The stambhA gene

 stmA gene was discovered at IARI based on paralysis phenotype (Shyngle and Sharma, 1985)

 Codes for a membrane bound lipase

 Regulates the activity of Phospholipase C- in the G-protein signaling pathway (Huang et al., 2004)

 Sequence conserved across large number of organisms ranging from yeast, Arabidopsis, rice and humans

Studies On A Lipase Coding Gene, Regulating G-protein Coupled PLC- Signaling In Drosophila melanogaster

Mridul Chakraborti Division of Genetics Indian Agricultural Research Institute

Objectives
1. To study and correlate allelic variation and phenotype of stmA gene

2. To evaluate the effect of stmA gene on development and olfaction

3. To analyze the functional interaction between stmA and inositol triphosphate receptor (itpr) genes

Materials
 stmA Alleles : stmA1, stmA2, stmAP1, stmAP6

 itpr alleles : ka1091 and ug3  stmA- eGFP fusion transgenic stock

 TM6Tb and Cy 3rd and 2nd chromosome balancer  CantonS wild type stock

Objective

Study and correlation of allelic variation and phenotype of stmA gene

Complementation Test by Paralysis profile of stmA Mutants

Genotypes
2 min

Flies out Water Paralysis time outlet

2.5 min 3 min

3.5 min 4 min

stmA1

0
1

50 50 50 0

50 50 25 50

0 0 25 50 0

0 0 0 0 0

stmP

P 6 stmWater inlet 0

stmA1/ stmP stmA1/ stmP

1 6

0 0

Flies in 100 0

The Sushi Cooker

No Complementation: stmAP 1 and stmAP are alleles of stmA1

DNA Based Polymorphism Study

Sequencing Strategy for stmA Alleles

 The 4.7 kb stmA gene was divided into 4 contigs  Primers for the contigs designed by using Primer-3 software

Contig A Contig B Contig C Contig D

Optimization of PCR Amplification Protocol for stmA Contigs

9 10 11 12

Optimum annealing temperature above Tm value was determined for different primer sets

Standardized PCR Amplification Protocol for stmA Contigs

Step 1 2 3 Primer set-A Primer set-B Primer set-C Primer set-D 4 5 6

Temp. 94C 94C 59.3C 63.5C 62.4C 57.8C 72C 72C 4C

Time 3 min. 30 s

Cycles 1

1 min.

35

2 min. 10 min. 1 Hold

Polymorphism among stmA Alleles

A
M A2 P
6

B
P
1

C
6

A2

A2

C
M P
6

D
P
1

A2

Possible mutational lesion near the primer binding site of stmAP

6

A contig

Sequence Analysis of Mutant Alleles

Objective

Effect of stmA gene on development and olfaction

Analysis of Olfactory Responses of Various StmA Mutants

 Simple behavioral assay  Odorant Response analysis by Parks Method

Odorant Response Index =

No. of flies [ODOR TRAP] No. of flies [CONTROL TRAP] Total No. of flies [BEAKER]

ODORANT TRAP

CONTROL TRAP

Standardization of Optimum Odorant Concentration in Wild Type Flies

Dilution Factor of Benzaldehyde Dilution Factor of Ethyl Acetate (aqueous solution) 1 1 Mean ORI SD 10-1 10-1 10-2 10-2 210-3 210-3 10-3 10-3 210-4 210-4

-0.35 -0.64 0.09 0.07

-0.72 -0.21 0.14 0.24

0.79 0.56

0.40 0.13

0.44 0.76

0.54 0.72

0.07 0.04

0.10

0.07 0.05

0.13 0.11

Odorant Response Index Towards Benzaldehyde (10-3 Dilution)

Odorant Response Index

1.00 0.50
stmAP1

WT

0.00 -0.50 -1.00

stmA2 stmAP6

Odorant Response Index Towards Ethyl Acetate (10-2 Dilution)

Odorant Response Index

1 0.8 0.6 0.4 0.2 0

stmA2 stmAP1 stmAP6

WT

Reduced odorant response in mutants suggests involvement of stmA in olfactory behaviour

Electroantennogram Alalysis for Olfactory Response Study

 Electroantennogram (EAG) pattern indicates the electrophysiological response in the antennae

 Genotypes showing reduced EAG are not able to recognize odorants

Comparison of Electroantennogram Pattern of stmA Mutants

14 12

stmA1/+

stmA1/ stmA1

Amplitude (mV)

10 8 6 4 2 0
Butanaol (10-2) Ethyl Acetate (10-2) Propionic Acid (10-2) Benzaldehyde (10-3) Isoamyl Acetate (10-2 )

Odorants (Concentration)

Gene Expression in Antennal Tissue

Histological study in antenna of stmA-eGFP transgenic line

EcoRI

Pm/I

eGFP

Confirmation of stmA expression in antenna

Objective
Analyses of functional interaction between stmA and itpr mutants

itpr

Generation of stmA-itpr Double Mutant Combination

Cy Sp ;

TM6Tb +

stmA2 stmA2 ;

+ +

stmA2 Cy stmA2 Cy ; ;

TM6Tb + TM6Tb

stmA2 Sp stmA2 Cy ; ;

Sb + TM6Tb Sb

Sb stmA2 stmA2 ;

TM6Tb Sb

Generation of stmA-itpr Double Mutant Combination

+ +
Cy ;

TM6Tb ka1091 TM6Tb ; ka1091 TM6Tb ; ka1091 stmA2 stmA2

Cy

TM6Tb ; Sb TM6Tb ; Sb TM6Tb ; Sb

Sp stmA2 Sp stmA2 stmA2

+
stmA2 Cy

TM6Tb ; ka1091

Generation of stmA-itpr Double Mutant Combination

stmA2 ; stmA2

ug3

stmA2

ka1091 ; TM6Tb

TM6Tb stmA2 ; stmA2 ug3 stmA2 ka1091

Reciprocal cross

New stock with itpr mutation in stmA2 background

Study of Pupation Pattern in stmA-itpr Mutant Combinations (25rC)

Percentage Pupation

90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00

ug3 (or ka1091)/Tb stmA2, ug3(or ka1091)/Tb ug3/ka1091 stmA2,ug3/ka1091

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Day of Culture
Delayed pupation in double mutants suggest interaction

Partial Lethality in stmA-itpr Double Mutants (25rC)

[stmA2; ug3/Tb] [stmA2; ka1091/ Tb]

stmA2; ug3 (or ka1091)/Tb No. of flies survived Mean Survival Value SD stmA2; ug3/ka1091

[+/+; ug3/Tb] [+/+; ka1091/ Tb]

+/+; ug3(or ka1091)/Tb +/+; ug3/ka1091

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

Maternal Activity of stmA Gene

 Reciprocal cross difference for embryonic lethality  GFP expression in the female germ line of stmA-eGFP transgenic flies

Conclusions
 Specific allelic variation is present for stmA gene  stmA is involved in olfaction of Drosophila  stmA and itpr gene interact during development

 Mutation at stmA leads to delayed pupation

 stmA is a developmentally indispensable maternally acting gene

The Road Ahead

 Determining the exact position and role of stmA in G-protein signaling  Interaction of stmA with all other components of G-protein pathway  Identifying specific SNPs for stmA gene  Developing stmA as a specific target for drug screening

The Road Ahead

Developing pest control strategy based on chemosensory response using Drosophila as a model

NATURE [Vol.444], 16 November2006

The Road Ahead

Developing odour sensors based on biological information on olfaction in Drosophila