Progress 08/01/06 to 07/31/07
Outputs
The specific aim of this grant was to purchase an automated DNA sequencer for use in a number of projects in our department. Using funds from the USDA and matching funds from MSU, we purchased an Applied Biosystems 3130 Genetic Analyzer, which is a multi-capillary automated DNA sequencing instrument. Since installation, this instrument has been used by nearly every lab in our department by providing rapid and reliable sequence analysis. The following are brief descriptions of several research projects utilizing the new sequencer. Quinn Lab: Activation of the complement system results in production of proinflammatory fragments C3a, C4a, and C5a. Among these, C5a plays the most important role in modulating phagocyte function. Thus, we recently cloned the bovine C5a receptor. We also performed molecular and functional characterization of the bovine C5a receptor. The new sequencer has greatly enhanced this project. We also used sequencing to screen for C5a receptor
mutations and polymorphisms in different cattle. Thus, the sequencer has greatly enhanced our project. Pascual Lab: Current livestock vaccines for brucellosis show variable protection in bison and in most incidences do not prevent development of infection in bison cows. To aid in identifying protective epitopes to develop a multivalent brucellosis subunit vaccine, we cloned several B. abortus genes. The sequencer was extremely beneficial to our vaccine discovery efforts. For example, we required DNA sequencing to verify the fidelity of our cloned B. abortus genes for subsequent DNA manipulations into DNA vectors or protein expression systems. Hardy Lab: Our research program focuses on understanding the interactions between enteric viruses (rotavirus and norovirus) and host cells that result in changes in gene expression related to establishment of the antiviral state. We also seek to understand the initial interactions that begin with virus binding to cells and what components of the
virus capsid are important for a productive infection. Much of our work is at the molecular level and we have constructed and characterized numerous cDNA clones both of cellular viral genes. We have performed relatively extensive site-directed mutational analyses of several different genes. Determination of the sequence of new viral genes, characterized cellular genes, and confirmation of introduced targeted mutations is critical to our work. Thus the automated DNA sequencer is invaluable to our program. Voyich Lab: Our lab has used the sequencer on several projects. We have used the sequencer to confirm cloning and sequence of the bovine chemokine CCL28. We are in the process of characterizing the role of this chemokine in bovine immunity to mastitis. The ability to rapidly sequence cloned products has allowed us to move forward with generating recombinant bovine CCL28 that we can use in functional assays. We have also used the sequencer to confirm cloning and sequence of
staphylococcal genes. The time saved by having the ability to sequence in-house has been invaluable and has definitely increased my ability to compete for funding.
Impacts
Enhanced research capability to study mechanisms of host-pathogen interaction in disease and host-defense processes used by livestock to combat pathogens will have a major impact on our understanding of livestock disease.
Publications
- Liu, M., Zhu, H., Zhang, J., and Lei, B. (2007) Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with group A streptococci. Infect. Immun. 75:3651-3657.
- Ammons, M.C.B., Siemsen, D.W., Nelson-Overton, L.K., Quinn, M.T., and Gauss, K.A. (2007) Binding of Pleomorphic Adenoma Gene-Like 2 to the TNF-alpha-responsive Region of the NCF2 Promoter Regulates p67phox Expression and NADPH Oxidase Activity. J. Biol. Chem. 282: 17941-17952.
- Gauss, K.A., Nelson-Overton, L.K., Siemsen, D.W., Gao, Y., DeLeo, F.R., and Quinn, M.T. (2007) Role of NF-kappaB in Transcriptional Regulation of the Phagocyte NADPH Oxidase by Tumor Necrosis Factor alpha. J. Leukoc. Biol. 82: 729-741.
- Ran, Y., Zhu, H., Liu, M., Fabian, M., Olson, J.S., Aranda, R., Phillips, G.N., Dooley, D.M., and Lei, B. (2007) Bis-Methionine ligation to heme iron in the streptococcal cell surface protein Shp facilitates rapid hemin transfer to HtsA of the HtsABC transporter. J. Biol. Chem. In press.
- Lochridge, V.P. and Hardy, M.E. (2007) A single amino acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization. J. Virology. In press.
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