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stmA gene was discovered at IARI based on paralysis
phenotype (Shyngle and Sharma, 1985)
stmA gene was discovered at IARI based on paralysis
phenotype (Shyngle and Sharma, 1985)
Sequence conserved across large number of organisms
ranging from yeast, Arabidopsis, rice and humans
Sequence conserved across large number of organisms
ranging from yeast, Arabidopsis, rice and humans
Regulates the activity of Phospholipase C- in the
G-protein signaling pathway (Huang et al., 2004)
Regulates the activity of Phospholipase C- in the
G-protein signaling pathway (Huang et al., 2004)
The stambhA geneThe stambhA gene
Codes for a membrane bound lipase Codes for a membrane bound lipase
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Division of Genetics
Indian Agricultural Research Institute
Division of Genetics
Indian Agricultural Research Institute
Studies On A Lipase Coding Gene,
Regulating G-protein Coupled
PLC- Signaling In
Drosophila melanogast er
Studies On A Lipase Coding Gene,
Regulating G-protein Coupled
PLC- Signaling In
Drosophila melanogast er
Mridul ChakrabortiMridul Chakraborti
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1. To study and correlate allelic variation and phenotype
of stmA gene
1. To study and correlate allelic variation and phenotype
of stmA gene
2. To evaluate the effect of stmA gene on development
and olfaction
2. To evaluate the effect of stmA gene on development
and olfaction
3. To analyze the functional interaction between stmA
and inositol triphosphat e r ece ptor (itpr ) genes
3. To analyze the functional interaction between stmA
and inositol triphosphat e r ece ptor (itpr ) genes
ObjectivesObjectives
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Study and
correlation of allelic
variation and
phenotype of
stmA gene
Study and
correlation of allelic
variation and
phenotype of
stmA gene
ObjectiveObjective
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stmA1 / stmP6
stmA1 / stmP1
stmP6
stmP1
stmA1
Genotypes
4 min3.5 min3 min2.5 min2 min
Paralysis time
0010000
0505000
02525500
0050500
0050500
Complementation Test by Paralysis
profile of stmA Mutants
Complementation Test by Paralysis
profile of stmA Mutants
Water inletWater inlet
Water
outlet
Water
outlet
Flies inFlies in
Flies outFlies out
The Sushi Cooker The Sushi Cooker
No Complementation: stmAP1 and stmAP6
are alleles of stmA1
No Complementation: stmAP1 and stmAP6
are alleles of stmA1
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Sequencing Strategy for stmA AllelesSequencing Strategy for stmA Alleles
The 4.7 kb stmA gene was divided into 4 contigs The 4.7 kb stmA gene was divided into 4 contigs
Primers for the contigs designed by using Primer-3
software
Primers for the contigs designed by using Primer-3
software
Contig A
Contig B
Contig C
Contig D
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M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12
Optimization of PCR Amplification Protocol
for stmA Contigs
Optimization of PCR Amplification Protocol
for stmA Contigs
Optimum annealing temperature above Tm value was
determined for different primer sets
Optimum annealing temperature above Tm value was
determined for different primer sets
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Standardized PCR Amplification Protocol
for stmA Contigs
Standardized PCR Amplification Protocol
for stmA Contigs
Hold4°C6
110 min.72°C5
2 min.72°C4
1 min.
59.3°C
63.5°C
62.4°C
57.8°C
3
Primer set-A
Primer set-B
Primer set-C
Primer set-D
35
30 s94°C2
13 min.94°C1
CyclesTimeTemp.Step
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Polymorphism among
stmA Alleles
Polymorphism among
stmA Alleles
Possible mutational lesion near the primer
binding site of stmAP6 ±A contig
Possible mutational lesion near the primer
binding site of stmAP6 ±A contig
M A2 P 6 P 1 A2 P 6 P 1 A2 M A2 P 6 P 1 A2 P 6 P 1 A2
M P 6 P 1 A2 P 6 P 1M P 6 P 1 A2 P 6 P 1
AA BB CC
CC DD
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Sequence Analysis of Mutant AllelesSequence Analysis of Mutant Alleles
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Effect of stmAgene on
development
and
olfaction
Effect of stmAgene on
development
and
olfaction
ObjectiveObjective
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Odorant Response analysis by ³Park¶s Method´ Odorant Response analysis by ³Park¶s Method´
No. of flies [ODOR TRAP] ± No. of flies [CONTROL TRAP]No. of flies [ODOR TRAP] ± No. of flies [CONTROL TRAP]
Odorant Response Index =Odorant Response Index =
Total No. of flies [BEAKER]Total No. of flies [BEAKER]
ODORANT
TRAP
ODORANT
TRAPCONTROL
TRAP
CONTROL
TRAP
Analysis of Olfactory Responses of
Various StmA Mutants
Analysis of Olfactory Responses of
Various StmA Mutants
Simple behavioral assay Simple behavioral assay
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Standardization of Optimum Odorant
Concentration in Wild Type Flies
Standardization of Optimum Odorant
Concentration in Wild Type Flies
Dilution Factor of Benzaldehyde
(aqueous solution)
2×10-410-32×10-310-210-11
0.110.050.100.040.240.07SD
0.720.760.130.56-0.21-0.64Mean
ORI
Dilution Factor of Ethyl Acetate
(aqueous solution)
2×10-410-32×10-310-210-11
0.130.070.100.070.140.09SD
0.540.440.400.79-0.72-0.35Mean
ORI
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W T W T
stmAP¨1stmAP¨1
stmA2 stmA2 stmAP¨6 stmAP¨6
-1.00-1.00
-0.50-0.50
0.000.00
0.500.50
1.001.00
O d o r
a n t R e s p o n s e
I n d e x
O d o r
a n t R e s p o n s e
I n d e x
Odorant Response Index Towards
Benzaldehyde (10-3 Dilution)
Odorant Response Index Towards
Benzaldehyde (10-3 Dilution)
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stmA2 stmA2 stmAP¨1stmAP¨1
stmAP¨6 stmAP¨6
W T W T
00
0.20.2
0.40.4
0.60.6
0.80.8
11
O d o
r a n t R e s p o n s e I n d e x
O d o
r a n t R e s p o n s e I n d e x
Odorant Response Index Towards
Ethyl Acetate (10-2 Dilution)
Odorant Response Index Towards
Ethyl Acetate (10-2 Dilution)
Reduced odorant response in mutants suggests involvement
of stmA in olfactory behaviour
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Electroantennogram (EAG) pattern indicates the
electrophysiological response in the antennae
Electroantennogram (EAG) pattern indicates the
electrophysiological response in the antennae
Electroantennogram Alalysis for Olfactory
Response Study
Electroantennogram Alalysis for Olfactory
Response Study
Genotypes showing reduced EAG are not able to
recognize odorants
Genotypes showing reduced EAG are not able to
recognize odorants
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Comparison of Electroantennogram
Pattern of stmA Mutants
Comparison of Electroantennogram
Pattern of stmA Mutants
00
22
44
66
88
1010
1212
1414
ButanaolButanaol
(10-2)(10-2)
Ethyl
Acetate
Ethyl
Acetate
(10-2)(10-2)
PropionicPropionic
Acid
(10-2)
Acid
(10-2)
Benzal-
dehyde
Benzal-
dehyde
(10-3)(10-3)
IsoamylIsoamyl
Acetate
(10-2 )
Acetate
(10-2 )
Odorants (Concentration)Odorants (Concentration)
A m p l i t u d e ( m
V )
A m p l i t u d e ( m
V )
stmA1 /+stmA1 /+ stmA1 / stmA1stmA1 / stmA1
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Histological study in antenna of stmA- eGF P transgenic lineHistological study in antenna of stmA- eGF P transgenic line
Gene Expression in Antennal TissueGene Expression in Antennal Tissue
Confirmation of stmA expression in antennaConfirmation of stmA expression in antenna
eGFPeGFP
E c oRIE c oRI Pm /IPm /I
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Analyses of functional interactionbetween stmA and itpr mutantsAnalyses of functional interactionbetween stmA and itpr mutants33
itpr
ObjectiveObjective
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Generation of stmA-itpr Double Mutant
Combination
Generation of stmA-itpr Double Mutant
Combination
Cy Cy
SpSp;;
TM 6 T bTM 6 T b
++
stmA2 stmA2
;;++
××
stmA2 stmA2
Cy Cy ;;
TM 6 T bTM 6 T b
++
stmA2 stmA2
SpSp;;
SbSb
++
stmA2 stmA2
++
stmA2 stmA2
Cy Cy ;;
TM 6 T bTM 6 T b
SbSb
stmA2 stmA2
Cy Cy ;;
TM 6 T bTM 6 T b
SbSb
××
××
stmA2 stmA2
stmA2 stmA2 ;;
TM 6 T bTM 6 T b
SbSb
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××
××
Cy Cy
SpSp;;
TM 6 T bTM 6 T b
SbSb××
Cy Cy
++;;
TM 6 T bTM 6 T b
k a1091k a1091
++;;
++
TM 6 T bTM 6 T b
k a1091k a1091
;;TM 6 T bTM 6 T b
SbSb
stmA2 stmA2
stmA2 stmA2
;;
TM 6 T bTM 6 T b
k a1091k a1091Cy Cy
;;
TM 6 T bTM 6 T b
SbSb
SpSp
stmA2 stmA2
stmA2 stmA2
stmA2 stmA2
;;
TM 6 T bTM 6 T b
k a1091k a1091stmA2 stmA2
Generation of stmA-itpr Double Mutant
Combination
Generation of stmA-itpr Double Mutant
Combination
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stmA2 stmA2
;
ug3ug3
TM 6 T bTM 6 T bstmA2
stmA2
stmA2 stmA2
;
k a1091k a1091
TM 6 T bTM 6 T bstmA2
stmA2
××
stmA2 stmA2
;;
k a1091k a1091
ug3ug3stmA2
stmA2
Reciprocal
cross
Reciprocal
cross
New stock with itpr mutation in stmA2
background
New stock with itpr mutation in stmA2
background
Generation of stmA-itpr Double Mutant
Combination
Generation of stmA-itpr Double Mutant
Combination
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Study of Pupation Pattern in stmA-itpr
Mutant Combinations (25rC)
Study of Pupation Pattern in stmA-itpr
Mutant Combinations (25rC)
0.000.00
10.0010.00
20.0020.0030.0030.00
40.0040.00
50.0050.00
60.0060.0070.0070.00
80.0080.00
90.0090.00
11 22 33 44 55 66 77 88 99 1010 11111212 1313 1414 15151616 1717 1818 1919 20202121
Day of CultureDay of Culture
ug3 (or k a1091)/ T bug3 (or k a1091)/ T b
stmA2 , ug3(or k a1091)/ T bstmA2 , ug3(or k a1091)/ T b
ug3/ k a1091ug3/ k a1091
stmA2 ,ug3/ k a1091stmA2 ,ug3/ k a1091
P e r c e n t a g e P u p a t i o n
P e r c e n t a g e P u p a t i o n
Delayed pupation in double mutants suggest interactionDelayed pupation in double mutants suggest interaction
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Partial Lethality in stmA-itpr Double
Mutants (25rC)
Partial Lethality in stmA-itpr Double
Mutants (25rC)
[stmA2 ; ug3/ T b] ×
[stmA2 ; k a1091/ T b]
[+/+; ug3/ T b] ×
[+/+; k a1091/ T b]
stmA2 ;
ug3 (or
k a1091)/ T b
stmA2 ;
ug3/ k a1091
+/+;
ug3(or
k a1091)/ T b
+/+;
ug3/ k a1091
No. of flies
survived
Mean Survival
Value
SD
666
112.50
7.42
216
75.00
14.83
1091
99.30
0.75
557
101.39
1.50
Lethality in stmA-itpr double mutant combination was
at pre-pupation (early) stage of development
Lethality in stmA-itpr double mutant combination was
at pre-pupation (early) stage of development
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Maternal Activity of stmA GeneMaternal Activity of stmA Gene
Reciprocal cross difference for embryonic lethality Reciprocal cross difference for embryonic lethality
GFP expression in the female germ line of stmA- eGF P
transgenic flies
GFP expression in the female germ line of stmA- eGF P
transgenic flies
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stmA is a developmentally indispensable maternally
acting gene
stmA is a developmentally indispensable maternally
acting gene
Mutation at stmA leads to delayed pupation Mutation at stmA leads to delayed pupation
stmA and itpr gene interact during development stmA and itpr gene interact during development
ConclusionsConclusions
stmA is involved in olfaction of Drosophila stmA is involved in olfaction of Drosophila
Specific allelic variation is present for stmA gene Specific allelic variation is present for stmA gene
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Determining the exact position and role of stmA in
G-protein signaling
Determining the exact position and role of stmA in
G-protein signaling
Interaction of stmA with all other components of
G-protein pathway
Interaction of stmA with all other components of
G-protein pathway
Identifying specific SNPs for stmA gene Identifying specific SNPs for stmA gene
Developing stmA as a specific target for drug
screening
Developing stmA as a specific target for drug
screening
The Road Ahead The Road Ahead
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Developing pest control strategy based on chemosensory
response using Drosophila as a model
Developing pest control strategy based on chemosensory
response using Drosophila as a model
NATURE [Vol.444], 16 November-
2006
The Road Ahead The Road Ahead
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Developing ³odour sensors´ based on
biological information
on olfaction in
Drosophila
Developing ³odour sensors´ based on
biological information
on olfaction in
Drosophila
The Road Ahead The Road Ahead
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