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

Protein Carboxyl Methyltransferase

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

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

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 days – Brain histologically normal –Accumulation of methyl- accepting substrates –Mating behaviors disrupted

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

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

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

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

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

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

Exelixis stocks Figure from Thibault et al, 2004

Figure from Flybase genome browser; Release 4.1 Feb 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

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

Exon 2 Exon 33 dna 2 noxE

Exon 2 Exon 33 dna 2 noxE

Exon 2 Exon 33 dna 2 noxE DICER RISC

Exon 2 Exon 33 dna 2 noxE DICER RISC RNAi

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

pUAST Vector Figure from A. Brand

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 bp EcoRI pCR bp NotIXhoI pLST bp EcoRI NotI 3 2 pLST bp EcoRI 3 2 NotI XhoINotI

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

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

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

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

Pcmt Specific Activity using a Constitutive Promoter

Pcmt Specific Activity at 29°C

Effect of Multiple Transgenic Copies of IRPcmt

Effects of Neuronal Drivers on Pcmt Expression

Northern Blot Analysis of Pcmt Expression

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

Parental Fly CG TAF6l Pcmt yellow mini-white UAS Kb Homolog to TAF6 Tissue specific transcription factor Expressed in primary spermatocytes Mutations render flies male sterile

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

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

Mating Scheme to Generate Deletions

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

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

Genes Deleted From 1.25e

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

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

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

8.2 Kbp 9.4 Kbp

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

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

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

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

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

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

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

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

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

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

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.