The University of Health Sciences Center

Labor atory for Genomics and Bioinformatics Vol. 4, No. 3 September 2006 http://microgen.ouhsc.edu

University of Oklahoma Health Sciences Center/405-271-2337

Technology seminars to illustrate new equipment

This fall, we are sponsoring three October 9, Dr. Ed Horton will discuss how technology seminars that will be offered you can take advantage of our Beckman during the regular seminar series for the Proteomelab (“Applying Intact Protein In this issue Department of Microbiology and Partitioning and Fractionation to Biomarker Immunology. These seminars, at noon on Discovery”; see related article on page 3). • Technology seminars 1 Mondays, are designed to bring you up to Lastly, Dr. Charles Greaves will discuss on • Featured Project: date on new technology that we offer in our October 30 a new microarray technology that Intergenetics SNP core facility or technology that we are we are considering (“Custom Microarrays for Genotyping 1 thinking about adding to our repertoire. On Application Specific Studies”). We hope

OK INBRE September 25, Dr. Wendy VanScyoc will that you will be able to attend, as we will be discuss the use of surface plasmon resonance sending around surveys after each seminar, • INBRE News 2 (“Next Generation Surface Plasmon assessing how useful this has been, and • INBRE Summer Resonance Biosensor Technology for the whether you are interested in having these Undergraduate Research Production of High Quality Kinetic technologies in the core facility. Thanks for Program 4 Information in Protein Interactions”). On your support! -Dave Dyer & Allison Gillaspy OUHSC COBRE Featured project: InterGenetics SNP Genotyping • COBRE News 4 InterGenetics Incorporated (IGI) is • COBRE operating platform as that for the only FDA Investigator Focus 2 offering a new service to provide high approved cystic fibrosis mutation carrier throughput single nucleotide polymorphism screening test. New Services! (SNP) genotyping to academic investigators One of the factors that makes this new and biotechnology companies. IGI is a service possible is access to robust, accurate • The Beckman biotechnology- and bioinformatics-based and timely DNA sequencing provided by the ProteomeLab 3 company in the PHF Research Park located OUHSC core facility. Development of adjacent to the OUHSC. IGI has significant accurate SNP genotyping assays using ASPE expertise in developing and implementing requires a modest sample of individuals for Services and rates 6 multiplexed SNP assays through its whom SNP genotypes have been determined Acknowledgements development of a test for genetic by a method other than ASPE. It also is The OUHSC Laboratory for predisposition to breast cancer. Over a critical to know the locations of nearby Genomics and Bioinformatics million individual SNP genotype SNPs, to avoid problems in PCR and/or is partially supported by determinations were made by IGI over the ASPE primer design. Direct DNA USPHS grants 5P20-RR-15564 course of this and other projects. IGI is sequencing is the gold standard for finding (COBRE) and 5P20-RR- making available this expertise in SNP and characterizing such SNPs. Thus, 016478 (INBRE), from the genotyping to other investigators. development of each new assay begins with a National Center for Research Initiating and operating a large-scale SNP small resequencing project. Resources of the National genotyping project can be expensive and If you are contemplating a SNP genotyping Institutes of Health. Additional time consuming. Capital equipment costs are project, IGI will deliver timely, efficient and support is provided by the large and technical operators usually require competitively priced user-friendly service. OUHSC Office of the Provost. extensive training. IGI allows you to avoid And, IGI technical staff will be glad to guide these costs, shortening the time required for you through the process of designing your data acquisition and permitting you to SNP genotyping application. Interested concentrate on data analysis and investigators can contact IGI at 405-271- interpretation. IGI’s core technology uses 1720 or visit their website at allele specific primer extension (ASPE) www.intergenetics.com. mounted onto a bead-based assay system Contributed by David Ralph, Ph.D. with a readout linked to a flow cytometer. CSO, IGI This robust technology uses the same

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INBRE News by Edgar Scott, M.S. This summer, I had the pleasure of refines a specific scoring matrix that models mentoring Craig Covey from the query sequence and sequence relatives Community College during the INBRE and can be used in successive searches to Summer Undergraduate Research Program find distantly related sequences. He then processed the two data sets separately with a (see page 5). His research focused on using bioinformatic tools to identify unique motif searching program called MEME sequence motifs in a family of bacterial (Maximum Expectation maximization for metal ion sensor proteins. The Ferric uptake Motif Elicitation). MEME uses the regulator (Fur) protein and the Zinc uptake expectation maximization algorithm to regulator (Zur) protein are related proteins identify conserved characters in a group of that regulate the uptake of iron and zinc, protein or DNA sequences. The program respectively. Their amino acid sequences are outputs conserved regions as local multiple so similar that it is difficult to distinguish sequence alignments with a bit score to between the proteins. Craig's project quantify the degree of conservation within attempted to identify unique amino acid that alignment. motifs from a group of Fur/Zur homologues This analysis showed that the Fur and Zur

that might be useful for distinguishing Fur proteins have a strong sequence motif that http://okinbre.org/ from Zur. overlaps both the DNA binding domain and Craig proceeded in several steps. He first portions of a metal-binding domain. acquired amino acid sequences of Unfortunately, the sequence logos of the experimentally-characterized Fur and Zur motifs for the two proteins did not proteins from the UniProt Knowledgebase. distinguish Fur from Zur. However, the exercise was good for Craig’s introduction To this, he added sequences homologous to Zur and Fur using Protein Specific Iterative to bioinformatics and suggested several BLAST (PSI-BLAST) at NCBI. Through possible avenues for additional several iterations, PSI-BLAST creates and investigation.

COBRE Investigator Focus: John West, Ph.D. Defining the impact of HIV-1 fitness on virus transmission from mother to child Infections with HIV-1 subtype C are survive in a given environment (fitness), and

responsible for more than 50% of new between fitness and disease progression. infections and this continues to increase, We use a combination of genetic, particularly in the developing countries of phylogenetic, molecular, immunological and sub-Saharan Africa, India and China. The biochemical approaches to investigate primary routes of infection are unprotected relationships between Env evolution and heterosexual contact and mother-to-child biological function, including replication or transmission. transmission fitness. We employ Untreated mothers transmit HIV-1 to their fluorescent-tagged HIVs (containing infants in approximately 30% of deliveries. variants of Env) in dual-infection Of the remaining 70%, 15% become infected competition experiments to define through breast-feeding. My laboratory replication fitness. We also are developing focuses on mother-to-child transmission organotypic culture systems to evaluate viral because this represents one of few instances transmission fitness across mucosal barriers.

where the donor, recipient, the direction and These experiments will facilitate our John West, Ph.D. timing of transmission are known. understanding of the determinants of HIV-1 Assistant Professor Transmission of HIV-1 is a function of the transmission, evolution and disease. Such OUHSC Department of envelope glycoprotein (Env) that mediates information will be essential in the design of Microbiology and Immunology fusion of the viral membrane with a target treatments or preventatives that limit or cell. Because the viral replicase lacks proof- redirect viral Env evolution such that lasting reading capability, the Env proteins are protection can be achieved. tremendously diverse, creating a swarm of For more information, contact: nearly identical but distinct individuals called John T. West, Ph.D. a ‘quasispecies’. This diversity lends Dept. of Microbiology and Immunology incredible plasticity to the virus to adapt to OU Health Sciences Center selective pressures. Recent data suggest a Email: [email protected] link between HIV Env and viral ability to Tel: 405-271-2570 OUHSC Laboratory for Genomics and Bioinformatics Page 3 The Beckman PF2D ProteomeLab in Practice In our last newsletter, we announced the be collected if the entire first dimension were availability of the Beckman PF2D fractionated, and because the fraction ProteomeLab for proteomics separations by collector is not large enough for that many our core facility. Bob Hurst’s lab has fractions, someone has to change out the full extensive experience with the PF2D, tubes for empty ones. This either means improving the throughput and accuracy of someone does this at 4AM, or the the instrument. Bob and coworkers have a fractionation requires some three days. recent paper published online with Biomed Running in “mapping mode,” we can Central’s “Proteome Science”: complete a separation in 24 hours. http://www.proteomesci.com/content/pdf/14 We then take our map and compare it to 77-5956-4-13.pdf. This article is a our control or controls to decide which peaks description of their experience, and advice need to be identified by mass spectroscopy on how to employ the PF2D for your own (MS). The identification of proteins by purposes. Bob writes: peptide mass fingerprinting is relatively “The power of proteomics is that it simple, and there is no in-gel recovery represents the working level of the cell. needed. We were most interested in peaks Important regulatory events involving post- that were unique to one sample in our study translational modification may not be of the effect of extracellular matrix on the reflected at all in the levels of transcripts. phenotype of bladder cancer cells. We The PF2D is certainly one of the most picked the fractions we wanted to be advanced proteomic displays around. The analyzed by MS and then re-ran the first dimension separation is by appropriate first-dimension fractions, chromatofocusing, which separates by pI. collecting the second-dimension fractions. The actual separation can be divided into We showed that even a small peak with an three groups of proteins. First are the highly absorbance of 0.02 could easily be identified. Beckman PF2D ProteomeLab basic proteins that either are eluted We found that many of these unique peaks proteomics separations platform immediately the by pH 8.5 wash or elute represented post-translational modifications slowly in the next several fractions. The and that large proteins are easily separated PF2D is set to wash out everything that by this technique. Two of our proteins were doesn’t bind strongly at pH 8.5, but some 10 >300 KD in size. The main source of error is or more fractions are collected. While many pH control. If two first-dimension fractions of these contain protein, most proteins are differ slightly in pH coverage, then the found in the first two fractions. Whatever second-dimension separations will be separation occurs at this pH is poorly different. For example, if sample A yields a understood, and consecutive fractions will be first-dimension fraction covering the range seen to contain the same bands. The next set of 7.0 to 7.3, whereas the equivalent fraction is collected during the pH gradient elution. from sample B covers 7.03 to 7.33, then the This is where resolution is best; most proteins eluting between 7.00 and 7.03 will proteins eluted in the gradient appear as be missing from the second-dimension well-resolved peaks in the second dimension. separation of sample B and will appear to be The third group of proteins is eluted at pH unique in sample A. However, those proteins 4.0 by a 1 M NaCl wash. These highly acidic will appear in the adjacent fraction in sample proteins are not well resolved, but the first B. It is therefore important to examine two fractions generally contain most of the closely the adjacent fractions and the pH of information. the first-dimension fractions before deciding To speed the proteomic display, we a protein is unique. routinely run only 25 of the 44 first- In summary, we first map a proteome and dimension fractions through the second then pick fractions containing interesting dimension. Also, to speed the separation, we proteins for subsequent examination. do not collect fractions but just discard the Clearly, it would be most exciting if the material. Generally, sufficient material is PF2D could be directly interfaced with the collected from each first-dimension fraction mass spectrometer. If anyone has questions to make two second dimension runs. Because as how to best use the PF2D, I will be glad to a total of about 4,000 fractions would need to share my experience.” Contributed by Robert Hurst, Ph.D.

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COBRE News by John Iandolo, Ph.D.

In late July (21-23), NCRR hosted the to maintain a balance in supply and demand. Institutional Development Award (IDeA) Following these talks, the meeting provided Program First Biennial Symposium. The extensive opportunities for researchers highlights of the meeting included supported by INBRE and COBRE funds to presentations by NIH Director Dr. Elias A. present and discuss the results of their work Zerhouni, who spoke on “NIH at the in comprehensive poster sections and at Crossroads: Strategies for the Future”; a specifically focused scientific sessions. Our presentation by NCRR Director Dr. Barbara COBRE was well represented with posters M. Alving, who provided an overview of presented by Dr. Ira Blader, Dr. Michael NCRR and lastly a presentation by Dr. Fred Sakalian, Dr. W. Michael McShan, Dr. Holly Taylor, who provided an historical account Hoffman-Roberts, Dr. Mark Lang and Dr.

OUHSC of the IDeA program. Dr. Zerhouni’s John West. Following the scientific Center of Biomedical Research message focused on the present budgetary sessions, the annual COBRE Principal Excellence problems at NIH and the plan to rescue the Investigator’s meeting was convened. NIH mission. The plan calls for utilizing Announcement of a permanent study section adaptive strategies to preserve NIH key to review proposals gave rise to a lively principles. These will include developing a discussion of the review process for COBRE balanced research portfolio with NIH competitive renewals. In the open discussion continuing to focus on basic research and the session, the future of the COBRE program private sector taking the lead in clinical and ranged over a number of topics. Among the translational research, by protecting the most interesting, follow-on programs to future with imaginative new programs for continue support of successful COBREs new and established investigators and by generated much interest. better management of available grant funds

INBRE Summer Undergraduate Research Program

The INBRE Summer Research Program the capitol this coming spring. Research for Undergraduate Students recently Day at the Capitol is an opportunity for concluded its fifth year of student undergraduate students to inform the participation. This year’s program was the Legislature and the public about high quality biggest yet, with thirty-one students research being conducted at Oklahoma’s participating from six regional universities colleges and universities. Also at the and three community colleges. The goal of luncheon, INBRE student Kelly Etherton the program was to expose undergraduate from Oklahoma City Community College students to the world of biomedical research received a $2000 transfer scholarship to and encourage them to pursue careers in the attend OU this fall. The transfer scholarship fields of science and technology. The is given to the community college student students were matched with mentors who with the best poster and presentation skills. share their same area of interest. Applications for the 2007 program will be

At the end of the eight-week program, the available this fall for both students and http://okinbre.org/ students presented a poster of their research mentors interested in the program. To learn findings and attended a luncheon in their more about the INBRE Summer Research honor at the OU Health Sciences Center. Program or the Oklahoma INBRE grant, Two award winners from the INBRE please visit the INBRE website at program were announced at this year’s www.OKINBRE.org. Students who luncheon. Michael Landoll from Cameron participated in this summer internship, their University was selected to represent OU mentors and projects are listed on page 5. Health Sciences Center at Research Day at Contributed by Sasha Smith

OUHSC Laboratory for Genomics and Bioinformatics Page 5

INBRE Mentor Host Project Student Institution Students from Northeastern Oklahoma State University Christopher Co x Dr. David Dyer OUHSC In silico Methods to Predict Genes That Encode sRNAs in Actinobacillus actinomycetemcomitans Students from Southeastern Oklahoma State University Josiah Schomer Dr. Daniel Carr OUHSC Nervous System Expression of CXCL10 Contributes Towards Resistance to Herpes Simplex Virus Type 1 Infection Students from Oklahoma City Community College David Ayadpoor Dr. Melville Vaughan UCO In vitro Aging on Myofibroblast Phenotype Frank Boyd Dr. Lurdes Queimado OUHSC Studying a Novel Link between DNA Repair and the Wnt Pathway Steven Craig Dr. David Dyer OUHSC Using Bioinformatic Techniques to Characterize Sub-Family Groups of the Fur Protein Family Covey Kelly Etherton Dr. James McGinnis OUHSC Analyzing the Molecular Basis for the Translocation of Rod Alpha Transducin Bao-Linh Dr. Michael Ihnat OUHSC Effect of Chronic Low Dose Arsenic and High Glucose on Hypoxic Signaling Nguyen Lauren Reeves Dr. Mauricio Sanchez UCO Abstract Title Not Available Clay Sandel Dr. Michael Centola OMRF Evidence for IL-23 Mediated Pathology of Rheumatoid Arthritis Jenny Stacey Dr. Stephen Marek OSU Phenotypic Characterization of T-DNA Tagged Mutants of Phoma medicaginis Students from Langston University George Kpeli Dr. Dee Wu OUHSC Cisplatin Pharmacokinetic/Pharmacodynamic for Tumor Therapy: Modeling and Meta-Analysis Contessa Majors Dr. Muna Naash OUHSC The Efficacy of Compacted-DNA Nanoparticles in Ocular Gene Delivery Students from Redlands Community College Taylor Arnold Dr. Garo Philip OUHSC USP 797 End Product Verification of Commercially Available Compounded Low-Risk Basmadjian Radiopharmaceuticals Colby Shepherd Dr. Ramamurthy OSU Analysis of an Arabidopsis T-DNA Insertion Line Distriputing the DCP2 Gene Mahalingam Students from Cameron University Linda Ash Dr. Carla Guthridge Cameron Characterization of P2RX7 Expression and Function in Human Keratinocytes Univ. Michael Landoll Dr. Jialing Lin OUHSC Mutagenesis Study of the Pore-Formation by Anti-Apoptotic Bcl-2 Callie Mosiman Dr. Sundararajan OSU Cell Colonization in Thermosetting Injectable Hydrogels Madihally Sandra Pope Dr. Leonidas Tsiokas OUHSC Co-Localization of TRPC1 and TRPC4 in Transfected Cells Lora Bailey-Repp Dr. Ira Blader OUHSC Oxygen Sensing in Toxoplasma Gondii Valerie Toodle Dr. Guangpu Li OUHSC The Specificity of TBC1D15 as a Rab GAP Gabriel Vidal Dr. Joel Guthridge OMRF Detecting Epistasis in Human Lupus: Application of Multifactor Dimentionality Reduction (MDR) Students from Southwestern Oklahoma State University Samuel Cropp Dr. Arden Aspedon SWOSU Osmoprotective Genes in Pseudomonas aeruginosa Adarsha Koirala Dr. Muatasem Ubeidat SWOSU Whole-Mount In Situ Hybridization of 5’-Nucleotidase Gene in Dictyostelium discoideum Cheri Lemons Dr. Randle Gallucci OUHSC Effects of Hyperglycemic Conditions on Interleukin 6 Receptor Function Andrew Nelson Dr. Jason Johnson SWOSU Mimicking Synchronization Signals in Carbamoyl Phosphate Synthetase Anna Nelson Dr. Doris Benbrook OUHSC Mitochondrial Effects of the Anti-Cancer Compound SHetA2 on Ovarian Cancer Cammi Valdez Dr. Han Wang OU Heme Deficiency Downregulates Expression of Exocrine Pancreas Genes in Zebrafish Students from the University of Abdiwahab Dr. Wei Chen UCO Immunological Effect of Laser Immunotherapy in Treatment of Metastatic Tumors in Mice Mohamed Danjela Dr. Darrin Akins OUHSC Expression of the E. coli OmpA Porin in Borrelia Burgdorferi: The Lyme Disease Spirochete Mojsilovic Vagan Dr. James Jarvis OUHSC Immunologic Function of STAT 1 in Trophoblast-Like JAR Cells Mushegyan Students from Comanche Nation College Michael Murrow Dr. Dennis Frisby Cameron Isolation of the NAG Promoter in Caenorhabditis elegans Univ.

Rate structure (partial listing; please check our web page for details)

The Uni versity of DNA sequencing (<96 samples) $5/reaction Library construction contact us Oklahom a DNA sequencing (>96 samples) $3/reaction Human genotyping $500/sample Health S ciences DNA fragment analysis $3/sample Membrane array fabrication $11/array Center HLA DNA sequencing $400/locus Microarray scanning $20/slide

Laborator y for DNA normalization, 96 samples $10.60 Bioinformatics support $45/hour Genomic s and Bioinformatics support Bioinform atics Stanton L . Young We offer bioinformatics support through our Informatics Core, directed by Jeremy Zaitshik.

Biomedic al Research The rate for support is $45/hour with a one-hour minimum charge. Please note: we do not Center, R oom 1106 provide Tier 1 IT support. For inquiries or work requests contact: Phone: 405-271-23 37 Jeremy Zaitshik ([email protected]) Tim Schmidt, M.S. (Timothy- Fax: Perl programming (for sequence analysis, s [email protected]) 405-271-1204 manipulation, etc.) Phylogenetic analysis (PAUP, PHYLIP, E-Mail: UNIX/Linux system administration MEGA, PAML, etc.) For general information: Security issues Comparative genomics (alignment, promoter [email protected] Sequence analysis software questions analysis, etc.) Allison- Data acquisition/automation Perl programming (data manipulation, data gillaspy@o uhsc.edu Database design (MySQL/PostgreSQL) mining, etc.) For inquiries r egarding DNA sequencing: Web design (HTML/CGI) Proteomic analysis (localization, structure, Nicole-bent [email protected] display with spdv, Rasmol, etc.) For inquiries r egarding protein Web design/HTML fractionation: [email protected] INBRE Multicampus Bioinformatics Specialist For inquiries a bout microarray fabrication an d hybridization: The INBRE MBE Specialist is responsible for fostering the development of bioinformatics Mandy-gips [email protected] education on 14 undergraduate campuses in the state of Oklahoma, and coordinating INBRE- For bioinformatics support: related bioinformatics activities with the INBRE Bioinformatics Core. For inquiries and Jeremy- INBRE information, please contact: zaitshik@o uhsc.edu Timothy- Edgar Scott, M.S. [email protected] ([email protected]) For billing information: Bioinformatics education [email protected] For inquiries regarding OK Computational biology education INBRE activities: Sequence analysis software questions [email protected] Data acquisition/automation Web design INBRE activity scheduling We’re on the Web! About us…

See us at: The OUHSC Laboratory for Genomics and Bioinformatics is a full-service genomics facility microgen.ouhsc.edu offering DNA sequencing (small- and large-scale projects), microarray design and hybridization and other services. The Director of the Laboratory for Genomics and Bioinformatics is David Dyer, Ph.D., a Professor in the Department of Microbiology and Immunology at the Health Sciences Center. Allison Gillaspy, Ph.D., is the Associate Director of the Laboratory for Genomics and Bioinformatics and a Research Assistant Professor in the Department of Microbiology and Immunology. The University of Oklahoma is an equal opportunity institution. This publication, printed by OU Printing services, is issued by the University of Oklahoma. 150 copies have been prepared and distributed at no cost to the taxpayers of the State of Oklahoma.