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
• 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.
Bacterial Kidney Disease (BKD)
Normal vs. diseased
juvenile salmon
Bacterial Kidney Disease
Focal abscess
Focal abscess
Image courtesy of:
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
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)
• 3179312 base pair
genome
• Bacterial genome:
circular
• Encodes more than
50 transposases
A simplified view of a bacterial genome
= Open reading frame
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
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 - 363 bp
• orfB - 831-888 bp, variable
• orfA and orfB separated by a single
stop codon
Transposase interrupts dipeptide-related
chain
Related regions in other organisms:
2
1
XXYYZZZ
3
4
~2000
1000
Primers
600
500
400
A
B
A
B
A
B
G
Our region of interest
• Expected:
X = Primers 1 and 2
X - 535 nt,
Y = Primers 3 and 4
Z = Primers 1 and 4
Y - 449 nt,
Zwith-1783 nt or Zwithout- 456 nt
• Possible contamination?
300
200
100
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#
109496 XbaI
14258
Transposase
1570
RRSA00337
472093 XbaI
348
Transposase
1854
RRSA02075
969223 XbaI
1921
Transposase
29263
RRSA03438
2353959 XbaI
21505
Transposase
1707
RRSA03708
3085404 XbaI
1340
Transposase
6143
RRSA01533
• These correspond closely to the five
segments in the Xba-I restriction digest.
Xba-I restriction digest:
• Summary and questions?