1. Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants. By Chris Brown Dr. Walt Ream’s Laboratory Microbiology department

2. Background Agrobacterium damages at least 1.4 million dollars worth of agriculture per year in California and Oregon alone.

3. 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

4. Agrobacterium tumefaciens and A. rhizogenes infect wounded plants and transfer plasmid DNA (T-DNA) and virulence (Vir) proteins into plant cells. http://www.nature.com/nature/jour nal/v433/n7026/images/433583a- f2.2.jpg http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg

5. Fig. 1.2 GALLS GALLS-CT GALLS C Terminal GALLS Full Length Understanding the GALLS proteins Repeat 1Repeat 2Repeat 3 GALLS-FL

6. Research Question: What causes the lower quantity of GALLS-FL relative to GALLS-CT ? Hypothesis: Two Causes 1. mRNA degradation 2. Codon bias FL 170 130 CT 100 GALLS

7. Average Ratio A to B 1.04 +0.37 Transcription of GALLS gene C to A.081+.0003 D to B.106+.009 Ratio of Sense RNA levels Ratio of Antisense to Sense RNA levels Repeat 1Repeat 2Repeat 3 Sense probe Antisense probe GALLS-CT probes GALLS-FL probes A D C B

8. 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 Facts about Codon Bias

9. Repeat 1Repeat 2Repeat 3 GALLS-FL Fig. 1.2 Location of rare leucine codons GALLS-CT CUA (83 codon)CUA (254 codon) CUA (495 codon) CUA (606 codon) CUA (740 codon) CUA (1139 codon) CUA (1347 codon) UUA (152 codon)

10. 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 Method for Overcoming Codon Bias PCR product pCR4-TOPO® loxP site

11. 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

12. 5`CATACA 3` 5`TGTATG 3` 3`ACATAC 5` 3`GTATGT 5` 5`CATACA 3` 3`GTATGT 5` 5`TGTATG 3` 5`CATACA 3` 3`GTATGT 5` 5`TGTATG 3` 3`GTATGT 5` 5`CATACA 3` 3`GTATGT 5` 5`CATACA 3` 5`TGTATG 3` 3`ACATAC 5` 3`GTATGT 5` 5`CATACA 3` 3`GTATGT 5` 5`CATACA 3` Cre-Lox

13. loxP site tRNA gene (CUA) Cre protein Mediated pCR4-TOPO vector Recombined the plasmid containing rare leucine tRNA gene corresponding to CUA with broad host range plasmid (compatible with A. tumefaciens) via Cre-Lox recombination

14. Method for Overcoming Codon Bias Cont. Transformed cointegrate (broad-host- range/tRNA containing plasmid) into A. tumafaciens Expressed gene and determine protein yield

15. 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 Method for overcoming Codon Bias Part 2 GALLS-CT PCR product UUA (154 codon)

16. A C D B 5` 3` 5` A A D D C B 1. RXN with primers A & C 1. RXN with primers B & D 2. Combination of DNA from Reactions in step one with out primers (self priming) RXN with Primers A & D 3`

17. Method for overcoming Codon Bias Part 2C Insert PCR product with leucine mutation into vector containing the flanking sequence of Galls PCR product Galls Flanking Vector GALLS-CT PCR product ΔUUA (154 codon)

18. Insert entire plasmid containing Galls-Δleu154 (UUA CUC) into an A. tumafaciens compatible vector Transform into A. tumafaciens Method for overcoming Codon Bias Part 2C

19. Results of tRNA over expression Repeat 1 Repeat 2 Repeat 3 GALLS-FL GALLS-CT CUA (83 codon)CUA (254 codon) CUA (495 codon) CUA (606 codon) CUA (740 codon) CUA (1139 codon) CUA (1347 codon) FL 170 kD 130 kD CT tRNA - +

20. 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

21. Future Work Use a regulated non- Agrobacterium promoter to allow control of GALLS-FL production

22. Acknowledgements HHMI COS Cripps Scholarship Fund Dr. Kevin Ahern Larry Hodges Dr. Walt Ream