ANALYSIS OF THE PHENOTYPES ANALYSIS OF THE PHENOTYPES ASSOCIATED WITH LOSS OF PROTEIN ASSOCIATED WITH LOSS OF PROTEIN

CARBOXYL METHYLTRANSFERASE CARBOXYL METHYLTRANSFERASE ACTIVITY IN ACTIVITY IN DROSOPHILA MELANOGASTERDROSOPHILA MELANOGASTER

Linda S. Tanini

Ph.D. Defense

December 19, 2005

Protein Carboxyl MethyltransferaseProtein Carboxyl Methyltransferase

L-Aspartyl

L-Asparaginyl

CH

CH2

C

C

O

NH NH

O

NH2

L-Isoaspartyl

CH CH2 C

C

O OH

NH NH

O

CH

CH2

C

C

O OH

NH NH

O

NH3

H2C

HC

C

O

N

C

OHN

L-Isoaspartyl methyl ester

CH CH2 C

C

O

NH NH

O

OCH3

PCMT

CH3OH

AdoMet

Conservation of PCMTConservation of PCMT

• Conserved within 3 branches of life• High similarity between species

– Drosophila PCMT

OverallConserved

Region

Human 50% 93%

Mouse 55% 90%

Transgenic ModelsTransgenic 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

Using Using DrosophilaDrosophila as Model Organism 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 PCMT and DrosophilaDrosophila

• Overexpression of Pcmt

• At 29ºC

– 30-40% increase in lifespan

• At 25ºC

– lifespan not altered

ObjectivesObjectives

• 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 OutlinePresentation Outline

Genome Disruption ProjectsGenome Disruption Projects

• 3 individually funded projects

• Systematically disrupt genome– Thousands of stocks– Contains single transposon disruption

• P element based

• piggyBac based

Different Types of TransposonsDifferent 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 stocksExelixis stocks

Figure from Thibault et al, 2004

A.

Parental Isogenicpmol/min•mg

( .se. )m

pBac( ){WH Pcm }tpmol/min•mg

( .se.m.).Female 0.92 0.51 (+/- 0.14)

Male 1.35 (+/- 0.29) 0.58 (+/- 0.03)

.B

Parent al Isogenicpmol/min•mg

( .se. )m

pBac( ){WH Pcm }tpmol/min•mg

( .se.m.).Female 1.11 (+/- 0.20) 0.51 (+/- 0.04)

Male 1.30 (+/- 0.05) 0.82 (+/- 0.09)

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

• 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 OutlinePresentation Outline

Exon 2 Exon 3 3 dna 2 noxE

Exon 2 Exon 3 3 dna 2 noxE

Exon 2 Exon 3 3 dna 2 noxE

DICER

RISC

Exon 2 Exon 3 3 dna 2 noxE

DICER

RISC

RNAi

GAL4 IRPcmtUAS

GAL4 protein

PCMT-silencing progeny

IRPcmtUAS

Parental driver strain Parental responder strain

X

Tissue-specific enhancer

GAL4

Tissue specificity of Tissue specificity of PcmtPcmt silencing is controlled by the silencing is controlled by the parental driver strainparental driver strain

Expression

pUAST VectorpUAST Vector

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

3’5’NotI

EcoRI

21 3 4

NotI

XhoI

XhoI

NotI

NotI

EcoRIXhoINotI

EcoRI32

NotI

NotI

EcoRI

Xho

I

XhoI

NotI

pUAST

Eco

RI

Not

I

BamHIBamHI

9050 bp

NotI

pLST-05

XhoI

EcoRI

BamHI

BamHI9607 bp

pLST-07

XhoI

NotI

EcoRI

BamHI

BamHI

10643 bp

EcoRIEcoRI

pCR.2.1

3900 bp

NotI XhoI

pLST-02

4457 bp

EcoRI

NotI3

2

pLST-01

4936 bp

EcoRI32

NotIXhoINotI

• 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 OutlinePresentation Outline

30 minutes

Collect Eggs

20 minutes

3 days

Transfer larvae to food vials3-5 days

Mate adults with w1118

10 days

Identify flies with red eyes

Segregation analysis of Transgenic LinesSegregation analysis of Transgenic Lines

GoInjected Fly X

[w+mC] Xw*

YPm

CyOBsbTM6G1

G2 Xw1118

w1118[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)

w1118

w1118

• 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 OutlinePresentation Outline

Control Genotype

Specific Activity

pmol/min*mg (s.e.m.)

Experimental Genotype

Specific Activity

pmol/min*mg

(s.e.m.)

% Reducion

w1118/IRPcmt X-1 0.40 (0.05) IRPcmt X-1/+:Act5C/+ 0.43 (0.01) 7%w1118/IRPcmt X-3 0.49 (0.05) IRPcmt X-3/+:Act5C/+ 0.40 (0.05) 18%w1118/IRPcmt X-4 0.52 (0.10) IRPcmt X-4/+:Act5C/+ 0.39 (0.01) 25%w1118/IRPcmt X-5 0.56 (0.04) IRPcmt X-5/+:Act5C/+ 0.10 (0.02) 82%

w1118/+;IRPcmt 2-1/+ 0.69 (0.07) Act5C/IRPcmt 2-1 0.14 (0.06) 80%w1118/+;IRPcmt 2-2/+ 0.60 (0.05) Act5C/IRPcmt 2-2 0.10 (0.01) 83%w1118/+;IRPcmt 2-4/+ 0.44 (0.00) Act5C/IRPcmt 2-4 0.08 (0.01) 82%w1118/+;IRPcmt 2-5/+ 0.51 (0.13) Act5C/IRPcmt 2-5 0.10 (0.01) 80%w1118/+;IRPcmt 2-6/+ 0.49 (0.02) Act5C/IRPcmt 2-6 0.09 (0.00) 82%

w1118/+;IRPcmt 3-1/+ 0.38 (0.03) Act5C/+; IRPcmt 3-1;+ 0.08 (0.01) 79%w1118/+;IRPcmt 3-2/+ 0.47 (0.13) Act5C/+; IRPcmt 3-2;+ 0.10 (0.01) 79%

Actin5C/+; w1118/+ 0.45 (0.00)

PcmtPcmt Specific Activity using a Constitutive Promoter Specific Activity using a Constitutive Promoter

PcmtPcmt Specific Activity at 29°C Specific Activity at 29°C

Control Genotype

Specific Activity

pmol/min*mg (s.e.m.)

Experimental Genotype

Specific Activity

pmol/min*mg (s.e.m.)

% Reduction

w1118/IRPcmt X-1 0.52 (0.01) IRPcmt X-1/+:Act5C/+ 0.47 (0.08) 11%w1118/IRPcmt X-3 0.38 (0.03) IRPcmt X-3/+:Act5C/+ 0.52 (0.08) 37%w1118/IRPcmt X-4 0.50 (0.03) IRPcmt X-4/+:Act5C/+ 0.46 (0.01) 8%w1118/IRPcmt X-5 0.33 (0.01) IRPcmt X-5/+:Act5C/+ 0.06 (ND) 82%

w1118/+;IRPcmt 2-1/+ 0.51 (0.02) Act5C/IRPcmt 2-1 0.20 (ND) 61%w1118/+;IRPcmt 2-2/+ 0.41 (0.07) Act5C/IRPcmt 2-2 0.09 (0.02) 78%w1118/+;IRPcmt 2-4/+ 0.57 (0.00) Act5C/IRPcmt 2-4 0.12 (0.00) 79%w1118/+;IRPcmt 2-5/+ 0.25 (ND) Act5C/IRPcmt 2-5 0.12 (0.01) 52%w1118/+;IRPcmt 2-6/+ 0.50 (0.00) Act5C/IRPcmt 2-6 0.12 (0.06) 76%

w1118/+;IRPcmt 3-1/+ 0.32 (0.01) Act5C/+; IRPcmt 3-1;+ 0.13 (0.00) 59%w1118/+;IRPcmt 3-2/+ 0.37 (0.01) Act5C/+; IRPcmt 3-2;+ 0.11 (0.00) 70%

Actin5C/+;w1118/+ 0.45 (0.00)

Effect of Multiple Transgenic Copies of IREffect of Multiple Transgenic Copies of IRPcmtPcmt

Genotype

SpecificActivity

pmol/m in*mg (s.e.m.)

PercentReduction

Compared toActin5C driver

PercentReduction

Compared toIR Pcmt

responderAct5C-GAL4/CyO; +/+ 1.12 (0.04) - -+/+;I RPcmt; IRPcmt 0.47 (0.05) - -Act5C-GAL4/Cyo;IRPcmt/TM3,Sb 0.25 (0.07) 78% 44%Act5C-GAL4/CyO;IRPcmt/ IRPcmt 0.13 (0.00) 88% 72%

Effects of Neuronal Drivers on Pcmt ExpressionEffects of Neuronal Drivers on Pcmt Expression

Genotype

Specific Activity

pmol/min*mg (s.e.m.)

Percent Reduction Compared

to elav driver

+/+; UAS- IRPcmt 3-1/UAS- IRPcmt 3-1 0.51 (0.03) -elav-Gal4/+; +/+ 0.74 (0.07) -elav-Gal4/+; UAS- IRPcmt 3-1/+ 0.67 (0.08) 9%elav-Gal4/elav-Gal4; IRPcmt 3-1/ IRPcmt 3-1 0.34 (0.09) 54%

Genotype

SpecificActivity

pmol/ min*mg (s.e.m.)

PercentReductionCompared

to GMRDriver

ninaE.GMR-GAL4ninaE.GMR-GAL4; +/+ 0.97 (0.15) - +/+;UAS-IRPcmt 3-1;UAS- IRPcmt 3-1 0.67 (0.05) -ninaE.GMR-GAL4 /+;UAS-IRPcmt3-1/+ 0.47 (0.01) 52%ninaE.GMR-GAL4/ ninaE.GMR-GAL4;UAS-IRPcmt 3-1/UAS- IRPcmt 3-1 0.43 (0.01) 56%

Northern Blot Analysis of Northern Blot Analysis of PcmtPcmt Expression 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 OutlinePresentation Outline

Parental FlyParental Fly

CG2519

1373 1374 1375 1376 1377 1378 1379 1380

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 DysgenesisModel of Hybrid Element Dysgenesis

A A B

A A B

C

C

A B C

A B C

A B

A

C

C

A

A

A B C

A B C

B

A B A B C

A

A B C

A A

C

B C

Imprecise ExcisionImprecise Excision

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

Mating Scheme to Generate DeletionsMating Scheme to Generate Deletions

y’w*;P{EPgy2}Taf6L

TM3,Sb’

Δ2-3,CyO ;BcEgfc

ee

Δ2-3,CyO ;

Y +X’y *w ;

TM3,Sb’TM6B

Red eye, wild typebody

Curly wi ,ng blackcell, ebony body

Curl y wi , ng wi ldtype, body dysgeni ceyes

Stubbl e bristles, whitee yee bony body

Majority of theprogeny

Normal Mendeliansegregation

Rare Recombinants

Select for non curly,ebony body and red eyes

Deletion Events

Select for non curly wing,wild type body and white

eyes

P

F1

F2

P{EPgy2}Taf6L

e

X

y’w*;

• 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 OutlinePresentation Outline

Flies Generated Through Site Specific RecombinationFlies Generated Through Site Specific Recombination

1000s of flies

36 possible lines

(~.45%)

12Lethal

24 Viable

1

Genes Deleted From 1.25eGenes Deleted From 1.25e

143014201410

CG14671 CG12746

140013901380SnmI Rheb CRMP Vha26

Pcmt Pi4KI Ialpha CG2926 noi eIF-5C CG2919 Rga

Taf6L CG2931

rev7 CG1427

• 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 OutlinePresentation Outline

Flies generated through imprecise excisionFlies generated through imprecise excision

1000s of flies

126 possible

lines

120Viable

6 Lethal

1

Specific Activity of Specific Activity of ΔΔPcmt FliesPcmt Flies

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Specific Activity (pmol/min*mg)

P{EPgy2}Taf6l P{EPgy2}Taf6l ΔPcmt ΔPcmt

8.2 Kbp

9.4 Kbp

9.48.2

4.1

1.8

117531

123531

• 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 OutlinePresentation Outline

Parental Males

Parental Females

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

ΔPcmt Canton-S P{EPgy2}Taf6l

Canton-S Canton-S ΔPcmt

Progeny of Progeny of ΔΔPcmtPcmt Compared to Parental Line Compared to Parental Line

100

150

200

250

300

350

400

450

Number of Eclosed Flies

Fecundity of Fecundity of ΔΔPcmtPcmt Compared to Parental Line Compared to Parental Line

Females Canton-S ΔPcmt P{EPgy2}Taf6l Canton-S

Males ΔPcmt Canton-S Canton-S Canton-S

Paraquat ResistanceParaquat 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

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0 5 10 15 20 25 30 35 40

Age (hours)

Percent Survival

Paraquat ResistanceParaquat Resistance

ΔPcmt treated with 2mM Paraquat

and 2% sucrose

ΔPcmt treated with 2% sucrose

P{EPgy2}Taf6l treated with 2mM Paraquat and 2% sucrose

P{EPgy2}Taf6l treated 2% sucrose

Mild Heat ShockMild 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 Shock5-day old and 17-day old Mild Heat Shock

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75

Age (days)

Percent Survival

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69

Age (Days)

Percent Survival

5-day old Flies 17-day old Flies

- P{EPgy2}Taf6l

- ΔPcmt

Lifespan at 29°CLifespan at 29°C

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47

Age (days)

Percent Survival

- Male ΔPcmt

- Male P{EPgy2}Taf6l

- Female ΔPcmt

Female P{EPgy2}Taf6l

Summary of ResultsSummary of Results• Aged ΔPcmt flies respond better to mild heat

shock• At increased temperature, ΔPcmt male flies live

longer than parental male flies

Possible ReasonsPossible Reasons• Pleiotrophic effect with Taf6l• Increase in HSPs

– Increase in damaged protein

– HSPs extending lifespan of ΔPcmt flies

Future ProjectsFuture Projects

• Investigate levels of HSPs in ΔPcmt flies

• Isogenize the ΔPcmt flies with w67c23

• Investigate expression levels of other genes in ΔPcmt 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.