Transcript Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants. By Chris Brown Dr.

Understanding expression of
Agrobacterium rhizogenes proteins
(GALLS) required for gene transfer
to plants.
By
Chris Brown
Dr. Walt Ream’s Laboratory
Microbiology department
Background
• Agrobacterium damages at least 1.4 million
dollars worth of agriculture per year in
California and Oregon alone.
• Agrobacterium is the only known prokaryote
to transfer genes to a eukaryote.
• Agrobacterium is used throughout
microbiology to transfer DNA into plant cells
Agrobacterium tumefaciens attached to a plant cell.
Image by Martha Hawes
• Agrobacterium tumefaciens and A. rhizogenes
infect wounded plants and transfer plasmid DNA
(T-DNA) and virulence (Vir) proteins into plant
cells.
http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg
http://www.nature.com/nature/jour
nal/v433/n7026/images/433583af2.2.jpg
Understanding the GALLS proteins
Fig. 1.2 GALLS
GALLS Full Length
Repeat 1
GALLS-FL
Repeat 2
Repeat 3
GALLS-CT
GALLS C Terminal
Research Question:
What causes the lower quantity of GALLS-FL
relative to GALLS-CT ?
GALLS
Hypothesis:
Two Causes
1. mRNA degradation
2. Codon bias
FL
170
130
CT
100
Transcription of GALLS gene
GALLS-CT probes
GALLS-FL probes
Sense probe
Antisense
probe
D
C
Repeat 1
Repeat 2
Repeat 3
A
B
Ratio of Sense RNA levels
Average
Ratio A to B
1.04 +0.37
Ratio of Antisense to Sense RNA levels
C to A
.081+.0003
D to B
.106+.009
Facts about Codon Bias
• Codon UUA is found in .075% of leucines in
GALLS
• CUA is found in 5% of leucines in GALLS
• Most common leucine in GALLS is CUC
• GALLS contains 8 of these rare codons
• 7 CUA 1 UUA
• GALLS-FL upstream of GALLS-CT contains
6 of the 8 rare codons including the single
UUA
Fig. 1.2
Location of rare leucine codons
CUA (606 codon)
CUA (1347 codon)
UUA (152 codon)
CUA (1139 codon)
CUA (495 codon)
CUA (740 codon)
Repeat 1
GALLS-FL
Repeat 2
GALLS-CT
CUA (83 codon) CUA (254 codon)
Repeat 3
Method for Overcoming Codon Bias
Over expression of tRNA gene corresponding to
CUA
• Located and amplified tRNA gene from
Agrobacterium rhizogenes genome using
Polymerase Chain Reaction (PCR)
• Inserted amplicon of tRNA corresponding to
leucine CUA into pCR4-TOPO® vector
• Inserted loxP site into plasmid
PCR
product
loxP site
pCR4-TOPO®
Method for Overcoming Codon Bias cont.
• Recombined the plasmid containing rare
leucine tRNA gene corresponding to CUA with
broad-host-range plasmid (compatible with
• A. tumefaciens) via Cre-Lox recombination
5`TGTATG 3`
3`ACATAC
5`5`TGTATG
3`
3`GTATGT
5`5`CATACA 3`
3`GTATGT 5`
5`CATACA 3`
3`ACATAC 5`
5`TGTATG 3`
5`CATACA 3`
5`CATACA
3`
3`GTATGT 5`
5`CATACA 3`
3`GTATGT 5`
5`TGTATG
3`
3`GTATGT 5`
5`CATACA 3`
5`CATACA 3`
3`GTATGT 5`
5`CATACA 3`
5`CATACA 3`
5`CATACA 3`
3`GTATGT 5`
3`GTATGT 5`
Cre-Lox
• Recombined the plasmid containing rare
leucine tRNA gene corresponding to CUA with
broad host range plasmid (compatible with A.
tumefaciens) via Cre-Lox recombination
Cre protein Mediated
pCR4-TOPO vector
tRNA gene (CUA)
loxP site
Method for Overcoming Codon Bias Cont.
• Transformed cointegrate (broad-hostrange/tRNA containing plasmid) into A.
tumafaciens
• Expressed gene and determine protein yield
Method for overcoming Codon Bias
Part 2
Replace rarest leucine codon (UUA) with most
common leucine codon (CUC)
•Create mutation in TTA (UUA) codon 152 of
Galls from Leu UUA to Leu CUC using overlap
extension Polymerase Chain reaction
UUA (154 codon)
PCR product
GALLS-CT
A
B
1. RXN with
primers A & C
A
C
3`
5`
5`
3`
5`
3`
D
3`
5`
C
1. RXN with
primers B & D
B
2. Combination of
DNA from Reactions
in step one with out
primers (self
priming)
5`
5`
3`
D 5`
3`
5`
5`
5`
3`
A
RXN with Primers A & D
3`
5`
D
Method for overcoming Codon Bias
Part 2C
• Insert PCR product with leucine mutation into
vector containing the flanking sequence of Galls
Vector
Vector
Galls
Flanking
PCR
product
ΔUUA (154 codon)
PCR product
GALLS-CT
Galls
Flanking
Method for overcoming Codon Bias
Part 2C
• Insert entire plasmid containing Galls-Δleu154
(UUA CUC) into an A. tumafaciens compatible
vector
• Transform into A. tumafaciens
Results of tRNA over expression
+
-
tRNA
FL
170 kD
130 kD
CT
CUA (1347 codon)
CUA (1139 codon)
CUA (606 codon)
CUA (495 codon)
CUA (740 codon)
GALLS-FL
Repeat
Repeat
GALLS-CT
1
2
CUA (83 codon) CUA (254 codon)
Repeat
3
Results of Codon 152 (Leu UUA to CUC) Mutation
• Transformation of DNA unsuccessful
• Co-transformation yielded all colonies
containing only plasmid the not containing
GALLS
• Possible toxicity to A. rhizogenes
Future Work
• Use a regulated non- Agrobacterium promoter
to allow control of GALLS-FL production
Acknowledgements
•
•
•
•
•
HHMI
COS Cripps Scholarship Fund
Dr. Kevin Ahern
Larry Hodges
Dr. Walt Ream