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

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

5. Bacterial Kidney Disease http://www.medvet.umontreal.ca/departements/patho_micro/ictyopat hologie/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&t bnw=120&prev=/images%3Fq%3Drenibacterium%2Bsalmoninarum %26svnum%3D10%26hl%3Den%26lr%3D%26sa%3 Image courtesy of: Focal abscess

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 = Open reading frame A simplified view of a bacterial genome

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 Our region of interest X X Y Y Z Z Z 14 2 Expected: X - 535 nt, Y - 449 nt, Z with -1783 nt or Z without - 456 nt Possible contamination? 600 500 400 300 200 100 ~2000 1000 3 X = Primers 1 and 2 Y = Primers 3 and 4 Z = Primers 1 and 4 A B A B A B G

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. Position/Enzyme before site (size )Function after site (size)ERGO ORF# 109496 XbaI14258Transposase1570RRSA00337 472093 XbaI348Transposase1854RRSA02075 969223 XbaI1921Transposase29263RRSA03438 2353959 XbaI21505Transposase1707RRSA03708 3085404 XbaI1340Transposase6143RRSA01533 These correspond closely to the five segments in the Xba-I restriction digest.

16. Xba-I restriction digest:

17. Summary and questions?