1. ANALYSIS OF THE PHENOTYPES ASSOCIATED WITH LOSS OF PROTEIN CARBOXYL METHYLTRANSFERASE ACTIVITY IN DROSOPHILA MELANOGASTER Linda S. Tanini Ph.D. Defense December 19, 2005

2. Protein Carboxyl Methyltransferase

3. L-Aspartyl L-Asparaginyl CH CH 2 C C O NH O NH 2 L-Isoaspartyl CH CH 2 C C O OH NH O CH CH 2 C C O OH NH O NH 3 H2CH2C HC C O N C O HN L-Isoaspartyl methyl ester CH CH 2 C C O NH O OCH 3 PCMT CH 3 OH AdoMet

4. Conservation of PCMT Conserved within 3 branches of life High similarity between species –Drosophila PCMT Overall Conserved Region Human50%93% Mouse55%90%

5. Transgenic Models E. coli –Viability decreased in stationary phase when environmentally stressed C. elegans –Survival decreased in dauer stage S. pombe –Reduced spore viability –Rescued with overexpression plasmid Mice – Die prematurely between 20-60 days – Brain histologically normal –Accumulation of methyl- accepting substrates –Mating behaviors disrupted

6. Using Drosophila as Model Organism Knockout mice die as juveniles Short life span Large populations Adult tissues are postmitotic Genetic tools Single PCMT transcript

7. Protein expression –Highest in adult flies –Lowest 3rd instar larvae In embryos –Highest in nerve cord and brain PCMT and Drosophila Overexpression of Pcmt At 29ºC –30-40% increase in lifespan At 25ºC – lifespan not altered

8. Objectives Generate Drosophila mutants with reduced expression of Pcmt –Stock collections –RNAi –P element mobilization Analyze the phenotypic consequences of reduced Pcmt expression

9. Search current stock collections for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

10. Genome Disruption Projects 3 individually funded projects Systematically disrupt genome –Thousands of stocks –Contains single transposon disruption P element based piggyBac based

11. Different Types of Transposons P element –31 bp inverted repeat –Generate 8bp direct repeat –Imprecise excision –Inserts into “hot spots” piggyBac –Non species specific –13 bp inverted repeat –Does not insert into “hot spots” –Does not excise imprecisely mobile piece of DNA

12. Exelixis stocks Figure from Thibault et al, 2004

14. Figure from Flybase genome browser; Release 4.1 Feb 2005 309 bp upstream of translation start site 210 bp upstream of TATA box 230 bp upstream of transcription start site 60 bp upstream of initiator sequence

15. Search current stock collection for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

16. Exon 2 Exon 33 dna 2 noxE

17. Exon 2 Exon 33 dna 2 noxE

18. Exon 2 Exon 33 dna 2 noxE DICER RISC

19. Exon 2 Exon 33 dna 2 noxE DICER RISC RNAi

20. GAL4 IRPcmt UAS GAL4 protein PCMT-silencing progeny IRPcmt UAS Parental driver strainParental responder strain X Tissue-specific enhancer GAL4 Tissue specificity of Pcmt silencing is controlled by the parental driver strain Expression

21. pUAST Vector Figure from A. Brand http://www.gurdon.cam.ac.uk/~brandlab/reagents/pUAST.html

22. 3’ 5’ NotI EcoRI 2134 NotI XhoI NotI EcoRI XhoI NotI EcoRI 3 2 NotI EcoRI XhoI NotI pUAST EcoRI NotI BamHI 9050 bp NotI pLST-05 XhoI EcoRI BamHI 9607 bp pLST-07 XhoI NotI EcoRI BamHI 10643 bp EcoRI pCR.2.1 3900 bp NotIXhoI pLST-02 4457 bp EcoRI NotI 3 2 pLST-01 4936 bp EcoRI 3 2 NotI XhoINotI

23. Search current stock collections for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

24. 30 minutes Collect Eggs 20 minutes 3 days Transfer larvae to food vials 3-5 days Mate adults with w 1118 10 days Identify flies with red eyes

25. Segregation analysis of Transgenic Lines GoGo Injected Fly X [w +mC ]X w*Yw*Y Pm CyO Bsb TM6 G1G1 G2G2 X w 1118 [w * ];CyO;Bsb Establish chromosome with P element insertion by segregation of mini-white gene with other markers X chromosome (only females have eye color) Chromosome 2 (eye color segregates with normal wings) Chromosome 3 (eye color segregates with normal bristle) w 1118

26. Search current stock collections for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

27. Pcmt Specific Activity using a Constitutive Promoter

28. Pcmt Specific Activity at 29°C

29. Effect of Multiple Transgenic Copies of IRPcmt

30. Effects of Neuronal Drivers on Pcmt Expression

31. Northern Blot Analysis of Pcmt Expression

32. Search current stock collections for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

33. Parental Fly CG2519 1373 1374 137513761377137813791380 TAF6l Pcmt yellow mini-white UAS Kb Homolog to TAF6 Tissue specific transcription factor Expressed in primary spermatocytes Mutations render flies male sterile

34. Model of Hybrid Element Dysgenesis AAB A AB C C A BC ABC A B A C C A A A BC ABC B AB A BC A ABC AA C BC

35. Imprecise Excision Figure from Adams, M.D. and J.J. Sekelsky

36. Mating Scheme to Generate Deletions

37. Search current stock collections for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

38. Flies Generated Through Site Specific Recombination 1000s of flies 36 possible lines (~.45%) 12 Lethal 24 Viable 1

39. Genes Deleted From 1.25e

40. Search current databases for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

41. Flies generated through imprecise excision 1000s of flies 126 possible lines 120 Viable 6 Lethal 1

42. Specific Activity of  Pcmt Flies P{EPgy2}Taf6l  Pcmt

43. 8.2 Kbp 9.4 Kbp

44. 9.4 8.2 4.1 1.8

45. 117531123531

46. Search current databases for Pcmt deletions –piggyBac{WH} Pcmt Silencing Pcmt using RNAi –Constructing RNAi plasmid –Generating IRPcmt flies –Silencing levels Construction of Pcmt deletion mutants using P element mobilization – Male site specific mobilization – Imprecise excision – Phenotypes associated with deletion Presentation Outline

47. Parental Males Parental Females  Pcmt Canton-SP{EPgy2}Taf6l Canton-S  Pcmt Progeny of  Pcmt Compared to Parental Line

48. Fecundity of  Pcmt Compared to Parental Line FemalesCanton-S  Pcmt P{EPgy2}Taf6lCanton-S Males  Pcmt Canton-S

50. Paraquat Resistance Increase ROS reduce lifespan Increase in lifespan in Drosophilia if SOD or catalase if overexpressed Increase ROS attributed to peroxidation, DNA modification, and carbonyl modification of proteins

51. Paraquat Resistance   Pcmt treated with 2mM Paraquat and 2% sucrose  Pcmt treated with 2% sucrose  P{EPgy2}Taf6l treated with 2mM Paraquat and 2% sucrose P{EPgy2}Taf6l treated 2% sucrose

52. Mild Heat Shock Elevated temperatures- PCMT protein is stable Aging progresses rapidly at elevated temperatures –Increase in protein unfolding, isomerization and deamidation Increase in HSP when exposed to high temps –Decreases when return to normal temps –Age dependent Increased hsp70 and hsp22 linked to increased lifespan Accumulation of damaged protein can increase HSP

53. 5-day old and 17-day old Mild Heat Shock 5-day old Flies 17-day old Flies - P{EPgy2}Taf6l -  Pcmt

54. Lifespan at 29°C - Male  Pcmt - Male P{EPgy2}Taf6l  - Female  Pcmt mFemale P{EPgy2}Taf6l

55. Summary of Results Aged  Pcmt flies respond better to mild heat shock At increased temperature,  Pcmt male flies live longer than parental male flies Possible Reasons Pleiotrophic effect with Taf6l Increase in HSPs –Increase in damaged protein –HSPs extending lifespan of  Pcmt flies

56. Future Projects Investigate levels of HSPs in  Pcmt flies Isogenize the  Pcmt flies with w 67c23 Investigate expression levels of other genes in  Pcmt flies through microarray

57. Acknowledgments Committee Members –Dr. Laura Hake –Dr. Marc Muskavitch Dr. Anne Stellwagen Dr. Annette Parks Thesis Advisor –Dr. Clare O’Connor Rob Jackson and Mary Roberts –Tufts University My Family Members of the Biology Dept.