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

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

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

R. salmoninarum: background

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

Bacterial Kidney Disease hologie/images/reni3.jpg&imgrefurl= /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

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

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

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

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.

Two Open Reading Frames (ORFs) orfA bp orfB bp, variable orfA and orfB separated by a single stop codon

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

Primers Our region of interest X X Y Y Z Z Z 14 2 Expected: X nt, Y nt, Z with nt or Z without nt Possible contamination? ~ X = Primers 1 and 2 Y = Primers 3 and 4 Z = Primers 1 and 4 A B A B A B G

Xba-I restriction digest:

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

The five segments smaller than 2.0 kb were identified. Position/Enzyme before site (size )Function after site (size)ERGO ORF# XbaI14258Transposase1570RRSA XbaI348Transposase1854RRSA XbaI1921Transposase29263RRSA XbaI21505Transposase1707RRSA XbaI1340Transposase6143RRSA01533 These correspond closely to the five segments in the Xba-I restriction digest.

Xba-I restriction digest:

Summary and questions?