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Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants.

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

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Understanding expression of Agrobacterium rhizogenes proteins (GALLS) required for gene transfer to plants.

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

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

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

  7. Transcription of GALLS gene Repeat 1 Repeat 2 Repeat 3 GALLS-CT probes Sense probe GALLS-FL probes Antisense probe D C A B Ratio of Antisense to Sense RNA levels Ratio of Sense RNA levels

  8. 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

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

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

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

  13. 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

  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. 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

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

  17. 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 Galls Flanking ΔUUA (154 codon) PCR product GALLS-CT

  18. 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

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

  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

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