2005 Annual Report
This report serves to document research conducted under a reimbursable agreement between ARS and USDA-CSREES-National Research Initiative-Competitive Grants Program (NRI-CGP). Additional details of research can be found in the report for the parent project 3625-32000-062-00D Understanding Host Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This agreement between ARS (NADC) and the NRI-CGP (subcontracted from the University of Minnesota) encompasses measures to ensure animal health through the control and management of Johne’s disease through genomic research on Mycobacterium avium subsp. paratuberculosis. This project has the objective of providing scientific core resources to other JDIP investigators. This includes development of a M. paratuberculosis (MAP) whole genome microarray, development of expression clones for many of the MAP genes, and characterization of transposon mutant libraries. Therefore, this project contributes to the National Program component of Animal Protection and also touches on Microbial Genomics. One of the tasks of this project is to develop an oligo (small nucleotide sequence) array representing each of the genes in the MAP genome. The MAP oligo array has now been designed at the National Animal Disease Center (NADC). One 70 nucleotide oligo was designed to represent each annotated MAP gene < 4000 bp by using ArrayOligoSelector scripts (http://arrayoligosel.sourceforge.net/). MAP genes > 4000 bp were split in half and one oligo was designed for each half. Finally, control oligos were designed for six A. thaliana sequences. A test set of 192 oligos (two 96 wells plates representing 96 ORFs called using two different software programs) was sent to NADC for evaluation of hybridization performance. Quality control assays checked out and the entire order was shipped to N.A.D.C. The oligo array is now ready to be printed and used in MAP experiments. Another task is to clone most of the important genes in the MAP genome and express them to form recombinant proteins. These proteins can then be used in functional and immunological assays. To date over fifty such proteins have been cloned, expressed and purified from E. coli.