A full body and nervous system specific knockdown of Alanyl-tRNA

synthetase in Drosophila melanogaster

AJ Sacks

University of Miami Miller School of Medicine

Department of Cellular and Molecular Pharmacology

Drosophila melanogaster

bull Large population numbers- stronger statistically

bull Short lifespan- can observe inheritance of many generations

bull High homology of genes

~60

bull Genetic manipulation

Carolina Drosophila Manual

Backgroundbull 6 Americans have undiagnosed diseases (many neurological)

ndash Sequence genome- elucidate potential causes

ndash Mutations identified- (alanyl t-RNA synthetase)

httpwwwscrippsedunewsscientificreportssr2004mb04schimmelhtml

Molecular Biology for MastersDr G R Kantharaj

httpmol-biol4mastersmastersgrkrajorghtmlRibose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthaseshtm

Alanyl t-RNA synthetase

Binds appropriate tRNAmolecule to the amino acid alanine in tRNA charging

Purposebull To observe the effects of the protein

knockdown of alanyl tRNA synthethase in the whole body and also only in the nervous system

ndash Loss of function development lifespan

httpwwwnaturecomnrgjournalv3n3boxnrg751_BX2html

Actin orC155 promoter

region

Gene for RNAi

Nature ReviewsThe art and design of genetic screens Drosophila melanogasterDaniel St JohnstonGal-4 UAS System

Father- has gene for Gal 4 protein after tissue specific promoter region

F1

Parents will mate and produce F1 generation

Mother- has UAS located right before the gene of interest (RNAi)

F1 generation expresses Gal 4 protein (show on left) which binds to the Upstream Activation Sequence (UAS) and drives the expression of the RNAi (shown on right)

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Drosophila melanogaster

bull Large population numbers- stronger statistically

bull Short lifespan- can observe inheritance of many generations

bull High homology of genes

~60

bull Genetic manipulation

Carolina Drosophila Manual

Backgroundbull 6 Americans have undiagnosed diseases (many neurological)

ndash Sequence genome- elucidate potential causes

ndash Mutations identified- (alanyl t-RNA synthetase)

httpwwwscrippsedunewsscientificreportssr2004mb04schimmelhtml

Molecular Biology for MastersDr G R Kantharaj

httpmol-biol4mastersmastersgrkrajorghtmlRibose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthaseshtm

Alanyl t-RNA synthetase

Binds appropriate tRNAmolecule to the amino acid alanine in tRNA charging

Purposebull To observe the effects of the protein

knockdown of alanyl tRNA synthethase in the whole body and also only in the nervous system

ndash Loss of function development lifespan

httpwwwnaturecomnrgjournalv3n3boxnrg751_BX2html

Actin orC155 promoter

region

Gene for RNAi

Nature ReviewsThe art and design of genetic screens Drosophila melanogasterDaniel St JohnstonGal-4 UAS System

Father- has gene for Gal 4 protein after tissue specific promoter region

F1

Parents will mate and produce F1 generation

Mother- has UAS located right before the gene of interest (RNAi)

F1 generation expresses Gal 4 protein (show on left) which binds to the Upstream Activation Sequence (UAS) and drives the expression of the RNAi (shown on right)

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Backgroundbull 6 Americans have undiagnosed diseases (many neurological)

ndash Sequence genome- elucidate potential causes

ndash Mutations identified- (alanyl t-RNA synthetase)

httpwwwscrippsedunewsscientificreportssr2004mb04schimmelhtml

Molecular Biology for MastersDr G R Kantharaj

httpmol-biol4mastersmastersgrkrajorghtmlRibose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthaseshtm

Alanyl t-RNA synthetase

Binds appropriate tRNAmolecule to the amino acid alanine in tRNA charging

Purposebull To observe the effects of the protein

knockdown of alanyl tRNA synthethase in the whole body and also only in the nervous system

ndash Loss of function development lifespan

httpwwwnaturecomnrgjournalv3n3boxnrg751_BX2html

Actin orC155 promoter

region

Gene for RNAi

Nature ReviewsThe art and design of genetic screens Drosophila melanogasterDaniel St JohnstonGal-4 UAS System

Father- has gene for Gal 4 protein after tissue specific promoter region

F1

Parents will mate and produce F1 generation

Mother- has UAS located right before the gene of interest (RNAi)

F1 generation expresses Gal 4 protein (show on left) which binds to the Upstream Activation Sequence (UAS) and drives the expression of the RNAi (shown on right)

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Purposebull To observe the effects of the protein

knockdown of alanyl tRNA synthethase in the whole body and also only in the nervous system

ndash Loss of function development lifespan

httpwwwnaturecomnrgjournalv3n3boxnrg751_BX2html

Actin orC155 promoter

region

Gene for RNAi

Nature ReviewsThe art and design of genetic screens Drosophila melanogasterDaniel St JohnstonGal-4 UAS System

Father- has gene for Gal 4 protein after tissue specific promoter region

F1

Parents will mate and produce F1 generation

Mother- has UAS located right before the gene of interest (RNAi)

F1 generation expresses Gal 4 protein (show on left) which binds to the Upstream Activation Sequence (UAS) and drives the expression of the RNAi (shown on right)

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

httpwwwnaturecomnrgjournalv3n3boxnrg751_BX2html

Actin orC155 promoter

region

Gene for RNAi

Nature ReviewsThe art and design of genetic screens Drosophila melanogasterDaniel St JohnstonGal-4 UAS System

Father- has gene for Gal 4 protein after tissue specific promoter region

F1

Parents will mate and produce F1 generation

Mother- has UAS located right before the gene of interest (RNAi)

F1 generation expresses Gal 4 protein (show on left) which binds to the Upstream Activation Sequence (UAS) and drives the expression of the RNAi (shown on right)

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Tissue Specific Promoter regions

bull Actin- whole body

ndash Actin- cytoskeleton protein involved in the structure and function of microfilaments

ndash Found in all eukaryotic cells

bull C155- nervous system

ndash Enhancer trap insertion into the elav locus on the X chromosome of Drosophila

ndash Only in nervous system cells

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Cross1 y-w-[ ]

+X

+RNAi

F1possibilities

Whole Body

Both will express Gal 4 which will drive the expression of the RNAi

Both Will NOT express RNAi because no Gal 4 protein instead will have curly wings

+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

Legend

CROSS 1 Whole Body

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

CROSS 2- Nervous System

Cross2 C155-gal4w-UAS-Dcr2+X ++RNAi

F1Possibilities

Legend+ =wild typey-= yellow bodyw-=white eyesCyo= curly wings

All flies will express RNAi because they all have Gal 4 protein to drive its expression

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Methods

bull Setting the Crossesndash 11 flies per vial (6 females 5 males)

bull Allowed to mate for 2 to 3 daysbull Transfer parents to new vial bull Parents in new vial left to lay eggs for 2 days (then parents

transferred out)

bull Culturing in vials with the standard cornmeal-agar-molasses-yeast medium at the bottom and a cotton swab at the top 25degC

bull Pupae counting- at the end of all eclosion the number of pupae were counted and recorded

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Results

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

Actin RNAi

0 0 0 - 0 - - - 0

Actin RNAi

0 0 0 - 0 - - - 0

CyoRNAi

21 21 23 - 4 - - - 1

CyoRNAi

17 19 25 - 17 - - - 3

Eclosion Cross Set 10012013

Date 1013 1014 1015 1016 1017 1018 1019 1020 1021

C155RNAi

0 0 0 - 0 - - - 0

C155 RNAi

0 0 0 - 0 - - - 0

CROSS 1 Whole body

CROSS 2 Nervous System

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

bull 154 empty pupae (154 flies to eclose)ndash All flies to eclose were curly= 154 curly flies eclosed

ndash 11 ratio inheritance 154 knockdown flies SHOULD have eclosed

bull 7 pupae with uneclosed fliesndash Must have been caused by knockdown

because pf the established 100 eclosion

rate of curly flies

bull 7154= 45 pupae lethal

bull 147154= 954 larvae or

embryonic lethal

Cross 1 Actin-Gal 4 X RNAi

httpwwwkillicoukgraphicsfruitfly10jpg

1 eclosed pupa2 uneclosed pupa3 larva

LEGEND

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Discussion

bull All flies with the fully body knockdown died before eclosion- significant lethality to knockdown especially early in developmentndash Pupa lethality rate- 45ndash larva or embryonic lethality rate 954

bull All flies with nervous system specific knockdown died before eclosionndash More pupae had formed on the vials of nervous system

knockdownndash Less severe as full body

bull Charcot-Marie-Tooth diseasendash Inherited neurological disorderndash Peripheral neuropathy

bull CMT caused by mutations in genes for proteins of myelin sheath

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Charcot Marie Tooth Disease

bull Recent study (McLaughlin HM et al) associated point mutation in gene for alanyl-tRNA synthethasendash P Arg329His reduces enzyme activity

bull Several mutations in genes of aminoacyl-tRNA synthethasehave been implicated in CMT

bull Another study (Motley et al) revealed that it is not the loss of function of GARS but instead the toxicity of the mutant tRNA synthethase

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Next Step

bull Human ldquorescuerdquo

bull Insert the human homolog into the flies simultaneously with knockdown of the protein

bull Tell whether the protein is conserved between flies and humans

ndash Whether we can successfully express this human protein in flies

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399

Acknowledgements and References bull Dr Grace Zhai and her lab at University of Miami especially Chris Bellobull Ms Jennifer Gordinierbull Referencesbull Zhao Z Hashiguchi A Hu J Sakiyama Y Okamoto Y Tokunaga S Zhu L Shen H

Takashima H 2012 Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy Neurology

bull McLaughlin HM Sakaguchi R Giblin W NISC Comparative Sequencing Program Wilson TE Biesecker L Lupski JR Talbot K Vance JM Zuumlchner S Lee YC Kennerson M Hou YM Nicholson G Antonellis A 2012 A recurrent loss-of-function alanyl-tRNA synthetase(AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

bull Duffy B Joseph 2002 GAL4 System in Drosophila A Fly Geneticistrsquos Swiss Army Knife Department of Biology Indiana University Bloomington Indiana

bull National Institute of Neurological Disorders and Stroke httpwwwnindsnihgovdisorderscharcot_marie_toothdetail_charcot_marie_toothhtm

bull Madame Curie BioScience Database Lluis Ribas de Pouplana Karin Musier-Forsyth and Paul Schimmel Alanyl tRNA synthetase

bull Raymond O Flagg Carolina Drosophila Manual 2005 Carolina Biological Supply Company

bull Motley WW Seburn KL Nawaz MH Miers KE Cheng J et al (2011) Charcot-Marie-ToothndashLinked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels PLoS Genet 7(12) e1002399 doi101371journalpgen1002399