http://digital.library.wisc.edu/1793/7278 http://minds.wisconsin.edu:80/retrieve/7095 http://digital.library.wisc.edu/1793/7278 http://digital.library.wisc.edu/1793/23549 MHC class I allele characterization in Indonesian cynomolgus macaques Pendley, Chad There has recently been increased interest in the use of cynomolgus macaques for the study of infectious diseases. However, little focus has been given to studying the immunogenetics of these animals. To improve researcher’s understanding and utilization of these model organisms, we characterized 48 full length MHC (Major Histocompability Complex) alleles in Indonesian cynomolgus macaques via cloning and sequencing. Furthermore, we found that three of these alleles were originally found in cynomolgus macaques of Mauritian origin, supporting the hypothesis that Mauritian animals originally came from Indonesia. The Mauritian alleles, Mafa-B*4501,Mafa-B*5101, and Mafa-B*1201 present in 19%, 7% and 4% of our cohort respectively according to PCRSSP results. 12 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8136 Decreased production of superoxide by isolated retinal ganglion cell mitochondria Hoegger, Mark J. YOU CANNOT OPEN THIS PAPER. In accordance with the author's wishes, it is not available to the public. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8134 Stable expression and purification of Juvenile Hormone Binding Protein from Drosophila melanogaster Schneider line-2 cells Tan, Si Hui Studies on the hemolymph Juvenile Hormone Binding Protein (hJHBP) and Juvenile Hormone (JH) interaction have been considerably limited by the availability of pure hJHBP. An expression vector was created by cloning the recombinant JHBP (rJHBP) gene into pMT/BiP/V5-HisB vector and stably transfecting it into Drosophila melanogaster Schneider line-2 cells. The cells can be induced by copper sulfate to produce large quantities of rJHBP that is secreted into the medium. The rJHBP can be purified by passing it through a His-Tag column. Subsequent treatment with recombinant Enterokinase yields a protein that is similar to hJHBP in size. Results from radioactive binding assay affirm the functionality of rJHBP because it is able to bind JH rather efficiently. These suggest that rJHBP is folded and glycosylated in a manner similar to that of native hJHBP in Manduca sexta. This is the first successful instance of inducible expression of rJHBP in vitro. 21 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8132 Identification of the Functional Domains of Arabidopsis FLS2 Jansen, Kristin Lynn Arabidopsis FLS2 is a leucine-rich repeat (LRR) transmernbrane receptor-kinase that recognizes bacterial flagellin and induces plant innate immune responses. Flg22 is a 22 amino acid peptide based on a sequence of flagellin that is highly conserved among bacteria. FLS2 directly binds flg22 and then elicits plant defense responses. A set of residues in the FLS2-LRR domain were chosen for study based on their degree of evolutionary conservation among FLS2 proteins from diverse Brassicaceae species. These residues were examined using site-directed mutagenesis to identify the functional regions ofFLS2. Surprisingly, none of these mutations strongly disrupted FLS2 function. This is in contrast to the significant impacts on defense that have been observed with some mutations in the ~-strand/~-tum region of the FLS2-LRR. Additionally, putative N-glycosylation sites were disrupted to determine the role of glycosylation in the structure and/or function ofFLS2. 16 of the 17 mutations did not strongly disrupt FLS2 function, suggesting that these are not crucial N-glycosylation sites. One of these 17 mutations was found to cause decreased flg22 response and abnormal FLS2 expression. The LRR structural motif is common in plant immune receptors, and also in mammalian immune systems and proteins that control other aspects of growth and development, making knowledge about protein interactions involving LRRs beneficial both for plant and mammalian systems. 43 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7985 Engineering of maltose-binding protein to employ a poly-arginine tag and improve protein purification Wochinski, Abby Maltose binding protein (MBP), a solubility-enhancing fusion protein, has demonstrated great success in the application of protein purification when fused to a protein of interest. It has already been engineered to include a poly-histidine tag to allow for purification via Immobilized Metal Affinity Chromatography (IMAC). I have engineered this protein to include a poly-arginine tag, which would promote a cation-exchange purification step while maintaining the solubility-enhancing property of the original MBP. However, the inclusion of the poly-arginine tag did not significantly impact the elution profile of MBP during cation-exchange chromatography. This lack of purification suggests that the interactions between the positively charged arginine residues and .the negatively charged compounds on the resin were insufficient to affect the salt dependent binding of MBP. 17 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7981 Identification of two binding partners for the bacterial actin homolog MreB Buske, Paul A distinguishing characteristic of prokaryotes was long thought to be the lack of a cytoskeleton similar to eukaryotes consisting of actin filaments, microtubules, and intermediate filaments. However, recent work has shown the existence of bacterial proteins homologous to the eukaryotic cytoskeleton. One such protein, MreB (murein cluster e), encircles the bacterial cell and exhibits structural homology to actin. Because of the homology between MreB and actin and the many interacting proteins with which actin has in its various cellular functions, we hypothesized MreB also interacts with many proteins in the bacterial cell. Preliminary interaction tests using light scattering fluorometry and co-sedimentation assays show interactions between purified E. coli MreB and the His-tagged proteins NusG and EF-Tu, with work still to be done on identification of other interacting partners and the biochemical nature of theses interactions. 25 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7973 A genetic approach to understanding co-regulated MAPK genes in Arabidopsis thaliana Ho, Bonnie Yiu Mitogen-activated protein kinase (MAPK) signaling cascades connect cellular signal and gene transcription in cells. The pathway can regulate cell division, cell growth, and other pathways in eukaryotes. We used the Comprehensive Systems Biology Project database to find out that MPK1, 2, and 6 have a similar co-response level, as do MPK8, 19 and 20. We hypothesized that the genes in each group serve similar functions and would respond to stress similarly in Arabidopsis. We created a homozygous knockout mutant in MPK1, 2 and 6, and another one in MPK8, 19 and 20. Under normal growth conditions, the triple mutant seedlings appeared identical to wildtype seedlings. We compared the mutant and wildtype seedlings in different stress conditions (0.3 uM kinetin, 2% sucrose, 5% sorbitol, ethylene and 14°C chilling). We measured root growth of light-grown seedlings in the first three and chill treatments and hypocotyls length for sucrose-treated and ethylene-treated dark-grown seedlings. Results showed that there was no significant difference in the root or hypocotyls length growth between wildtype and mutant seedlings in all the experimental conditions. Thus, MPK1, 2, and 6 are not required in responding to stress based on the MPK1, 2, 6 triple mutant; MPK8, 19 and 20 are not required in the stress response cased on the MPK8, 19, 20 triple mutant. Future experiments have to be done to give information of their functions. This project shows a reverse genetic approach to study gene function. 16 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7965 Characterization of ZIIR element of BZLF1 promoter in the context of the whole Epstein - Barr Virus genome Lim, Hui Jun Latent infection of Epstein-Barr virus (EBV) is associated with many human cancers. Induction of lytic replication of EBV may lead to destruction of these tumor cells. The viral immediate-early gene, BZLF1, plays a key role in switching EBV infection from latent to lytic replication. Expression of the BZLF1 gene is down-regulated by a potent silencer element, ZIIR, located in the BZLF1 promoter (Zp). I introduced a 6-bp substitution mutation into the ZIIR element of Zp by mutagenesis of the genome of EBV strain B95.8, and studied the phenotype of this mutation in the context of the whole EBV genome. The human embryonic kidney cell line 293 was transfected with ZIIR mutant (ZIIRmt) EBV DNA, and independent clones of latently infected cells were grown out into cell lines. Strikingly, 293 cells latently infected with ZIIRmt spontaneously replicated EBV DNA and produced infectious virus, while cells latently infected with wild -type (WT) did not. Immunoblot analyses showed that ZIIRmt -infected 293 cells accumulated at least 30-fold more viral immediate-early proteins Zta and Rta and early protein BMRF1 than the WT-infected cells. Immunofluorescence staining confirmed the abundant presence of Zta, Rta, BMRF1 and viral late proteins gp125 and gp350 in ZIIRmt-infected cells, while these proteins were not detectable in WT-infected cells. Thus, the ZIIR element of Zp is crucial in the regulation of EBV's switch from latency to lytic replication. 32 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7957 Isolation and characterization of Drosophila melanogaster central processing mutants Tang, Grace The aim of this study is to isolate and characterize mutant fruit flies (Drosophila melanogaster) which are defective in central processing, with the ultimate goals of! finding the genes necessary for this process to ftInction, investigating what these genes do, and elucidating the mechanisms of central processing. Mutants that are deficient in central processing, or Type I mutants, are flies that are unable to process any form of sensory information that they receive from their environment, although their sensory receptors are intact. Twenty potential Type I mutants were found, but only one passed the Type I trait to the Fl generation. Further work is being done on this strain, M13, to map the mutant gene and characterize it, i.e. determine its response to other sensory modalities, measure its motility and geotaxis, etc. Assays for measuring responses to various sensory modalities, namely light, benzaldehyde and heat, have been developed, and responses of wild type flies in these assays were recorded. Future work in this study will include finding more Type I mutants, characterizing them, and mapping the genes involved in central processing, as well as development of further assays. 32 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7955 Optimization of recombinant E. coli protein expression in growth medium Duvnjak, Petar With the onset of novel genomic sequence data, much effort is now being taken to discover the role of many genes and the corresponding proteins that remain unknown. One of the limiting steps in such efforts has been the production of recombinant proteins in E. coli using bacterial expression systems. In this current work, several studies were initiated to address the issues affecting recombinant protein expression. These studies specifically focus on the aspects of protein expression and solubility levels, bacterial growth medium design, and expression vector design. While earlier attempts were aimed at comparing commercial expression systems (T5 and T7 RNA polymerase-based systems), later work on vector design showed that vectors with lower lac repressor dosing were optimal for the expression of higher levels of protein in E. coli. Similar collaborative work focused on a factorial-designed growth medium experiment. Results showed optimal growth medium conditions for the expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein (eGFP). Our current work is being directed at the comparison of expression levels and solubility for proteins expressed in vectors containing the Trigger Factor versus maltose binding protein fusion proteins. 46 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7953 Tanscriptome comparison of ionizing radiation resistant strain of Escherichia coli, CB2000, and naturally occurring strain of Escherichia coli, Founder Tan, Meng Kwang Marcus CB2000 is a strain of Escherichia coli (E. coli) that is extremely resistant to ionizing radiation. It was derived from a colony, referred to as the founder strain, isolated from naturally occurring Escherichia coli K12 MG1655. Using microarray technology developed by Affymetrix, a transcriptome comparison was done on CB2000 and Founder to determine the gene(s) that are differentially expressed in CB2000. A large number of genes which are involved in metabolism, transport and stress response were identified to display changes in expression levels in CB2000 when compared to Founder. 15 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7301 The effect of neuropeptides on growth and function of bovine articular chondrocytes Herzog, Amanda Cartilage injury is a central issue in orthopedics to which there is no known physiological treatment. Neuropeptides playa role in the proliferative and reparative processes of many tissue types, but little is known about their effects on articular cartilage. The purpose ofthis study was to investigate the effects ofthe neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP) on the growth and metabolism of bovine chondrocytes cultivated in monolayer culture. Proliferation and DMMB assays were conducted over an eight day period. Insulin-like growth factor-l was utilized as a positive control to validate the monolayer model. While CGRP showed little effect and NPY showed an inhibitive effect on proliferation, VIP demonstrated some ability to positively augment cartilage (n=8). Finally, SP showed the greatest reparative potential due to the significant increase in both proliferation and glycosaminoglycan production after SP 5 ug/ml. treatment. 25 p. Sun, 29 Oct 2006 22:58:59 GMT