StmA Gene Drosophila

8/3/2019 StmA Gene Drosophila

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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



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



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



Study and

correlation of allelic

variation and

phenotype of

stmA gene

Study and

correlation of allelic

variation and

phenotype of

stmA gene

ObjectiveObjective



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



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



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



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



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



Sequence Analysis of Mutant AllelesSequence Analysis of Mutant Alleles



Effect of stmAgene on

development

and

olfaction

Effect of stmAgene on

development

and

olfaction

ObjectiveObjective



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



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



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)


Benzaldehyde (10-3 Dilution)



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


Ethyl Acetate (10-2 Dilution)


Ethyl Acetate (10-2 Dilution)

Reduced odorant response in mutants suggests involvement

of stmA in olfactory behaviour



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



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



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



Analyses of functional interactionbetween stmA and itpr mutantsAnalyses of functional interactionbetween stmA and itpr mutants33

itpr

ObjectiveObjective



Generation of stmA-itpr Double Mutant

Combination


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



××

××

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


Combination


Combination



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


Combination


Combination



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



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



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



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



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



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




Developing ³odour sensors´ based on

biological information

on olfaction in

Drosophila

Developing ³odour sensors´ based on

biological information

on olfaction in

Drosophila


StmA Gene Drosophila

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