CURRICULUM VITAE PERSONAL INFORMATION Name: Paul Alan Kapke Ph.D. Address: 1420 28th Street, Ames, Iowa, 50010 Telephone: 515-294-6114 work 515-233-3697 home Birthdate: September 23, 1952 EDUCATION A.A. 1973 North Iowa Area Community College, Mason City, Iowa B.S. 1975 B.S. in Microbiology General Science, University of Iowa, Iowa City, IA M.S. 1979 Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky Area of Expertise: Microbial Physiology Thesis:Identification and Partial Characterization of a Phosphoenolpyruvate Carboxykinase from Capnocytophaga ochracea Ph.D. 1985 Department of Microbiology, University of Tennessee, Knoxville, Tennessee Area of Expertise: Molecular Virology Dissertation: cDNA Cloning and Partial Sequence Analysis of the Porcine Transmissible Gastroenteritis Coronavirus Genome WORK EXPERIENCE 1995-present Research Associate, Department of Animal Science, Iowa State University, Ames, Iowa 1992-1995 Research Associate, Veterinary Medical Research Institute (VMRI) Ames, IA 1990-1991 Project Leader, Fort Dodge Laboratories,Fort Dodge,Iowa. 1987-1990 Staff Microbiologist, Bacteriology Department National Animal Disease Center, Ames, IA 1985-1987 Postdoctoral research associate, Virology Department, National Animal Disease Center, Ames, IA 1980-1985 Graduate research assistant, Department of Microbiology, University of Tennessee, Knoxville, Tennessee. 1979-1980 Graduate teaching assistant, Department of Microbiology, University of Tennessee, Knoxville, Tennessee. Taught laboratory courses in general microbiology, veterinary virology and veterinary immunology. 1976-1979 Graduate teaching assistant, Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington Kentucky. Taught laboratory courses in general microbiology, pathogenic bacteriology, and immunology. 1975-1976 Graduate teaching assistant, Department of Chemistry University of Iowa, Iowa City, Iowa. Taught laboratory course in inorganic chemistry. PUBLICATIONS Kapke, Paul A., Albert T. Brown, and Thomas T. Lillich. Carbon Dioxide Metabolism by Capnocytophaga ochracea: Identification, Characterization, and Regulation of a Phosphoenolpyruvate Carboxykinase. Infection and Immunity 29(3):756-766 1980.. Brian, David A., Brenda Hogue, William Lapps, Barbara Potts, and Paul Kapke. Comparative Structure of Coronaviruses. In "Proceedings from the Fourth International Symposium on Neonatal Diarrhea." (Ed. S.D. Acres) University of Saskatoon, Saskatoon, Saskatchewan, Canada. 1983. Kapke, Paul A. and David A. Brian. Sequence Analysis of the Porcine Transmissible Gastroenteritis Coronavirus Nucleocapsid Protein Gene. Virology 151:41-49, 1986. Shockley, L.J., P.A. Kapke, W. Lapps, D.A. Brian, L.N.D. Potgieter, and R.D. Woods. Diagnosis of Porcine and Bovine Enteric Coronavirus Infections Using Cloned cDNA Probes. Journal of Clinical Microbiology 25(9):1592-1596, 1987. Woods, R.D., R.D. Wesley, and P.A. Kapke. Neutralization of Porcine Transmissible Gastroenteritis Virus by Complement-dependent Monoclonal Antibodies. American Journal of Veterinary Research 49(3):300-304, 1988. Kapke, P.A., F.Y.T. Tung, B.G. Hogue, D.A. Brian, R.D. Woods, R. Wesley. The Amino-Terminal Signal Peptide on the Porcine Transmissible Gastroenteritis Coronavirus Matrix Protein is not an Absolute Requirement for Translocation and Glycosylation. Virology 165:367-376, 1988. Kapke, P.A., F.Y.T. Tung, D.A. Brian, and R.D. Wesley. Nucleotide Sequence Between the Peplomer and Matrix Protein Genes of the Porcine Transmissible Gastroenteritis Coronavirus Identifies Three Open Reading Frames. Virus Genes 2:3,293-294, 1988. Whipple, D.L., P.A. Kapke, and R.E. Andrews. Analysis of Restriction Endonuclease Fragment Patterns of DNA from Mycobacterium Paratuberculosis. Vet. Microbiol. 19:189-194,1989. Whipple, D. L., P. A. Kapke, and C. P. Vary. Identification of Restriction Fragment Length Polymorphisms in DNA from Mycobacterium Paratuberculosis. Journal of Clinical Microbiology. 28(11):2561-2564, 1990. Whipple, D. L., P. A. Kapke, P. R. Andersen. Comparison of a commercial DNA probe test and three cultivation procedures for detection of Mycobacterium paratuberculosis in bovine feces. J. Vet. Diagn. Invest. 4: 23-27, 1992. Kapke, P.A., K.D. Knudtson, and F.C. Minion. Transformation of Mollicutes with single-stranded Tn4001 DNA. Plasmid 32:85-88, 1994. Jian, C., P.A. Kapke, and F. C. Minion. Transformation of Mycoplasma gallisepticum with Tn916, Tn4001, and integrative plasmid vectors. J. Bact: 176:4459-4462, 1994. Kapke, P. A., K. Jarville-Taylor, J. Cao, and F. C. Minion. Alternate methods for transforming mycoplasmas. manuscript in preparation. Kapke, P, L. Wang, J. Helm, and M. F. Rothschild. Integration of the PiGMaP and USDA maps for porcine chromosome 14. (in press Animal Genetics). Kapke, P, R. Wales, K. Siggens, G. Plastow and M. Rothschild. Rapid Communication: Mapping of an X-Linked Porcine Microsatellite. (in press Journal of Animal Science). BOOK CHAPTERS Kapke, Paul A., Frank Y.C. Tung, David A. Brian, Roger D. Woods, and Ronald D. Nucleotide Sequence of the Porcine Transmissible Coronavirus Matrix Protein Gene. In Advances in Experimental Medicine and Biology. vol. 218 (Proceedings of the Third International Coronavirus Symposium, held September 14-18, 1986 in Asilomar, California). ed. Lai and Stohlman. pages 117-122, 1987. Woods, Roger D., Ronald D. Wesley, and Paul A. Kapke. Complement-Dependent Neutralization of Transmissible Gastroenteritis Virus by Monoclonal Antibodies. In Advances in Experimental Medicine and Biology. vol. 218 (Proceedings of the Third International Coronavirus Symposium, held September 14-18, 1986 in Asilomar, California). ed. Lai and Stohlman. pages 493-500, 1987. Wesley, Ronald, Roger Woods, Paul Kapke. Antibody Response in Swine to Individual Transmissible Gastroenteritis Virus (TGEV) Proteins. In Advances in Experimental Medicine and Biology. vol. 218 (Proceedings of the Third International Coronavirus Symposium, held September 14-18, in Asilomar, California). ed. Lai and Stohlman. pages 475-481, 1986 ABSTRACTS Lillich, T., P. Kapke, W. Gorman, and R. Calmes. Glucose Metabolism in a Gram Negative, Gliding, Bacterium Isolated from Human Gingival Crevices. Int. Congress Bacteriology XII, Munich, West Germany, 1978. Kapke, P.A., A.T. Brown, and T.T. Lillich. The Role of Phosphoenolpyruvate Carboxykinase in Bicarbonate Metabolism by a Gliding Anaerobic Bacterium Isolated from Human Juvenile Periodontitis. Regional ASM meeting, Louisville, Kentucky, 1978. Kapke, P.A., and D. Brian. Oligonucleotide Fingerprints of Porcine Coronaviruses. National Meeting of the American Society for Microbiology, Atlanta, Georgia, 1982. Kapke, P.A. and D.A. Brian. Cloning and Sequence Analysis of the 3' end of the Porcine Transmissible Gastroenteritis Coronavirus Genome. Annual Meeting for The American Society for Virology, Albuquerque, New Mexico, 1985. Kapke, P.A., C.Y. Tung, R.D. Wesley, and D.A. Brian. Sequence Analysis of the Matrix Protein Gene of the Porcine Transmissible Gastroenteritis Coronavirus.Third International Coronavirus Symposium, Asilomar, Calf., 1986. Kapke, P.A. Sequence Analyses of TGEV Genes. The 21st UJNR (United States and Japanese Natural Resources) Mycoplasmosis Meeting. November 4-5. NADC Ames, IA., 1986 Kapke, P.A., S. L. Whipple and L.S. Hahn. Genetic Relatedness Among Mycobacterium paratuberculosis Isolates. 68th Annual Meeting of the Conference of Research Workers in Animal Disease. November 16-17, Chicago, IL. 1987 Kapke, P.A., F. Tung, D. Brian, and R. Wesley. Sequence Analysis of a large Open Reading Frame Residing between the Peplomer and Matrix Protein Genes of the Porcine Transmissible Gastroenteritis Coronavirus. American Society for Microbiology 88th annual meeting. May 8-13, Miami Beach, FL. 1988. Kapke, P. A., D. L. Whipple, and C. P. Vary. Initial Results: Evaluation of a DNA Based Probe Test for Mycobacteium paratuberculosis. 70th Annual Conference of Research Workers in Animal Disease. November 6-7, 1989. Whipple, D. L., P. A. Kapke, and R. L. Zuerner. Isolation and preliminary characterization of extrachromosomal DNA from Mycobacterium paratuberculosis. Vth International Symposium World Association of Veterinary Laboratory Diagnosticians. 1990. Kapke, P. A. and F. Chris Minion. Transformation of Mycoplasma pulmonis using Single-Stranded DNA harboring a gram positive transposon. Center for Immunity Enhancement in Domestic Animals Industrial Affiliates Annual Meeting. May 20-21. Ames, IA. 1992. Kapke, P. A., K. K. Knudtson, and F. Chris Minion. Transposition of Tn4001 Using Single-Stranded DNA. American Society for Microbiology 93th annual meeting. May 16-20. Atlanta, GA. 1993. Minion, F. Chris, J. Cao, and Paul A. Kapke. Genetic Manipulation of Mycoplasma gallisepticum. American Society for Microbiology 94th annual meeting. May Las Vegas NV. 1994 Paul Kapke, Cao Jian, and F. Chris Minion. Transformation studies with Mycoplasma gallisepticum strongly suggest that M. gallisepticum possesses an active DNA restriction system. Meeting of the International Organization for Mycoplasmology, Bordeaux, France. July 19-25, 1994. Artiushin, Sergey, Paul Kapke, and F. Chris Minion. Alternate Methods of Transforming Mycoplasmas. Meeting of the Organization for Mycoplasmology, Bordeaux, France. July 19-25, 1994. Extension Articles Kapke, Paul, Lizhen Wang, Jeannine Helm, Max F. Rothschild. Construction of a Chromosome 14 Consensus Map. 1995 Swine Research Report. pp. 43-45. Iowa State Press. Kapke, Paul, Hans Peter Jorgensen and Max Rothschild. Genotyping the last Mule Footed Pig Herd Reveals a High Degree of Genetic Relatedness. Swine Research Report 1996. Iowa State Press. (in preparation) Accomplishments while in Max Rothschild's lab 1.) I was responsible for mailing out microsatellite primers for the U. S. Pig Genome Coordinator and maintaining an updated data sheet listing the primers and appropriate reference material on each one. 2.) developed an integrated genome map for porcine chromosome 14 by mapping USDA markers to the PiGMaP mapping reference families. 3.) mapped a sex linked marker 4.) completed a genotyping project examining the genetic relatedness of animals purchased for a foundation herd where no information in parentage was available. 5.) established fluorescently based genotyping in the lab. Accomplishments while at VMRI 1.) demonstrating single stranded transposon DNA is functional in different mycoplasmal species 2.) established improved PEG transformation conditions for use with mycoplasmas and acholeplasmas. 3.) established electroporation as viable alternative to PEG mediated transformations. 4.) identified and isolated a promoter that was functional in Escherichia coli, Acholaplasma laidlawii, and Mycoplasma gallisepticum. 5.) developed a universal recipient strain of M. gallisepticum for cloning foreign genes in mycoplasmas 6.) developed an expression vector for use in mycoplasmas. 7.) cloned a herpes viral glycoprotein gene in mycoplasmas. Some of my work at VMRI was funded by private industry sources and is still under active investigation. I left when funds for my support were no longer available Accomplishments while at Fort Dodge Laboratories Fort Dodge Laboratories wanted to establish a molecular biology department. In the five months I worked at FDL, I was involved in the following: 1) electropherotyping of rotavirus master seed stock and 5X passage stock to ensure genetic stability of the virus with tissue culture passage. 2) restriction mapping of parvovirus KF11 and neopar isolates 3) subcloning gene regulatory elements to increase FIV expression, 4) cloning the p10 region of the FIV genome, and 5) designing oligos for site directed mutagenesis. While at FDL, I was able to accomplish the following: - completed the electropherotyping of rotavirus isolates. - restriction mapped parvovirus isolates KF11 and Neopar. - subcloned a regulatory element into a eucaryotic expression vector which was going to be used to coinfect FIV infected cells. - designed a cloning strategy of the FIV genome by PCR amplification. - PCR amplified the p10 gene for site directed mutagenesis. - cloned the p10 gene into vector psl1190. - designed oligonucleotides for site directed mutagenesis that would introduce new restriction sites in the p10 gene and simultaneously alter the desired amino acid residues. The new restriction sites would allow for easy screening of recombinant plasmids following mutagenesis rather than screening by sequencing the recombinant plasmids. SUPERVISORY EXPERIENCE I have been responsible for making work assignments and reviewing the results of one technician for over four years. MEMBERSHIPS The American Society for Microbiology American Association for the Advancement of Science HONORS AND AWARDS Member of the ALPHA CHAPTER OF IACCVTI (Iowa Area Community College and Vocational Technical Institute Honor Society) Science Alliance Fellowship given to outstanding graduate students in the life sciences (1985 University of Tennessee) USDA Certificate of Merit REFERENCES Dr. Max Rothschild Depart. of Animal Science, Iowa State University 515-294-6202 Dr. Chris Minion Veterinary Medical Research Institute, phone 515-294-6347 Dr. Carol Bolin National Animal Disease Center, phone 515-239-8200 Master's Thesis Abstract Capnocytophaga ochracea is a gram negative, anaerobic, capnophillic, rod shaped bacterium. It is a member of the normal oral flora that has also been implicated as a causative agent of periodontal disease. The interactions between a host and its normal oral flora are incompletely understood. Any , whether ultimately resulting in disease or maintenance of the status quo, must involve a host response to the microorganisms metabolizing and growing in specific anatomical sites such as the gingival crevice. It is, therefore, necessary to understand how the normal oral flora members grow and reproduce before understanding the molecular basis of disease yet very little is known about their (members of the normal oral flora including C. ochracea) metabolism. The role of CO2 (the bicarbonate anion) in the metabolism of C. ochracea was investigated. Resting cell suspensions incorporated bicarbonate into succinic acid. Phosphoenolpyruvate carboxykinase (PEPCK) was found to be the enzyme responsible for this reaction.. Partially purified PEPCK has a pH optimum of 6.0 and is specific for PEP, ADP, CO3= and a divalent cation. The enzyme showed the sigmoidal kinetics indicative of allosterism. Hill plot analysis established that the enzyme has at least 2 binding sites for PEP and ADP, and 3 binding sites for bicarbonate and a divalent cation. Several glycolytic intermediates, amino acids and mono, di, and trinucleoside 5' phosphates were tested as possible effectors of PEPCK acitvity. ATP was the only compound shown to have any effect on the enzyme. At concentrations below .1 mM, ATP is a positive effector. At concentrations above .1 mM, ATP is a negative effector. Dissertation Abstract Two approaches were taken to examine the genome structure of the porcine transmssible gastroenteritis coronavirus (TGEV). The first approach was designed to compare the degree of nucleotide sequence homology between the genomes of TGEV and the bovine enteric coronavirus (BCV), a member of an antigenically distinct coronavirus subgroup. For this study, the RNA genomes of TGEV and BCV were separately metabolically labeled with 32P-orthophosphate, and analyzed by two dimensional T1 oligonucleotide fingerprinting. From an analysis of comigrating oligonucleotides in a mixing experiment, it was determined that TGEV and BCV share a sequence homology of approximately 91%. This degree of homology supports the notion that the two viruses, while antigenically dissimilar, arose from a common ancestor. It further raises the question of whether conserved sequences might be confined to restricted regions of the genome allowing identification of conserved functional domains. The second approach was designed to systematically examine the primary structure of the TGEV genome thereby allowing rigorous genetic analyses. For this approach, the 3' end of the 20 KB TGEV genome was cloned and sequenced, and the properties of two genes were deduced and analyzed. TGEV genomic RNA was copied into cDNA after priming with oligo(dT) and the double stranded product was cloned into the Pst 1 site of the pUC9 vector. One clone of 2.0 Kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 Kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential nonoverlapping genes were identified witin the 3' terminal 1663 bases from an examination of open reading frames. The first gene encodes a 382 amino acid protein of 43,426 molecular weight, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second gene encodes a hypothetical 78 amino acid protein of 9101 molecular weight that is hydrophobic at both ends. A 3' non-coding sequence of 273 bases was also determined and a sequence of 9 nucleotides near the poly A tail was found to be conserved for TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.