRNAs (substrates for NMD) were investigated in response to activating the NMD pathway to determine if cytoplasmic nonsense mRNAs are immune to NMD in the yeast system. The results of these studies demonstrated that activation of NMD caused rapid and immediate degradation of both ...


Genetic And Biochemical Analysis Of The Activation Mechanism Of The Saccharomyces Cerevisiae Pheromone Receptor, Gul H. Bukusoglu Jan 1998

Genetic And Biochemical Analysis Of The Activation Mechanism Of The Saccharomyces Cerevisiae Pheromone Receptor, Gul H. Bukusoglu

GSBS Dissertations and Theses

Activation mechanism of the α-factor pheromone receptor of Saccharomyces cerevisiae was analyzed using biochemical and genetic techniques. An in vitro partial proteolysis assay was developed to determine the conformational change of the receptor that occurs upon binding of agonist. The activation specific cleavages were established by comparing cleavage products with antagonist versus agonist occupied receptor. Of the changes in peptide pattern that were revealed by trypsinization, the fragment resulting from the exposure of the third loop to the protease was found to be agonist specific and to be G-protein independent. A low-affinity binding receptor mutant was isolated which failed to ...


In Vivo Functional Analysis Of The Saccharomyces Cerevisiae Swi/Snf Complex: A Dissertation, Loree Griffin Burns Jul 1997

In Vivo Functional Analysis Of The Saccharomyces Cerevisiae Swi/Snf Complex: A Dissertation, Loree Griffin Burns

GSBS Dissertations and Theses

Chromatin remodeling is crucial to transcriptional regulation in vivo and a number of protein complexes capable of altering genomic architecture in the budding yeast Saccaromyces cerevisiaehave been identified. Among these, the SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characterized. The SWI/SNF complex is required for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not all, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was ...


Mismatch Repair Acts As A Barrier To Homeologous Recombination In Saccharomyces Cerevisiae: A Dissertation, Erica Marie Selva Jul 1996

Mismatch Repair Acts As A Barrier To Homeologous Recombination In Saccharomyces Cerevisiae: A Dissertation, Erica Marie Selva

GSBS Dissertations and Theses

Homeologous recombination refers to genetic exchanges between DNA partners containing similar but not identical DNA sequences. Heteroduplex intermediates in such exchanges are expected to contain multiple DNA mismatches at positions of sequence divergence and hence are potential targets for mismatch correction. Thus recombination of this type is of particular interest in understanding the role of DNA mismatch correction on recombination fidelity. Previous studies that examined this question in prokaryotic systems suggested that mismatch repair acts as a barrier to recombination between diverged sequences. The central hypothesis of this thesis is that mismatch correction acts as a barrier to homeologous recombination ...


Transcriptional Regulation By The Saccharomyces Cerevisiae Centromere-Binding Protein Cp1: A Dissertation, Kevin F. O'Connell Jun 1994

Transcriptional Regulation By The Saccharomyces Cerevisiae Centromere-Binding Protein Cp1: A Dissertation, Kevin F. O'Connell

GSBS Dissertations and Theses

CP1 (encoded by the gene CEP1) is a sequence-specific DNA-binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 are viable but exhibit defects in growth, chromosome segregation, and methionine biosynthesis. To investigate the basis of the methionine requirement, a YEp24-based yeast genomic DNA library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. PHO4 encodes a factor which positively regulates transcription of genes involved in phosphate metabolism via an ...


Genetic Analysis Of The Saccharomyces Cerevisiae Centromere-Binding Protein Cp1: A Thesis, Daniel C. Masison Mar 1993

Genetic Analysis Of The Saccharomyces Cerevisiae Centromere-Binding Protein Cp1: A Thesis, Daniel C. Masison

GSBS Dissertations and Theses

CP1 is a sequence specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved centromere DNA element I (CDEI) of yeast centromeres. The gene encoding CP1, which was designated CEP1 for centromere protein 1, was cloned and sequenced. CEP1 encodes a highly acidic protein of molecular weight 39,400. CEP1 was mapped to a position 4.6 centiMorgans centromere distal to SUP4 on the right arm of chromosome X. Phenotypic analysis of cep1 mutants demonstrated that yeast strains lacking CP1 are viable but have a 35% increase in cell doubling time, a ninefold increase in the rate ...


Genetic Analysis Of The Saccharomyces Cerevisiae Pheromone Response Pathway: A Thesis, Dmitry B. Blinder May 1990

Genetic Analysis Of The Saccharomyces Cerevisiae Pheromone Response Pathway: A Thesis, Dmitry B. Blinder

GSBS Dissertations and Theses

The cell division of Saccharomyces cerevisiae is controlled by the action of pheromones at the G1 phase of the cell cycle. A general method was developed for the isolation of constitutive mutants in the pheromone response pathway. Recessive alleles of the SCG1 gene (encoding the α subunit of a G protein) were isolated as well as a dominant mutation in the STE4 gene (encoding the β subunit of a G protein). Analysis of double mutants suggested that the STE4 gene product functions after the SCG1 product but before the STE5 gene product. Double mutants carrying either scg1 or STE4Hp1 ...


Structural Determinants Of Mrna Turnover In Yeast: A Thesis, David Herrick Jan 1989

Structural Determinants Of Mrna Turnover In Yeast: A Thesis, David Herrick

GSBS Dissertations and Theses

Large differences exist in the decay rates of individual mRNAs yet the molecular basis for such differences is substantially unknown. We have developed a procedure for the measurement of individual mRNAs in the yeast Saccharomyces cerevisiae which utilizes northern or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant strain (RY260). To assess the reliability of half-life measurements obtained in this manner, we have compared the results of this procedure to results obtained by three other procedures (pulse-chase analysis, approach to steady-state labeling, and inhibition of transcription with ...