Genomics of IS 994 : Transposase Genes in Renibacterium salmoninarum

1. Genomics of IS994: Transposase Genes in Renibacterium salmoninarum Gina Capri Dr. Daniel Rockey, Department of Biomedical Sciences

2. Acknowledgements • Howard Hughes Medical Institute • Undergraduate Research, Innovation, Scholarship, Creativity (URISC) • Dr. Daniel Rockey, OSU Department of Biomedical Sciences • The Rockey Lab (Jae Dugan, Sara Weeks, Hency Chu, Damir Alzhanov, Brendan Jeffrey) • Dr. Kevin Ahern • Dr. Mark Strom, Dr. Greg Wiens • USDA/NRI Genome Sequencing program

3. R. salmoninarum: background • Bacterium, causes bacterial kidney disease (BKD) in salmonids • Discovered in the 1930’s • First isolated and cultured in 1956 • Affects both cultured and wild salmonids, forming focal abscesses in the kidney, spleen and liver.

4. Bacterial Kidney Disease (BKD) Normal vs. diseased juvenile salmon

5. Bacterial Kidney Disease Focal abscess Focal abscess Image courtesy of: http://www.medvet.umontreal.ca/departements/patho_micro/ictyopathologie/images/reni3.jpg&imgrefurl=http://www.medvet.umontreal.ca/departements/patho_micro/ictyopathologie/traitements.htm&h=294&w=400&sz=93&hl=en&start=3&tbnid=oDiDHhdui2i8IM:&tbnh=88&tbnw=120&prev=/images%3Fq%3Drenibacterium%2Bsalmoninarum%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3

6. Renibacterium salmoninarum • Gram-positive • Non-sporulating • Non-motile rod • Grows best at 15-18°C and not at all at 25°C • Slow growing (2-3 weeks on KDM2, can take up to 8 or 12 weeks) • Survives inside eggs and phagocytes

7. R. salmoninarum genome • 3607 identified open reading frames (ORFs) • 3179312 base pair genome • Bacterial genome: circular • Encodes more than 50 transposases A simplified view of a bacterial genome = Open reading frame

8. Infected Kidney Samples • Approximately 20 kidney samples were received for study from ODFW • So far, I have extracted DNA from two samples • “A” - 2002 CC00002 6/5/06 • “B” - 2003 CC00481 6/7/06 • Qiagen tissue protocol, followed by Qiagen genomic-tip protocol

9. IS994: Insertion Sequence • IS3 family insertion sequence • 60+ IS994 sequences in the genome • First IS identified from R. salmoninarum • Common element 1330, 1331 or 1345 bp in length • Two open reading frames: orfA and orfB, indicated in ERGO as arrows.

10. Two Open Reading Frames (ORFs) • orfA - 363 bp • orfB - 831-888 bp, variable • orfA and orfB separated by a single stop codon

11. Transposase interrupts dipeptide-related chain Related regions in other organisms:

12. Primers X X Y Y Z Z Z 3 2 ~2000 1 4 1000 600 500 400 A B A B A B G • Expected: • X - 535 nt, • Y - 449 nt, • Zwith-1783 nt or Zwithout- 456 nt • Possible contamination? 300 Our region of interest 200 X = Primers 1 and 2 Y = Primers 3 and 4 Z = Primers 1 and 4 100

13. Xba-I restriction digest:

14. Where precisely are the restriction fragments <2kb located in the genome?

15. The five segments smaller than 2.0 kb were identified. • These correspond closely to the five segments in the Xba-I restriction digest.

16. Xba-I restriction digest:

17. Summary and questions?