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ANALYSIS OF THE PHENOTYPES ASSOCIATED WITH LOSS OF PROTEIN CARBOXYL METHYLTRANSFERASE ACTIVITY IN...
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Transcript of ANALYSIS OF THE PHENOTYPES ASSOCIATED WITH LOSS OF PROTEIN CARBOXYL METHYLTRANSFERASE ACTIVITY IN...
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.