Browsing by Subject "Genetic Processes"
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Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Analysis of Yeast Genes Influencing the Lethality of DNA Damage Related Checkpoint Mutants(2009-05-01) Kim, Eunmi; Siede, WolframThe purpose of this study was to determine the functions of Hug1 and Srl3. It has been reported that HUG1 or SRL3 deletion rescues the lethality of a DNA damage checkpoint gene deleted mutant, mec1Δ. It is known that the lethality of mec1Δ can be rescued by high dNTP levels. To elucidate the functions of these proteins, the phenotypes of hug1Δ and srl3Δuvs, as well as the transcript profile of hug1Δ were analyzed. Novel phenotypes of hug1Δ were uncovered: resistance to oxidative stress or heat shock, earlier arrest in G1/G0 phase, defect in hydroxyurea-induced filamentation, and slow growth inresponse to combined stresses of hydroxyurea and reduced dextrose content. These phenotypes correlate with a transcription profile that indicated altered stress responses in hug1Δ as compared to WT. We assumed that the reason for many constitutively expressed stress-related transcripts is a higher dNTP level in hug1Δ compared to WT. The similarities in the phenotypes of dif1Δ and sml1Δ to those of hug1Δ support the assumption. The phenotypes of dif1Δ and sml1Δ were studied since Dif1 and Sml1 are known inhibitors of ribonucleotide reductase activity. Furthermore, Dif1, Sml1, and Hug1 are considered proteins that evolved from the same ancestor protein. Initially, Srl3 was a protein of special interest because the commercially available srl3Δ mutant causes high spontaneous mutation rates and sensitivity to UV light. However, during the course of this study, it was found that the two phenotypes originated from a second, unrelated mutation in srl3Δ strain. Through complementation test and sequencing, this mutation was identified as a nonsense mutation of MMS2, a gene involved in post-replication repair.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Differentiation of Vascular Smooth Muscle Cells(2004-05-01) Deaton, Rebecca Ann; Dillon, Glenn; Shepard, Allan; Mallet, Robert T.Rebecca Ann Deaton, Characterization of the role of PKN in TGF-beta 1-mediated differentiation of vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), May 2004, 178 pp, 5 tables, 34 illustrations, references, 197 titles. Differentiated vascular smooth cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors. SMCs retain the ability to de-differentiate into an “immature” proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanism involved in maintain SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-B1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-B1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs. These characteristics are indicative of the differentiated phenotype. The effects of TGF-B1 can be blocked by pretreatment of the cells with either HA1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-B1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM a-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF-GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-B1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK ½. Co-expression of dominant negative p38 MAPK is sufficient to abolish PNK-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-B1 activates RhoA and PKN to promote differentiation of SMCs.Item Crime Scene Investigation: TV versus Reality(2013-08-01) ; Warren, Joseph; Budowle, Bruce; Eisenberg, Arthur J.; Pullin, Mike; Milligan, JessieJoseph Warren, Bruce Budowle and Arthur J. Eisenberg speak about crime scene investigation and forensic science as portrayed in popular television. They discuss how the shows distort and overstate the ways in which forensic scientists help solve crimes and identify victims, and they describe potential impacts on jurors' expectations. They also appreciate how these shows drive curiosity and bring better grant funding and more students to forensic science.Item DNA-Prokids: Forensic Genetics and Human Trafficking(2013-08-01) Eisenberg, Arthur J.DNA - ProKids is a program jointly run by the Universidad de Granada (UGR) and the University of North Texas Health Science Center (UNTHSC). The Goal of DNA - Prokids is to return trafficked individuals, specifically young people and children to their families in their home countries. The FBI estimates that there are more than 100,000 children and teens in the United States that are being trafficked in the sex trade. Since Texas is a portal into the U.S. for Central and South America, the state government has been proactive in trying to address the problem. Part of the solution is the partnership between UNTHSC and UGR to work on DNA-Prokids. DNA-Prokids is an international humanitarian effort to help identify missing children and if possible to reunite abducted and homeless children with their parents, and to provide law enforcement agencies a scientific methodology to help deter the human trafficking of children. The organization also works with law enforcement to ensure that the children are returned to their families or placed in safe environments. Since this video, 697 parent-child associations have been made, with the vast majority of those children being returned to their families. Out of those 697, approximately 12 children have not been able to return home due to crime, violence, and drugs in their biological families. DNA-Prokids has also detected and avoided 221 illegal adoptions, where the woman presenting the baby to be adopted was not the child's biological mother. The success of the program deters human trafficking and can prevent child abuse and slave labor by increasing coordination between efforts like DNA-Prokids and authorities.Item Dr. Arthur Eisenberg(2013-08-01) Eisenberg, Arthur J.; Milligan, JessieItem Dr. John Planz(2013-08-01)John V. Planz holds a B.S degree in Biology and Zoology (double major) from the State University of New York (Oswego, NY), a M.S. degree in behavioral ecology from Shippensburg University (Shippensburg, PA) and a Ph.D. in molecular evolutionary genetics and population genetics from the University of North Texas (Denton, TX). Dr. Planz studied as a postdoctoral fellow at the Carnegie Museum of Natural History, Section of Mammals (Pittsburgh, PA) in mammalian phylogenetic systematics. Dr. Planz entered the forensics field in 1993 at the Southwestern Institute of Forensic Sciences in Dallas, TX. He later served as the Director of Identity Testing at GeneScreen, Inc. in Dallas, TX and Biosynthesis, Inc. in Lewisville TX adding mitochondrial DNA typing and SNP development to the testing performed by those laboratories. Dr. Planz joined the faculty of the University of North Texas Health Science Center at Fort Worth in January 2000 were he serves as an Associate Professor in the Department of Forensic and Investigative Genetics and is the Associate Director of the UNT Center for Human Identification.Item Dr. Joseph Warren(2013-08-02)Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.Item Evaluation of a Novel Multiplex Ministr System for Analysis of Degraded and Low Copy DNA Samples(2006-05-01) Orcutt, Joseph L.; Arthur Eisenberg; Joseph Warren; John PlanzOrcutt, Joseph L., Evaluation of a Novel Multiplex MiniSTR System for Analysis of Degraded and Low Copy DNA Samples. Masters of Science (Forensic Genetics), May, 2006, 78 pp., 25 tables, 14 figures, bibliography, 20 titles. The goal was to evaluate the performance of a novel miniSTR multiplex system for the analysis of degraded and low quantity DNA samples. Three studies were designed to evaluate this new miniSTR kit: 1. A concordance study to insure that the profiles generated are identical to those with currently used STR kits; 2. A dilution study to identify the sensitivity limits of the multiplex system, and 3. The ability to generate profiles from DNA isolated from skeletal remains which had previously given incomplete profiles using conventional STR kits. The results indicate that the Applied Biosystems new miniSTR multiplex system will provide a valuable tool for forensic scientists to obtain genetic data from challenging casework samples.Item Evaluation of Y-STR Data Using a Duplex Gender Real-Time PCR Assay on an ABI Prism 7000 SDS Followed by Amplification with Applied Biosystems AmpFLSTR Yfiler PCR Amplification Kit(2007-08-01) Miller, Jennifer J.; John Planz; Arthur Eisenberg; Joseph WarrenQuantification is the process of determining the concentration of DNA in a sample and plays an extremely important role in the processes of amplification and STR typing. A method of quantification is mandated for a laboratory conducting forensic DNA analysis by National Standard 9.3 (1). Furthermore, anytime a forensic laboratory chooses to implement a new or novel methodology for any step in DNA analysis, a laboratory must conduct an internal validation to ensure the quality of method and any results generated on the equipment used within that laboratory are reliable, reproducible, and accurate before the method is utilized for casework analysis (1). Prior to an internal validation, the method or technology must undergo a developmental validation by the developer or manufacturer to determine conditions or limitations of the method or technology on DNA analysis of forensic samples (2). A study has shown that Y-STR results can be obtained even when the quantification of samples yields a value of 0.00ng/μl (4). The issue of the absolute lowest limit of detection in the quantification process versus input DNA concentrations of the unknown samples to yield any valuable Y-STR typing data has not been addressed. A duplex gender assay developed by Nicklas and Buel (3) has a reported detection limit of 0.5pg for the Alu probe of the duplex assay and quantification will be evaluated on a different qPCR platform than originally reported and followed by amplification using Applied Biosystems’ AmpFLSTR Yfiler PCR Amplification Kit to assess quantification limits. The goal of this internship project was to complete a preliminary evaluation of the sensitivity of a quantification methodology on a different qPCR platform under different detection parameters utilizing Y-chromosome DNA in correlation to Y-STR typing results and evaluate the data qualitatively.Item Evaluation or the Gold-Plated Silver Sample Block on the GeneAmp PCR System 9700 Thermal Cycler(2003-05-01) Moore, Melody Ann; Arthur Eisenberg; Joseph Warren; John PlanzMoore, Melody Ann. Evaluation of the Gold-plated Silver Sample Block on the GeneAmp PCR System 9700 Thermal Cycler. Master of Science (Forensic Genetics), May 2003, 49 pages, 9 figures, 3 tables, 2 appendices, 24 references. The GeneAmp PRC System 9700 Thermal Cycler has been introduced with interchangeable silver and gold-plated silver sample blocks. To validate the new gold-plated silver sample block on the System 9700, amplifications were performed on both the gold-plated silver and the previously validated silver sample blocks. PCR amplifications using the AmpFLSTR Profiler Plus ID COfiler, Identifiler and SGM Plus typing kits (Applied Biosystems, Forster City, CA) were performed. Electrophoretic characteristics such as allele concordance, peak heights, and peak height ratios were used to discern any differences in the amplification capabilities of the two sample blocks. The results demonstrate that the PCR reactions on both silver and gold-plated silver blocks are equivalent. In addition, the data obtained (allele cells, peak heights, peak height ratios) from both Macintosh and Windows NT platforms were identical for each amplification kit, indicating concordance between the software packages of each operating system. The results of this study demonstrate the interchangeability of the silver and gold-plated sample blocks.Item Exercise-Evoked Metabolic Adaptations in Canine Myocardium(1999-12-01) Stuewe, Steven Richard; Robert Mallet; Neeraj Agarwal; Patricia GwirtzStuewe, Steven Richard, Exercise-Evoked Metabolic Adaptations in Canine Myocardium. Doctor of Philosophy (Biomedical Sciences), November 1999; 128 pp; 4 tables; 17 figures; bibliography, 130 titles. Aerobic exercise training evokes adaptations in the myocardial contractile machinery that enhance cardiac functional capabilities, and the myocardium’s capacity to consume energy. Despite considerable investigative effort, the effects of exercise training on myocardial intermediary metabolism, the source of energy for cardiac function, have not been defined. The investigations described herein were undertaken to delineate the effects of aerobic exercise training on key rate-controlling enzymes of myocardial intermediary metabolism and energy transport, and to characterize the effects of acute exercise on cardiac messenger RNA transcripts encoding metabolic enzymes. To address these questions, dogs were conditioned by a 9 wk treadmill running program or cage rested for 4 wk. Exercise conditioning was documented by a significant decrease in heart rate at rest and during submaximal exercise. A panel of glycolytic and oxidative enzymes was measured in myocardial extracts. It was demonstrated that aerobic exercise training of dogs selectively increased capacities of key rate-controlling enzymes of each of the major pathways of intermediary metabolism in ventricular myocardium. In addition, it appeared that the training-evoked increases in enzyme activities were due to increased enzyme contents, not to changes in substrate affinity. The same training program was implemented to investigate the effects of aerobic exercise training on the myocardium’s energy shuttling system. Total creatine kinase (CK) activity and content of the CKMB isoenzyme were measured in canine myocardial extracts. It was demonstrated that aerobic exercise training increased total myocardial CK activity and CKMB content, although the CKMB isoenzyme remained minor component of the myocardial CK system. A third investigation was conducted to examine the effects of aerobic exercise on the abundance of messenger RNA (mRNA) encoding key enzymes involved in myocardial energy production and transport. Left ventricular myocardium was sampled 30 min after an exercise bout, and messenger RNA transcripts were analyzed by reverse transcriptase polymerase chain reaction. Exercise increased in the myocardial abundance of mRNA transcripts encoding glyceraldeheyde 3-phosphate dehydrogenase, citrate synthase, and the CK-M subunit. These mRNA enhancements could be responsible, at least in part, for exercise-evoked adaptations in myocardial metabolic enzymes demonstrated in the first two investigations.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item How do you get a job in Forensic Science?(2013-08-01)Item Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning(1996-07-01) Price, Nancy E.; Neeraj Agarwal; Robert Easom; Stephen R. GrantPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.Item Other Applications of Forensic Genetics(2013-08-01)Item Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B(2003-12-01) Confer, Nils Forgard; Alvarez, Rafael; Ben S. Aar…; Wu, MingehiConfer, Nils Forgard, Protein-Protein Interactions Between Poly(ADP-ribose) Polymerase-1 and DNA Polymerase B. Doctor of Philosophy (Biomedical Sciences), December 2003, 114 Pages, 22 Figures, 1 Graph, and 80 References. The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase B (pol B) function. Activity gels were used to measure the DNA polymerase activity of pol B following protein-(ADP-ribosyl)ation. However, the fraction of pol B molecules (ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose) Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol B to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol B, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol B activity. Moreover, I also observed the specific molecular association of PARP-1 is specifically proteolyzed into peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either capase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol B as supported by co-immunoprecipitation/immune-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol B that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degredation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol B, and inhibits DNA repair associated pol B activity to irreversibly switch the fate of cell from BER toward chromatin degradation and, eventually, programmed cell death.Item Regulation of Nucleo-Cytoplasmic Trafficking of Human Annexin II(2005-12-01) Liu, Jie; Jamboor K. Vishwanatha; Myoung Kim; Jerry SimeckaAnnexin II is a molecule having diversified functions in the cell. It contains a specific N terminus and a C terminal core domain that is homologous with other molecules in annexin family. To date, whether annexin II is a tumor promoter or suppressor is still a question of controversy. So the study of the functions of annexin II according to the specific cancer is of utmost importance for further manipulating this molecule to achieve the control of cancer. Nuclear export signal (NES) was found in the N terminus of annexin II, and nuclear localization of annexin II is cell cycle dependent. In addition, nuclear annexin II was shown to be phosphorylated. Based on these observations along with the previous finding that anexin II is a subunit of primer recognition proteins (PRP), it was proposed that nucleo-cytoplasmic trafficking of annexin II is a phosphorylation-regulated process which is relevant to cancer cell growth. Annexin II-null LNCaP cell line was used as a model to study the intracellular localization of annexin II without the effects of background endogenous annexin II. The stable clones expressing GFP alone, GFP fused wild type annexin II and GFP fused NES mutant annexin II were established. We observed that consistent nuclear localizing of NES mutant annexin II resulted in a decreased proliferation rate of LNCaP cells, while expression of wild type annexin II had no effect on the cell proliferation. However, the expression of wild type annexin II changed the morphology and decreased matrigel migration of LNCaP cells. These observations suggest that annexin II play a role in regulating cell proliferation, and re-expression of this molecule in annexin II-null cells can decrease the malignancy of the parental LNCaP cells. In order to investigate the effect phosphorylation on the subcellular localization of annexin II, we used site directed mutagenesis to convert the putative phosphorylation sites on the N terminus of annexin II to other amino acid to resemble the unphosphorylated/phosphorylated status of the residues. We found that double mutation of serine 11 and 25, which mimics the double phosphorylation of these residues, resulted in inhibition of nuclear entry of annexin II. So, there might be other phosphorylation sites responsible for the nuclear retention of annexin II. In highly malignant cancer cells, we found that annexin II is usually over-expressed and present in both the nucleus and the cytoplasm, i.e. although there is NES, annexin II can not be exported from the nucleus by CRM1 mediated pathway. Hela, DU-145 and PC-3 cell lines also have the same pattern of annexin II distribution. We confirmed this observation by using immunocytochemistry, Western blotting and immunoprecipitation. Using different antibody recognizing different domains of annexin II, we found that C terminus is masked in the nucleus. In order to know the relationship between the masking of C terminus and the nuclear retention of annexin II, truncation mutation was used to delete the C terminal core domain of annexin II. After we expressed the truncated annexin II in Hela, Du-145 and PC-3, we found that C terminal masking is not responsible for the nuclear retention of annexin II. So, we proposed that there are other nuclear factors interacting with the N terminus that prevent the binding of CRM1 and nuclear export of annexin II.