Browsing by Subject "proteins"
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Item Altering the oligomerization state of GFP-like purple protein to enhance protein taggin ability(2009) Kuhn, Samantha; Scott StevensSince its discovery and purification in the 1960s, Green Fluorescent Protein has quickly become an extremely useful scientific tool for analyzing protein expression and dynamics. GFP was originally discovered in a species of jellyfish, Aequorea victoria, apparent only in response to a blue flash of bioluminescence. Upon examining its structure, scientists soon found that the source of GFP’s color was an autocatalytically formed chromophore at the center of its β-barrel structure. Because of this autocatalytic mechanism of fluorescence, the GFP gene alone can be attached to proteins of interest and used to track expression and movement in vivo. While GFP itself was a breakthrough in the biotechnology world, it is hardly the end of the line as far as fluorescent proteins go. In recent years, scientists have been searching for and finding more members of the GFP-family that have the same autocatalytic property but display different colors. The introduction of new colors to the library of fluorescent proteins offers great advantages in multiple protein tagging and studying protein interactions. However, most fluorescent proteins that have been found to date have been tetramers, consisting of four identical subunits. This quaternary structure makes them ineffective as protein tags. Researchers have recently been exploring ways to alter the oligomer ization state of fluorescent proteins and create dimeric and monomeric forms the can be useable tags. In my experiment, I attempted to do so in a GFP family protein from species Galaxea fascicularis and Montipora efflorescens known as purple protein. There is not a protein tag that emits is colored purple currently, and so my goal was to reduce the oligomerization state of purple protein, assuming that it is a tetramer, while maintaining its purple color. To do this, I used site-directed mutagenesis techniques to change specific amino acids in the protein that were predicted to be involved in formation of tetramers. All three mutations made in the sequence affected the spectral qualities, as the cells they that the mutants were transformed into showed no purple expression. One mutation resulted in a protein that migrated at half the relative molecular weight of the wild type protein in a gel filtration column, and thus we were successful in reducing the oligomerization state of the purple protein through that mutation. While it is not currently useful as tag, the successful mutant should be used in future experiments to isolate a functional monomer of the purple protein, adding to the library of protein tag colors.Item Cloning of wheat germ eukaryotic initiation factor eIF2(2009) Parekh, Priyanka; Karen BrowningEukaryotic Initiation Factor 2 (eIF2) is a protein complex found in eukaryotes involved in the initiation of translation. eIF2 consists of three non-identical subunits (α, β, and γ) and is required for the translation of virtually all mRNA transcripts. eIF2 forms a ternary complex with GTP and Met-tRNAi Met, which then associates with the 40S ribosomal subunit. This 43S preinitiation complex scans the mRNA transcript for the AUG initiation codon, at which time eIF2’s GTP is hydrolyzed to GDP and eIF2-GDP dissociates from the 40S subunit. A guanine nucleotide exchange factor called eIF2B is required to reform eIF2-GTP, which can now facilitate a new round of translation initiation. In many eukaryotic systems, eIF2 has been documented as a site of regulation of translation via phosphorylation at a conserved serine residue on the α subunit. These studies have found that phosphorylated eIF2 (eIF2[αP]) binds eIF2B with a much higher affinity than non-phosphorylated eIF2. Since eIF2B is typically present at less than half the cellular concentration of eIF2, the result of this strong binding is sequestration of eIF2B and inhibition of its nucleotide exchange activity. Because only eIF2-GTP can bind Met-tRNAi Met, the result is inhibition of eIF2’s ternary complex formation, and thus inhibition of protein synthesis. Most studies exploring this mechanism of translational control have focused on yeast and mammalian systems. However, in higher plants, regulation of translation by phosphorylation of eIF2α has not been shown to occur in vivo to date. Although homologous genes of all five eIF2B subunits have been identified in plants, the eIF2B protein complex has not yet been isolated from plants. Similarly, though a gene with homology to an eIF2α-kinase from yeast (gcn2) has been identified in plants, phosphorylation of eIF2 in a plant translational lysate system has not yet been shown to inhibit translation. The difference of the binding affinity of eIF2 for GDP and GTP is not nearly as severe as that found in other eukaryotic systems, suggesting that a recycling factor (eIF2B) and phosphorylation of eIF2α may play a less critical regulatory role in plants. There are significant differences between plants and animals in their responses to environmental stress, and therefore methods of translational regulation may also be different. In order to explore the extent of regulation via phosphorylation of eIF2α in plants, and to uncover other plant-specific regulation on eIF2, it is necessary to be able to express the eIF2 complex in a manipulable bacterial system. This will allow mutagenesis of the three subunits to probe structure and function. This thesis reports the use of recombinant methods to construct a single expression vector containing all three genes of eIF2 from T. aestivum (wheat), with the goal of subsequent expression of eIF2α, β, and γ as a functional complex in E. coli.Item Effect of nucleotide binding on Rad50 conformational state, multimeric state, and DNA binding ability(2009-05) Estrin, Eric; Tanya T. PaullThe ability of an organism to swiftly repair double-stranded breaks (DSBs) in DNA is a crucial process that, if absent, can result in genomic instability. The Mre11/Rad50 protein complex is highly conserved and plays a key role in sensing, processing, and repairing DNA DSBs. Rad50 is known to be necessary for the DNA end-bridging catalyzed by the MRN complex, and association of the broken DNA ends is essential to prevent loss of chromosome fragments in vivo. The Rad50 protein contains a large coiled-coil domain and ATP-binding motifs, with an overall structure similar to the Structural Maintenance of Chromosomes (SMC) family of proteins. Rad50 undergoes ATP-dependent homodimerization, which creates a potential DNA binding cleft. Recently, Rad50 has been shown to have adenylate kinase activity in addition to the previously known ATPase activity. It is still unknown what role ATP-induced dimerization and conformational changes play in pfRad50’s various activities. We hypothesize that dimerization is needed for DNA binding, and that Rad50 requires large conformational changes for proper function in its part in pfMR exonuclease activity. Here, we use site-directed mutagenesis to create Rad50 mutants that have cysteines placed in structurally relevant portions of the protein. With these cysteines, we used disulfide crosslinking, fluorescence, and Fluorescence Resonance Energy Transfer (FRET) to detect if changes in conformation or multimeric state of Rad50 are necessary for adenylate kinase activity, ATPase activity, and DNA binding.Item Expression and purification of recombinant wheat translation initiation factor eIF2(2010) Tzeng, Shinetin; Karen BrowningProteins carry out the activities for cellular growth and maintenance in a timely and space-specific manner, so the formation of proteins from their genetic counterparts, or translation, must be tightly controlled. Regulation occurs primarily during the initiation step of translation, a highly involved process that properly positions messenger RNA, methionine-charged initiator transfer RNA, and ribosomes via a family of proteins known as eukaryotic initiation factors (eIFs). One of these proteins is eIF2, a trimeric complex made of eIF2α, eIF2β, and eIF2γ. In mammalian and yeast systems, the phosphorylation of eIF2α by specific kinases and its downstream effects down-regulate global protein synthesis, and the mechanism has been widely studied. However, in plants, evidence suggests that different regulatory pathways may be involved. To ultimately describe the molecular and biochemical basis by which higher plants regulate protein synthesis, studies of mutant eIF2α and the effects of phosphorylation are necessary. To achieve this goal, genes for the three subunits of wheat eIF2 were synthesized de novo by overlapping oligonucleotides that were optimized for E. coli codon usage, and then the genes were cloned into an operon for expression of the complex in E. coli. This particular study focuses on optimizing the conditions for expression and purification of soluble eIF2 complexes. Alterations in the strain of E. coli, growth condition and media, and operon construct were tested to enhance eIF2 expression and solubility, and different methods of purification were applied. However, at this point, the expression and purification of eIF2 still remains unsuccessful.Item Functional and genetic interactions of Ribosomal-like Protein 24(2008-05) Helmke, Kara; Arlen JohnsonNearly all the processes within a cell are mediated by proteins: metabolism, movement, growth, and even death. These proteins are generated by a type of intracellular machinery called the ribosome which translates the cell’s genetic code into a functional protein. But where and how are ribosomes generated? Current research indicates that ribosomes are first made into two separate parts near the genetic material stored in the cell’s nucleus. The new subunits are passed through a discriminating gateway called the Nuclear Pore Complex (NPC) that separates the nucleus from the remainder of the cell’s interior, or cytoplasm. The ribosomes are very large, however, and cannot pass through the NPC on their own. It has been found that some proteins function to “chaperone” the ribosomal subunits out of the nucleus by assisting interaction between the ribosome and the NPC. We suspect a certain protein in yeast, Ribosomal-like Protein 24 (Rlp24), is involved in this export pathway. Its specific function is not yet known, but it has been previously determined that it binds to the ribosome in the nucleus and then is unloaded in the cytoplasm. In order to determine if this protein has a role in ribosome export, several genetic and functional tests were performed. In the first experiment, an altered form of Rlp24 with a molecular tag was expressed in cells that are deficient in specific proteins that comprise the Nuclear Pore Complex. A change in growth with these mutations suggests a functional interaction between the two proteins. Next, a screen for a gene that would restore normal growth in these mutants was done, suggesting that any of the potential genes also interact in the pathway. Finally, a special screen called a Yeast Two-Hybrid assay was used to identify proteins that actually bind to and function with Rlp24. From this series of tests, it was determined that Rlp24 does in fact interact functionally with specific components of the Nuclear Pore Complex, several of which have previously been identified as interacting in known export pathways. Additionally, the other screens identified one novel genetic interaction as well as two functional interactors with Rlp24. Though one physical interactor, a protein called Nog1, has already been cited in literature, the role of the other interactors in export and their relationships with Rlp24 are not yet known. However, the information gathered highly suggests that Rlp24 has a role in export and provides a basis for further testing and characterization.Item High resolution transmission electron microscopy of green fluorescent protein(2009) Knobles, Micah; Brown, R. Malcolm, Jr.Using fluorescence and transmission electron microscopy (TEM), this study investigated the structure of a novel recombinant protein used in tagging molecules. This molecule, green fluorescent protein (GFP) exhibited unexpected aggregation phenomena. The fluorescence, an indicator of protein integrity, could be used to test exposure to conditions of the electron microscope. Negative staining with uranyl acetate was explored to demonstrate how TEM might be employed to observe biological samples with high resolution. Extensive circumstantial evidence suggests that discrete GFP molecules were resolved. This study demonstrated that protein structure can be observed with relatively simple negative staining techniques using uranyl acetate. It was determined that the electron beam was primarily responsible for the degradation of protein structure. Crystal formations with uranyl acetate and GFP produced highly ordered monolayers and complex aggregates. These aided in the analysis of the TEM images. Low dose electron beam exposure and the uranyl acetate stain likely protected the GFP sample. His tag labeling of the recombinant GFP sample with nickel-nitrilotriacetic acid Nanogold (Ni-NTA-Nanogold) was used to definitively identify GFP molecules with inconclusive results. Our findings indicate a newfound use of TEM and negative staining in high resolution transmission electron microcopy studies of globular proteins, biological polymers, and small molecules. With further refinements, these techniques would offer a valuable addition to the contributions of cryo-electron microscopy and X-ray crystallography. The work may help us to interpret complex aggregation phenomena and to increase our understanding of amyloid and prion diseases whose insoluble protein aggregates consisting of tightly packed beta sheets are not amenable to conventional structural biology techniques.Item The HOPS complex and Vps33 in Dictyostelium discoideum(2009) Caffarelli, Nicolas; Arturo De LozanneIn order for cells to eat, they must detect food at their cell surface and pull it in to form a vesicle known as an endosome. This endosome then becomes acidified to become a lysosome and eventually becomes neutral again, as a postlysosome, so that waste can be expelled from the cell. Collectively, this is referred to as the endocytic pathway. Several proteins are involved in this process – Rabs are known to mediate specificity of fusing vesicles, and SNARES catalyze the actual vesicle fusion. In this study we look at the protein Vps33, a subunit of the HOPS complex. The HOPS complex is known to interact with Rabs and SNARES, and we are interested as to where it acts within the cell. By tagging Vps33 with Green Fluorescent Protein (GFP), we can visualize its localization under the microscope. We observe here that Vps33 localizes primarily to the cytoplasm, with sparse localization to intracellular vesicles.Item Independent Inactivation of Arginine Decarboxylase Genes by Nonsense and Missense Mutations Led to Pseudogene Formation in Chlamydia Trachomatis Serovar L2 and D Strains(2009-07) Giles, Teresa N.; Fisher, Derek J.; Graham, David E.; Giles, Teresa N.; Graham, David E.Chlamydia have reduced genomes that reflect their obligately parasitic lifestyle. Despite their different tissue tropisms, chlamydial strains share a large number of common genes and have few recognized pseudogenes, indicating genomic stability. All of the Chlamydiaceae have homologs of the aaxABC gene cluster that encodes a functional arginine: agmatine exchange system in Chlamydia (Chlamydophila) pneumoniae. However, Chlamydia trachomatis serovar L2 strains have a nonsense mutation in their aaxB genes, and C. trachomatis serovar A and B strains have frameshift mutations in their aaxC homologs, suggesting that relaxed selection may have enabled the evolution of aax pseudogenes. Biochemical experiments were performed to determine whether the aaxABC genes from C. trachomatis strains were transcribed, and mutagenesis was used to identify nucleotide substitutions that prevent protein maturation and activity. Molecular evolution techniques were applied to determine the relaxation of selection and the scope of aax gene inactivation in the Chlamydiales. Results: The aaxABC genes were co-transcribed in C. trachomatis L2/434, during the mid-late stage of cellular infection. However, a stop codon in the aaxB gene from this strain prevented the heterologous production of an active pyruvoyl-dependent arginine decarboxylase. Replacing that ochre codon with its ancestral tryptophan codon rescued the activity of this self-cleaving enzyme. The aaxB gene from C. trachomatis D/UW-3 was heterologously expressed as a proenzyme that failed to cleave and form the catalytic pyruvoyl cofactor. This inactive protein could be rescued by replacing the arginine-115 codon with an ancestral glycine codon. The aaxC gene from the D/UW-3 strain encoded an active arginine: agmatine antiporter protein, while the L2/434 homolog was unexpectedly inactive. Yet the frequencies of nonsynonymous versus synonymous nucleotide substitutions show no signs of relaxed selection, consistent with the recent inactivation of these genes. Conclusion: The ancestor of the Chlamydiaceae had a functional arginine: agmatine exchange system that is decaying through independent, parallel processes in the C. trachomatis lineage. Differences in arginine metabolism among Chlamydiaceae species may be partly associated with their tissue tropism, possibly due to the protection conferred by a functional arginine-agmatine exchange system against host nitric oxide production and innate immunity. The independent loss of AaxB activity in all sequenced C. trachomatis strains indicates continual gene inactivation and illustrates the difficulty of recognizing recent bacterial pseudogenes from sequence comparison, transcriptional profiling or the analysis of nucleotide substitution rates.Item Letter to H.B. Stenzel from P.E. Hare on 1966-03-14(1966-03-14) Hare, P.E.Item Letter to P.E. Hare from H.B. Stenzel on 1966-02-28(1966-02-28) Stenzel, Henryk B.Item The Look-Ahead Effect of Phenotypic Mutations(2008-05) Whitehead, Dion J.; Wilke, Claus O.; Vernazobres, David; Bornberg-Bauer, Erich; Wilke, Claus O.Background: The evolution of complex molecular traits such as disulphide bridges often requires multiple mutations. The intermediate steps in such evolutionary trajectories are likely to be selectively neutral or deleterious. Therefore, large populations and long times may be required to evolve such traits. Results: We propose that errors in transcription and translation may allow selection for the intermediate mutations, if the final trait provides a large enough selective advantage. We test this hypothesis using a population based model of protein evolution. Conclusion: If an individual acquires one of two mutations needed for a novel trait, the second mutation can be introduced into the phenotype due to transcription and translation errors. If the novel trait is advantageous enough, the allele with only one mutation will spread through the population, even though the gene sequence does not yet code for the complettrait. Thus, errors allow protein sequences to "look-ahead" for a more direct path to a complex trait. Reviewers: This article was reviewed by Eugene Koonin, Subhajyoti De (nomimated by Madan Babu), and David Krakauer.Item A New Biological Ruler Toolkit: The BDP-A Chemical Crosslinker with Biotin Handle Allows for Modular Crosslinker Synthesis and Protein Interaction Studies(2021-03) Selinidis, Malyn; Marcotte, EdwardThis thesis details the synthesis of the BDP-A crosslinker as well as how this synthesis toolkit can be used to synthesize novel crosslinkers. In this research, BDP-A was successfully synthesized and used to characterize interactions between various peptides. This methodology used a microwave peptide synthesizer in addition to biochemical and organic synthesis techniques. Each synthesis step was confirmed using mass spectrometry. A novel crosslinker based off of the BDP-A model known as the PCA crosslinker was partially synthesized using this method. The PCA crosslinker uses a 2-pyridinylcarboxyaldehyde functional group such that it exclusively targets the N-terminus of proteins and therefore is tailored to exclusively characterize intercrosslinks between complexed proteins.Item Predicting Combinatorial Binding of Transcription Factors to Regulatory Elements in the Human Genome by Association Rule Mining(2007-11) Morgan, Xochitl C.; Ni, Sshulin; Miranker, Daniel P.; Iyer, Vishwanath R.; Morgan, Xochitl C.; Ni, Sshulin; Miranker, Daniel P.; Iyer, Vishwanath R.Cis-acting transcriptional regulatory elements in mammalian genomes typically contain specific combinations of binding sites for various transcription factors. Although some cisregulatory elements have been well studied, the combinations of transcription factors that regulate normal expression levels for the vast majority of the 20,000 genes in the human genome are unknown. We hypothesized that it should be possible to discover transcription factor combinations that regulate gene expression in concert by identifying over-represented combinations of sequence motifs that occur together in the genome. In order to detect combinations of transcription factor binding motifs, we developed a data mining approach based on the use of association rules, which are typically used in market basket analysis. We scored each segment of the genome for the presence or absence of each of 83 transcription factor binding motifs, then used association rule mining algorithms to mine this dataset, thus identifying frequently occurring pairs of distinct motifs within a segment. Results: Support for most pairs of transcription factor binding motifs was highly correlated across different chromosomes although pair significance varied. Known true positive motif pairs showed higher association rule support, confidence, and significance than background. Our subsets of high-confidence, high-significance mined pairs of transcription factors showed enrichment for co-citation in PubMed abstracts relative to all pairs, and the predicted associations were often readily verifiable in the literature. Conclusion: Functional elements in the genome where transcription factors bind to regulate expression in a combinatorial manner are more likely to be predicted by identifying statistically and biologically significant combinations of transcription factor binding motifs than by simply scanning the genome for the occurrence of binding sites for a single transcription factor.Item Protein interactome mapping of GTPBP9 (OLA1) to determine role in ribosome biogenesis(2009) Hsu, Stephanie; Scott StevensGTPBP9, originally only a predicted protein, is a poorly understood protein. GTPBP9 has only recently begun to be more specifically classified via its close relation to other better understood proteins, namely those of the Obg-like ATPase family. The yeast homologue of GTPBP9 has been implicated in ribosome biogenesis; as such, we were interested in the study of its function in metazoan cells. Traditionally, protein function is determined through analysis of its folding pattern, primary amino acid sequence and other characteristics unique to the protein. We will instead determine the probable function of our target protein GTPBP9 (OLA1) through protein interactome mapping. Rather than study a protein in isolation, the process of interactome mapping identifies protein activity by analyzing in vivo protein complexes within which a protein functions. Proteins of similar activity cluster together to create these complexes that provide greater functional efficiency. Classifying the interacting proteins in a complex helps with our understanding of our target protein’s probable activity. To identify and purify our target protein in complex, we utilized several novel techniques available for targeted gene manipulation. CLEP tagging is the insertion of an epitope tag, or an antibody target region, via targeted sequence insertion upstream of a gene’s stop codon. CLEP tagging differs from previous epitope tagging techniques in that it minimally alters the native gene sequence by inserting the tagging sequence just prior to the stop codon rather than replacing the entire gene sequence with one intronless homologue. Our lab has previously demonstrated success with insertion of TAP-tag sequences for protein identification and isolation. Thus, we chose to use a TAP-tag construct containing neomycin resistance as our tagging construct. Utilizing the bacterial Red DNA repair system enzymes, we inserted our TAP-tag construct into a BAC vector host via a process called recombineering. Recombineering utilizes the Red DNA repair system to initiate homologous recombination between transgenes and bacterial chromosomes. The use of recombineering allowed us to insert our TAP-tag construct into the Gallus gallus GTPBP9 sequence hosted in a BAC vector. Electroporation of chicken DT40 pre-B cells in the presence of these altered BAC vectors triggered a second round of homologous recombination, this time between the Gg regions of the BAC vectors and the DT40 cell chromosome. This resulted in DT40 cells expressing TAP-tagged GTPBP9, confirmed via Western Blot analysis. The expression of the TAP-tag on our target protein allowed for the easy isolation of our protein in complex from cell culture via binding affinity and column chromatography. Fractionation analysis and mass spectrometry analyses of both GTPBP9 and the proteins isolated in complex with it would allow for greater understanding of GTPBP9’s probable role in cellular functioning. However, by the time we were able to perform such analyses on our samples, the cells had undergone gene silencing; thus our construct was unable to be isolated. Because of our success in inserting the construct into metazoan cell lines and the nature of our time constraints, we hope that future attempts will experience greater success in maintaining TAP-tag expression.Item Stability of C-Reactive Protein in saliva using an ELISA test(2008) Peddaiahgari, Rajesh; John McDevittThis project was done in order to measure the stability of proteins in general; in particular C-Reactive Protein (CRP) which is very important in cardiovascular studies. Current methods of handling CRP is to freeze the sample at -80°C for long term use, or -20°C for medium term use, or 4°C for short term use. This experiment was done in order to measure how much CRP will degrade after a certain time with different matrices. From this project, no conclusive data was gathered to give a certain approximation on the stability of CRP. The data that was gathered did tell us that CRP is most stable with PBS with 0.5% Albumin (PBSA) than saliva, which is more stable than in nanopure water. Furthermore, the data showed that samples kept at -20°C were more stable than those kept at 4°C.Item Synthetic Aminopyrrolic Receptors have Apoptosis Inducing Activity(2015-09) Park, Seong-Hyun; Choi, Yoon Pyo; Park, Jihong; Share, Andrew; Francesconi, Oscar; Nativi, Cristina; Namkung, Wan; Sessler, Jonathan L.; Roelens, Stefano; Shin, Injae; Sessler, Jonathan L.We report two synthetic aminopyrrolic compounds that induce apoptotic cell death. These compounds have been previously shown to act as receptors for mannosides. The extent of receptor-induced cell death is greater in cells expressing a high level of high-mannose oligosaccharides than in cells producing lower levels of high-mannose glycans. The ability of synthetic receptors to induce cell death is attenuated in the presence of external mannosides. The present results provide support for the suggestion that the observed cell death reflects an ability of the receptors to bind mannose displayed on the cell surface. Signaling pathway studies indicate that the synthetic receptors of the present study promote JNK activation, induce Bax translocation to the mitochondria, and cause cytochrome c release from the mitochondria into the cytosol, thus promoting caspase-dependent apoptosis. Such effects are also observed in cells treated with mannose-binding ConA. The present results thus serve to highlight what may be an attractive new approach to triggering apoptosis via modes of action that differ from those normally used to promote apoptosis.Item Using molecular modeling to engineer proteins with novel functions(2007) Codrea, Vlad; Andrew D. EllingtonThe RCSB Protein Data Bank currently stores over 30,000 X-ray crystal structures, information that has proven invaluable in various studies that have been undertaken to analyze these proteins. My goal is to apply molecular modeling techniques in order to add new functionality to enzymes whose 3D structures are available. The standard processes for modifying the functions of proteins have traditionally remained centered on experimental work, with a heavy reliance on directed evolution. One such approach has been used by Professor Peter Schultz from the Scripps Research Institute to introduce the unnatural amino acid 3-(2-naphthyl)-L-alanine into the amber stop codon of E. coli. This approach uses positive and negative selection to wean the cells into taking up 3-(2-naphthyl)-L-alanine. In contrast, I have used a computational method to predict mutations in aminoacyl-tRNA synthetases, the enzymes responsible for joining amino acids with their respective tRNAs, that will change the affinities of those proteins. My goal is to arrive at aminoacyl-tRNA synthetases that are capable of incorporating analogs of arginine, cysteine, phenylalanine, and tryptophan instead of the natural residues. As a follow-up, I have prepared a mutant tyrosyl-tRNA synthetase and tRNA to test how well an analog of tyrosine is incorporated into proteins. I have used an in-silico protein modeling framework developed by Professor Homme Hellinga from Duke University to tackle another outstanding problem in molecular biology: how to recreate the high affinity of the streptavidin/biotin complex. The specificity of streptavidin is changed so that it binds preferentially to biotin derivatives. Streptavidin is a tetrameric protein, similar to the avidin protein found in egg whites, but made by the Streptomyces avidinii bacteria. Streptavidin binds tightly to the vitamin D-biotin, and forms one of the strongest naturally-occurring non-covalent interactions between a protein and an organic ligand, with a dissociation constant (Kd) on the magnitude of 10-15 M. Because of this strong binding, the streptavidin/biotin combination has been used extensively in molecular and bioengineering studies that require the joining of different molecules that would not normally come together. One of the current limitations with using this combination is that it is only possible to specifically bring together two compounds (namely the compound attached to biotin and the compound attached to streptavidin) at any one step of an assay. The aim is to engineer orthologous pairs of mutant streptavidins and biotin analogs, each of which can be covalently attached to a distinct molecular payload depending on the end-user’s intended application. The members of one orthologous pair will not cross-react with the complementary member of another orthologous pair – in other words, a mutant streptavidin should have a relatively poor binding affinity to biotin. This provides an element of selectivity to parallel reactions performed in the same environment.Item What makes Bright a proto-oncogene?(2009-05) Mathur, Shawn; Philip W. TuckerCancer is now the second leading cause of death in the developed world and accounts for 1 out of every 4 deaths in the US (American Cancer Society). The fundamental defect of cancer is unregulated cell division. One way this can happen is from an increase in the activity of proteins which signal cell growth. These proteins, called proto-oncogenes, possess normal functions in cells but can induce unregulated cell division when permanently activated by mutation. Understanding the molecular biology behind proto-oncogenes may lead to insight into how cancer develops and aid in the development of better treatment options for cancer patients. We are interested in studying Bright, a proto-oncogene expressed in mouse B-cells, white blood cells which are an essential part of the immune system. Bright can induce proliferation when overexpressed in normal cells. Our ultimate goal is to understand the mechanism by which Bright causes cells to become cancer-like. Bright’s oncogenic activity is correlated with an increase in function of E2F1, a protein considered to be a master regulator of cell division. E2F1 controls the transcription of many genes needed to drive the cell cycle. Specifically, we are interested in understanding how Bright upregulates the ability of E2F1 to activate transcription of its target genes. Various lines of evidence suggest that Bright may disrupt the action of pRb/E2F1 complexes which normally repress E2F1 production at the E2F1 promoter. Several biological studies have been performed to investigate this possibility and to learn more about the action of Bright. In our first experiment, we determine which properties of Bright are necessary to transform cells. Second, we show that Bright can associate with pRb, suggesting that Bright may play a role in E2F1 regulation. Finally, chromatin immunoprecipitation assays suggest that Bright associates with the E2F1 promoter and may indeed affect the function of pRb/E2F1 repressive complexes. Thus, we provide evidence in support of a novel model for Bright’s oncogenic properties. Our findings are particularly relevant for patients with Activated B cell-like Diffuse Large B-cell Lymphoma, a highly pernicious cancer that appears to involve the human version of Bright. Further studies to explore the nature of Bright's interaction with the Rb/E2F1 protein complex may lead to a better prognosis for these patients.