2008 REU Research Projects and Mentors
Microtubule formation in Foraminifera
Our laboratory studies aspects of cell motility, both basic and applied. We use an ancient group of single-celled organisms, called Foraminifera, as a model system. These protists occur worldwide, but those best suited for our studies are giant ones from Antarctica. Our goal is to understand how these organisms accomplish remarkable architectural feats in building their shells, and to exploit their 800+ million-year evolutionary history for biotechnological and medical uses.
One unique feature of the Foraminifera is their ability to assemble tubulin -- the protein that forms microtubules -- into helical filaments. In a nutshell, foraminifera reversibly transform their microtubules into springs, and we think that this transition can do useful work (like a nano-scale biomolecular solenoid). We now have genomic DNA from species representing the full taxonomic range of foraminifera, and have developed PCR primers that specifically amplify their tubulin genes. Our immediate goal is to map sequence divergence in foraminiferal tubulin in order to better understand the molecular details of helical filament formation. A summer student involved in this project will become acquainted with methods ranging from PCR and molecular modeling to scanning electron microscopy. They will also get hands-on experience working with some of Nature's most amazing cells.
Virus assembly and the host-pathogen interface
Many viruses have evolved mechanisms to not only counter, but even exploit the cellular reaction to infection. During infection, several stress pathways are activated in response to incoming nucleic acids, changes in the amount of unfolded proteins, and/or the oxidation state of the cell. We have recently shown that Herpes Simplex Virus Type-1 (HSV-1) has evolved mechanisms to sequester various stress-activated chaperone molecules within discrete foci in the infected cell nucleus. We are interested in 1) whether these chaperone molecules are required for virus-specific mechanisms, such as virus assembly or DNA encapsidation, and 2) whether the redistribution of these stress factors results in a temporary anti-apoptotic state that is beneficial for virus production. Our research will provide information about chaperone-dependent viral processes and may reveal connections between viral infection and the activation of cancer-related anti-apoptotic pathways. Moreover, unique interactions made between viral proteins and cellular chaperone molecules may represent a new frontier of targets for antiviral therapies directly aimed at the host-pathogen interface. REU students would likely learn fluorescence microscopy, virus yield assays, tissue culture of mammalian cells, and Western Blotting.
Relevant references:
- Burch A.D., and Weller S.K. (2005). Herpes Simplex Virus Type 1 DNA polymerase requires the mammalian chaperone Hsp90 for proper localization to the nucleus. J Virol. Aug; 79(16):10740-9.
- Burch A.D., and Weller S.K. (2004). Nuclear sequestration of cellular chaperone and proteasomal machinery during Herpes Simplex Virus Type 1 infection. J Virol. Jul; 78(13):7175-85.
Using yeast to identify new targets for the treatment of AIDS
Human immunodeficiency virus-1 (HIV-1) is a retrovirus that is the causative agent of the AIDS global pandemic. Although a variety of antiretroviral treatments directed against HIV-1 proteins have been developed, the majority of infected people become resistant to these drugs over time. Consequently, there is no cure for AIDS. In my laboratory, we are interested in identifying cellular genes that may serve as novel drug targets for AIDS treatments. Because cellular genes do not mutate rapidly and are not transferred between individuals, they may have the potential to be more effective targets for antiretroviral drugs.
As a model system to understand how the host cell controls retroviruses such as HIV-1, we use a retrotransposon in brewer's yeast known as Ty1. The Ty1 retrotransposon replicates by the same mechanism as HIV-1 and other retroviruses, but it is not infectious. In the last several years, we have identified a large number of candidate genes that control the replication of Ty1, and several of these have been implicated in the control of retroviruses in human cells. Hence, these are potential target genes for the development of novel antiretroviral therapies. The mentor and student will choose several of these genes, and the student will perform genetic and biochemical assays to understand how Ty1 replication is altered when the genes are deleted in yeast. Different steps in Ty1 replication will be compared in mutant and wild-type strains. Assays to be used may include Western blotting to measure Ty1 protein levels, flow cytometry and fluorescent microscopy to quantify and localize Ty1 proteins, Southern blotting to measure Ty1 cDNA and quantitative PCR assays to measure cDNA integration. The student's findings will validate these genes as regulators of retrotransposon activity and provide a paradigm for the potential role of homologous human genes in HIV-1 replication.
Mycobacterial Conjugation
Lateral gene transfer (LGT) plays a critical role in the dissemination of genes associated with drug resistance and virulence among bacteria. LGT is mediated by one of three processes, conjugation, transformation and transduction, examples of which have been described for almost all bacterial species. We have identified a novel conjugal DNA transfer process in the non-pathogenic Mycobacterial species Mycobacterium smegmatis, and are using M. smegmatis as a model organism to study this process. This will allow a better understanding of the mechanism of genetic exchange and its contributions to the spread of drug resistance among the mycobacterial pathogens, including Mycobacterium tuberculosis. M. tuberculosis accounts for more deaths worldwide than any other infectious agent; it infects over 1/3 of the world's population and is estimated to cause ~1.6 x106 deaths annually. The development of new treatments for M. tuberculosis requires an understanding of the biology of these bacteria and the ability to manipulate their genomes to determine the genetic basis of pathogenesis and drug resistance.
This project involves the characterization of DNA transfer between strains of mycobacteria. In particular, we wish to identify the genes and DNA sequences required for DNA transfer and its regulation. This information will allow us to determine its prevalence among the Mycobacterial pathogens and possibly utilize conjugation as a molecular genetic tool to manipulate their genomes. We have already established a set of donor and recipient genes necessary for DNA transfer in M. smegmatis, many of which are conserved in M. tuberculosis. This REU project will involve their further characterization and will involve a variety of molecular techniques including, transformation, electroporation, conjugation, cloning, DNA sequence analysis and transposon mutagenesis of mycobacteria and general bacterial genetics in E. coli.
Relevant references:
- Parsons, L. M., Jankowski, C, and Derbyshire, K.M. (1998) "Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis." Molecular Microbiology 28, 571-582.
- Wang, J., Parsons, L.M., and Derbyshire, K.M. (2003) "Unconventional conjugal DNA transfer in mycobacteria". Nat Genet 34: 80-84.
- Flint, J., Kowalski, J., Karnati, P. and Derbyshire, K.M. (2004) "The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis" Proc. Natl. Acad. Sci. U.S.A. 101, 12598-12603
Cellular Biology of Glycoprotein Hormone Signaling
Glycoprotein hormones produced by the pituitary gland, including the pituitary hormone follitropin, control reproduction. Follitropin or follicle stimulating hormone (FSH) is produced in the pituitary gland, courses through the blood and acts at granulosa cells of the ovary in females and Sertoli cells of the testis in males. FSH acts by binding to its receptor, causing the release of a stimulatory G-protein, which can activate adenylate cyclase. This leads to the production of a second messenger, cAMP. The cAMP produced can bind to protein kinase A, and this causes the kinase to be activated. The activated kinase can then phosphorylate downstream targets, controlling cellular processes such as proliferation and steroid production. We are interested in proteins in the cytoplasm that are involved in FSH mediated signal transduction. We think that alternative pathways of hormone regulation exist besides the canonical adenylate cyclase/cyclic AMP/protein kinase A pathway. The incoming student will work with scientists in the lab, to help us understand how some of the proteins that we have identified, modulate FSH action. Methods learned will include western blots, molecular cloning, and cell culture. These studies should provide a better understanding of G-protein coupled receptor-signaling pathways and also clarify how FSH controls gonadal cell proliferation and steroidogenesis.
References:
- Nechamen CA, Thomas RM, Dias JA (2007) APPL1, APPL2, Akt2 and FOXO1a interact with FSHR in a potential signaling complex. Mol Cell Endocrinol. 2006 Oct 7; [Epub ahead of print]). PMID: 17030088
- Ulloa-Aguirre A, Uribe A, Zarinan T, Bustos-Jaimes I, Perez-Solis MA, Dias JA. (2007) Role of the intracellular domains of the human FSH receptor in G(alphaS) protein coupling and receptor expression. Mol Cell Endocrinol. 2007 Jan 2;260-262:153-62. Epub 2006 Oct 12. PMID: 17045734
- Thomas,RM, Nechamen CA, Mazurkiewicz, JM, Muda, M, Palmer, S. And Dias, JA. FSH Receptor Forms Oligomers and Shows Evidence of C-terminal Proteolytic Processing Endocrinology 2007 148(5):1987-95. PMID: 17272391
Function of the Makorin gene family
Dr. Todd GrayThe Makorin gene family encodes proteins with a characteristic arrangement of zinc fin-ger motifs. While a definitive function for the makorin1 protein in development remains elusive, it may be involved in regulating telomere length or erythropoiesis. We are using mouse and tissue culture models to define its regulation and function. To this end, we are evaluating potential miRNA regulation of Mkrn1 transcripts and developing siRNAs to knockdown Mkrn1 levels in tissue culture. This project encompasses techniques such as cloning, qRT-PCR, transfection, western blotting, and siRNA mediated knock-down.
A Makorin derivative is also found in poxviruses, where it acts as an important virulence factor. We hypothesize that the virulence function of poxviral Makorins stems from its activity as an ubiquitin ligase to covalently modify specific proteins. In ectromelia virus, the agent of mousepox, the Makorin-derived gene is known as p28. We are combining this mousepox model system with a novel proteomics approach to identify the targets of p28-mediated ubiquination. This project includes generating ectromelia virus-infected cell extracts, purification of ubiquitinated proteins by affinity chromatography, and mass spectometry analyses.
Regulation of RNA Polymerase II by Ess1 in Eukaryotes
My laboratory studies regulation of RNA polymerase II (RNA pol II), the enzyme responsible for transcription of protein-coding genes in all eukaryotes. Precise regulation of RNA pol II activity underlies all aspects of cell growth and embryonic development, and misregulation of RNA pol II activity is responsible for many human diseases. This project focuses on the function of a gene called ESS1, which is conserved in all eukaryotes including mammals (called PIN1 in humans). We showed that the S. cerevisiae ESS1 gene encodes a prolyl isomerase that binds to the carboxy-terminal domain (CTD) of RNA pol II, potentially altering its function. Mutations in ESS1 cause cell cycle arrest and chromosome fragmentation, but the exact defect remains a mystery. Misregulation of the human homolog, PIN1, has been associated with a wide variety of disorders ranging from cancer to Alzheimer's disease. Our goal is to understand the mechanism by which Ess1-induced conformational changes in the CTD affect RNA pol II activity, and to identify what genes are misregulated in ESS1 mutants. This knowledge may be useful for devising medical interventions to counteract diseases associated with alterations in PIN1.
A number of different projects are available. These will use molecular, genetic, and genomic approaches to investigate Ess1 function. Projects will be selected, in part, based on student interest. [For a review on the topic see P.E. Shaw, EMBO Reports, vol.8, p40-45 (2007).]
The functional analysis of murine virgin and memory T cells
The interests of this laboratory center upon the study of memory T lymphocyte development. The aim is to characterize fully the differences between T cells which have never been exposed to foreign pathogens (virgin T cells) and memory T cells at the developmental, phenotypic, and functional levels. The student will be involved in projects which examine either a) cell differentiation processes as virgin T cells are stimulated to become memory cells during aging, infection, or in mutant knockout mice; b) responses of virgin and memory T cells to Staphylococcal enterotoxin B (SEB). Immunization of mice with SEB leads to a state of T cell unresponsiveness (tolerance). We are interested in determining the involvement of virgin and memory T cells in tolerance induction, as an abrogation of tolerance may lead to autoimmunity. The student will learn and use the tools of the cellular immunologist: flow cytometry and cell sorting, ELISAs, tissue culture techniques, in vivo and in vitro measurements of lymphocyte activation.
Structure and function studies of proteins related to bacterial or viral infection and host response
My group focuses on structural biology to determine the atomic structures of biological macromolecules. Current projects include structural and functional studies on bacterial and viral superantigens, signaling molecules involved in apoptosis, transcription factors involved in regulating stem cell growth, and rational drug design against key viral enzymes. The first project involves study of superantigens, which are a group of bacterial toxins that have the ability to stimulate the activation of large number of T cells bearing particular TCR Vb domains (Li et al (1999) Ann. Rev. Immunol. 17 , 435-466; Zhao et al. (2004) Structure 12, 277-88; Wang et al. (2007), Nat. Struct. Mol. Biol. 14 , 169-171). They have been implicated in many human diseases. The second project is to study the structure and function of a protein kinase and its interaction with signaling proteins involved in apoptosis. In a third project, we will investigate the transcriptional network regulating stem cell initiation and growth. The fourth project is to investigate the structural and functional properties of essential enzymes encoded by West Nile virus, an emerging infectious pathogen. These enzymes include methyltransferase and polymerase (Ray et al. (2006), J. Virol., 80, 8362-70; Zhou et al. (2007), J. Virol. 81. 3891-3903; Dong et al. (2007) J. Virol. 81. 4412-4421). The REU student will work on one of the projects. He/She will gain hands-on experience of PCR, molecular cloning, bacterial transformation, protein expression and purification. He/She may also learn how to grow protein crystals and possibly how to collect X-ray diffraction data.
To see the published work of a former REU student, see http://www.jyi.org/research/re.php?id=847.
Gene and antibody therapies for neurological diseases
My neurogenetics laboratory is involved in novel therapeutic approaches to brain diseases. One major goal is to develop gene and protein therapies for neurodegenerative diseases caused by abnormal protein accumulation. The method develops intracellular antibodies called intrabodies that can specifically re-fold or retarget pathogenic proteins. We have two that appear to work against Huntington's Disease protein. These are currently being tested using transgenic drosophila and mice, and gene delivery vectors in mouse brain. A project taking a similar approach to Parkinson's Disease is at an earlier stage. We are also doing more basic studies on gene therapy vectors for delivery of intrabodies and other protein reagents in both adult and pediatric brain diseases, using mutants that affect cerebellar development as models. This has implications for many disorders, ranging from hypothyroidism to autism. The most likely student project for this summer will be to work on transfer and testing of anti-synuclein intrabodies in Drosophila (fruit fly) models of Parkinson's disease. Methods may include DNA analysis, protein analysis, microscopy, and Drosophila genetics and behavior. Other projects that include more cell culture or animal pathology and behavior are also possible.
Chromatin structure and gene activation in yeast
My lab uses the yeast Saccharomyces cerevisiae as a model organism to understand the mechanisms by which changes in chromatin structure that are associated with transcription take place, and to discover how these changes help to regulate gene activity. Genes whose products are involved in modification and remodeling of chromatin have been implicated in a number of human diseases, including cancer, so understanding these processes has implications for human health. Because the proteins associated with chromatin and transcription are highly conserved among eukaryotic organisms, our work has broad applicability to higher organisms, including humans.
A student working in the lab would likely work on one of two projects, though others are possible. In one, the student will help set up a system in which a reporter gene is introduced into several hundred mutant yeast strains in parallel in order to test the involvement of candidate genes in chromatin remodeling and gene activation. The second project is a genetic screen to identify proteins that regulate transcription through interactions with histone H3. We have identified a histone H3 mutant that causes the yeast CHA1 gene to be expressed under conditions where it is normally off. The mutation also causes temperature sensitivity. By identifying mutations that suppress these effects, we will identify genetic partners of histone H3 that regulate transcription. The student will gain experience in some of the following techniques, depending on the project they choose: yeast genetics, molecular cloning, chromatin immunoprecipitation, growth and transformation of yeast and bacteria, Southern blotting, and/or real time PCR.
Structure-function studies of specialized cellular and viral polymerases
Our primary research area is the study of lesion-bypass DNA polymerases. These enzymes are capable of synthesizing DNA opposite damaged template bases, sites that cause replicative DNA polymerases to stall. This allows genome replication to continue, but can also lead to mutation of the genetic information since the lesion-bypass polymerases are highly error-prone enzymes. Human cells contain several different lesion bypass polymerases, each specific for different types of DNA damage. We have determined the crystal structure of one lesion-bypass polymerase, Dbh, from Sulfolobus solfataricus and have shown how it can make single-base deletion mutations at a high rate (Wilson & Pata, in press). These types of mutations are particularly detrimental to cells because they cause -1 frameshift mutations when they occur in protein coding regions. The human homolog of this protein, polymerase kappa, is highly expressed in some lung cancer cells and may contribute to tumorigenesis because of the mutations it makes.
A summer student could either work on additional aspects of Dbh, such as how the polymerase bypasses DNA damage, or work on human polymerase kappa. This work would provided experience in a number of techniques, including molecular cloning, site-directed mutagenesis, protein expression and purification, in vitro polymerase activity assays, and X-ray crystallography.
Relevant references:
- Wilson, RC and Pata, JD. Structural insights into the generation of single-base deletions by the Y-family polymerase Dbh. Molecular Cell, in press.
Increasing the drug sensitivity of pancreatic tumor cells by using a dominant negative inhibitor of thymidylate synthase
Pancreatic cancer is the fourth leading cause of cancer death in the US. It is also one of the deadliest tumors with a median survival of less than six months and few effective treatment options. Among the few chemotherapeutic drugs that show some efficacy is 5-fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor that has been in use for nearly 50 years. Studies have shown that high tumor levels of TS are a poor prognostic indicator, and are associated with resistance to 5-FU. Conversely, reducing cellular TS levels sensitizes tumor cells to 5-FU and other TS inhibitors, thereby improving the drugs' efficacy. A novel way to suppress TS activity has been identified that is based on the finding that an active-site double-mutant of TS functions as a dominant-negative inhibitor of TS when it interacts with the wild-type enzyme. When this double-mutant of TS is introduced into Escherichia coli the bacteria die, because they are dependent on this enzyme's activity for DNA precursor synthesis. Similarly, when the analogous human double-mutant TS is expressed in human cancer cells, the cells become more sensitive to 5-FU. We propose to exploit these findings to enhance the drug sensitivity of pancreatic cancer by impairing this key enzyme. The hypothesis is that the selective attenuation of TS activity in tumor cells by a dominant-negative double-mutant TS will sensitize the cells to 5-FU and other TS inhibitors, thereby increasing the effectiveness of the chemotherapy. Short dominant-negative peptides will be identified that effectively inhibit the cellular TS activity, and ways of efficiently introducing these peptides into pancreatic tumor cells will be explored. This approach will be tested in pancreatic tumor cell lines, as well as in actual pancreatic tumor tissue explants in culture. It is expected that treating tumors with the dominant-negative peptide will increase the efficacy of TS inhibitor-based chemotherapy, both in single- and in combination-drug regimens. These experiments are expected to provide proof-of-principle that the dominant-negative inhibition of TS can be used to sensitize pancreatic cancer cells to chemotherapy, and lay the groundwork for the further development of this treatment modality for use in vivo in animal models and ultimately in humans.
Cholesterol trafficking in a mouse model
My laboratory works with a mouse model of Niemann-Pick type C (NPC) disease, which is caused by loss of either of two genes (NPC1 or NPC2). Loss of either gene results in defective intracellular cholesterol trafficking, leading to, among other features, adult neurodegeneration and neonatal respiratory failure. Specific projects currently focus on identifying genes that modulate disease severity (e.g. age of onset of neurodegeneration), as these may have a role in neurodegenerative diseases such as Alzheimer's disease, and control the frequency of respiratory failure. We are also interested in why a cholesterol trafficking defect might cause respiratory failure.
Genome-wide transcriptional regulation in bacteria
My lab is interested in the regulation of gene expression in bacteria. Transcription is the first step in gene expression and is the major regulatory target. We study the mechanisms of transcriptional regulation in the model organism, Escherichia coli. We also study how transcription is regulated in the pathogenic bacterium Yersinia pestis (causative agent of plague) during the course of infection. Work in the lab involves the use of basic molecular biology tools as well as genomic approaches involving microarrays.
Bacteria encode many transcription factors that regulate transcription by binding directly to DNA sequences in promoter regions. These transcription factors regulate a wide range of cellular processes. Hence, it is important to know which DNA sequences are bound by each transcription factor, yet we know little or nothing about the targets of most transcription factors even in E. coli. The goal of the project would be to identify all of the DNA sequences in the E. coli genome that are bound by a particular transcription factor. This can be determined using a method called ChIP-chip that couples chromatin immunoprecipitation and microarrays. The student will then determine whether the transcription factor is regulating transcription positively or negatively at each of the target sites, which cellular processes the transcription factor controls, and what DNA sequence motifs are bound by the transcription factor. This project will involve the use of basic molecular biology techniques as well as the use of microarrays and analysis of microarray datasets.
Relevant references:
- Wade, J. T., Struhl, K., Busby, S. J. and Grainger, D. C. (2007). Genomic analysis of protein-DNA interactions in bacteria: insights into transcription and chromosome organization. Mol. Microbiol. 65, 21-26.
- Wade, J. T., Reppas, N. B., Church, G. M. and Struhl, K. (2005). Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites. Genes Dev. 19, 2619-2630.
Host defense and intracellular bacteria
Our laboratory studies host defense against intracellular bacteria, and we study infections caused by mycobacteria and ehrlichia. The Ehrlichiae are tick-transmitted bacteria that can cause a number of serious diseases in humans. The bacteria typically reside in monocytes and macrophages, and are not thought to survive long outside of host cells. We have developed a mouse model for ehrlichia infection and are using it to determine the cellular and molecular basis for resistance and susceptibility to disease. The model system is relatively tractable, and allows us to address a number of different basic questions about host defense that will likely be relevant during many intracellular bacterial infections. One question of particular importance is the role of dendritic cells (DCs) in host defense. DCs are known to be the primary antigen presenting cells during bacterial infections, and it is likely that DCs are responsible for the production of IL-12 and the development of helper T cells. DCs may be triggered after engulfing bacteria via endocytosis. or may be targets of infection. Flow cytometry. immunohistochemical, and molecular detection techniques will therefore be used to DC activation in response to ehrlichia infection. Our studies of tuberculosis address basic questions regarding the genesis and maintenance of protective CD4 T cell responses. Possible projects will involve the role of antigen-specific polarized CD4 T cell subsets during infection. A summer student involved in any of these projects will therefore have the opportunity to become acquainted with both basic immunology and infectious disease research.
Relevant references:
- Bitsaktsis, C. and G. Winslow. 2006. Fatal recall responses mediated by CD8 T cells during intracellular bacteria infection. J. Immunol. 177:4644-4651.
- Yager, E., C. Bitsaktsis and G. Winslow. 2005. An essential role for humoral immunity during ehrlichia infection in immunocompetent mice. Inf. Immun. 73:8009.
- Bitsaktsis, C., J. Huntington and G. M. Winslow. 2004. Production of Interferon-g by CD4 T cells is essential for resolving ehrlichia infection. J. Immunol. 172:6894.
- Winslow, G. M., A. D. Roberts, M. A. Blackman and D. L. Woodland. 2003. Persistence and turnover of antigen-specific CD4 T cells during chronic tuberculosis infection in the mouse. J. Immunol. 170:2046.