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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
• Large population
numbers- stronger
statistically
• Short lifespan- can
observe inheritance of
many generations
• High homology of genes
~60%

• Genetic manipulation
Carolina Drosophila Manual
Background
• 6% Americans have undiagnosed diseases (many neurological)
– Sequence genome- elucidate potential causes
– Mutations identified- (alanyl t-RNA synthetase)
Alanyl t-RNA synthetase
Molecular Biology for Masters
Dr. G. R. Kantharaj

Binds appropriate tRNA
molecule to the amino acid
alanine in tRNA charging
http://www.scripps.edu/news/scientificreports/sr
2004/mb04schimmel.html

http://mol-biol4masters.masters.grkraj.org/html/Ribose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthases.htm
Purpose
• To observe the effects of the protein
knockdown of alanyl tRNA synthethase in the
whole body and also only in the nervous
system
– Loss of function, development, lifespan
Nature Reviews
The art and design of genetic screens:
Drosophila melanogaster
Daniel St Johnston

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

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

Parents will mate and
produce F1 generation
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)

F1

Actin or
C155 promoter
region

http://www.nature.com/nrg/journal/v3/n3/box/nrg751_BX2.html

Gene for
RNAi
Tissue Specific Promoter regions
• Actin- whole body
– Actin- cytoskeleton protein involved in the
structure and function of microfilaments
– Found in all eukaryotic cells

• C155- nervous system
– Enhancer trap insertion into the elav locus on the
X chromosome of Drosophila
– Only in nervous system cells
CROSS 1: Whole Body
y-w-

Cross 1:

;

[

]
,

Whole Body

;+ X

.

; + ; RNAi

Legend
+ =wild type
y-= yellow body
w-=white eyes
Cyo= curly wings

F1 possibilities:
;

;

.

;

;

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
CROSS 2- Nervous System
Cross 2:

C155-gal4, w- ; UAS-Dcr 2 ; + X

+ ; + ; RNAi

F1 Possibilities:
,

;

;

,
.

;

;

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

Legend
+ =wild type
y-= yellow body
w-=white eyes
Cyo= curly wings
Methods
• Setting the Crosses:
– 11 flies per vial (6 females, 5 males)
• Allowed to mate for 2 to 3 days
• Transfer parents to new vial
• Parents in new vial left to lay eggs for 2 days (then parents
transferred out)

• Culturing: in vials with the standard cornmealagar-molasses-yeast medium at the bottom and a
cotton swab at the top, 25°C
• Pupae counting- at the end of all eclosion the
number of pupae were counted and recorded
Results:
Eclosion:

Cross Set: 10/01/2013

CROSS 1: Whole body
Date:

10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21

Actin,
RNAi

0

0

0

-

0

-

-

-

0

Actin,
RNAi

0

0

0

-

0

-

-

-

0

Cyo,
RNAi

21

21

23

-

4

-

-

-

1

Cyo,
RNAi

17

19

25

-

17

-

-

-

3

CROSS 2: Nervous System

Date:

10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21

C155
RNAi

0

0

0

-

0

-

-

-

0

C155
RNAi

0

0

0

-

0

-

-

-

0
Cross 1: Actin-Gal 4 X RNAi
• 154 empty pupae (154 flies to eclose)
– All flies to eclose were curly= 154 curly flies eclosed
– 1:1 ratio inheritance 154 knockdown flies SHOULD have eclosed

• 7 pupae with uneclosed flies
– Must have been caused by knockdown
because pf the established 100% eclosion
rate of curly flies

• 7/154= 4.5% pupae lethal
• 147/154= 95.4% larvae or
embryonic lethal

LEGEND
1: eclosed pupa
2: uneclosed pupa
3: larva

http://www.killi.co.uk/graphics/fruitfly10.jpg
Discussion
• All flies with the fully body knockdown died before
eclosion- significant lethality to knockdown, especially early
in development
– Pupa lethality rate- 4.5%
– larva or embryonic lethality rate 95.4%

• All flies with nervous system specific knockdown died
before eclosion
– More pupae had formed on the vials of nervous system
knockdown
– Less severe as full body

• Charcot-Marie-Tooth disease
– Inherited neurological disorder
– Peripheral neuropathy
• CMT caused by mutations in genes for proteins of myelin sheath
Charcot Marie Tooth Disease
• Recent study (McLaughlin HM et al.) associated point
mutation in gene for alanyl-tRNA synthethase
– P. Arg329His reduces enzyme activity

• Several mutations in genes of aminoacyl-tRNA synthethase
have been implicated in CMT
• 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
• Human “rescue”
• Insert the human homolog into the flies
simultaneously with knockdown of the protein
• Tell whether the protein is conserved between
flies and humans
– Whether we can successfully express this human
protein in flies
•
•
•
•
•

•
•
•
•
•

Acknowledgements and References

Dr. Grace Zhai, and her lab at University of Miami, especially Chris Bello
Ms. Jennifer Gordinier
References:
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
McLaughlin HM, Sakaguchi R, Giblin W; NISC Comparative Sequencing Program, Wilson
TE, Biesecker L, Lupski JR, Talbot K, Vance JM, Züchner 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).
Duffy B. Joseph, 2002 GAL4 System in Drosophila: A Fly Geneticist’s Swiss Army Knife,
Department of Biology, Indiana University, Bloomington, Indiana
National Institute of Neurological Disorders and Stroke:
http://www.ninds.nih.gov/disorders/charcot_marie_tooth/detail_charcot_marie_tooth
.htm
Madame Curie BioScience Database, Lluis Ribas de Pouplana, Karin Musier-Forsyth and
Paul Schimmel, Alanyl tRNA synthetase
Raymond O. Flagg, Carolina Drosophila Manual, 2005 Carolina Biological Supply
Company
Motley WW, Seburn KL, Nawaz MH, Miers KE, Cheng J, et al. (2011) Charcot-MarieTooth–Linked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type
GARS Levels. PLoS Genet 7(12): e1002399. doi:10.1371/journal.pgen.1002399

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Sigma xi 2014 for slides

  • 1. 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
  • 2. Drosophila melanogaster • Large population numbers- stronger statistically • Short lifespan- can observe inheritance of many generations • High homology of genes ~60% • Genetic manipulation Carolina Drosophila Manual
  • 3. Background • 6% Americans have undiagnosed diseases (many neurological) – Sequence genome- elucidate potential causes – Mutations identified- (alanyl t-RNA synthetase) Alanyl t-RNA synthetase Molecular Biology for Masters Dr. G. R. Kantharaj Binds appropriate tRNA molecule to the amino acid alanine in tRNA charging http://www.scripps.edu/news/scientificreports/sr 2004/mb04schimmel.html http://mol-biol4masters.masters.grkraj.org/html/Ribose_Nucleic_Acid3B-Aminoacyl_tRNA_Synthases.htm
  • 4. Purpose • To observe the effects of the protein knockdown of alanyl tRNA synthethase in the whole body and also only in the nervous system – Loss of function, development, lifespan
  • 5. Nature Reviews The art and design of genetic screens: Drosophila melanogaster Daniel St Johnston Gal-4 UAS System Father- has gene for Gal 4 protein after tissue specific promoter region Mother- has UAS located right before the gene of interest (RNAi) Parents will mate and produce F1 generation 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) F1 Actin or C155 promoter region http://www.nature.com/nrg/journal/v3/n3/box/nrg751_BX2.html Gene for RNAi
  • 6. Tissue Specific Promoter regions • Actin- whole body – Actin- cytoskeleton protein involved in the structure and function of microfilaments – Found in all eukaryotic cells • C155- nervous system – Enhancer trap insertion into the elav locus on the X chromosome of Drosophila – Only in nervous system cells
  • 7. CROSS 1: Whole Body y-w- Cross 1: ; [ ] , Whole Body ;+ X . ; + ; RNAi Legend + =wild type y-= yellow body w-=white eyes Cyo= curly wings F1 possibilities: ; ; . ; ; 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
  • 8. CROSS 2- Nervous System Cross 2: C155-gal4, w- ; UAS-Dcr 2 ; + X + ; + ; RNAi F1 Possibilities: , ; ; , . ; ; All flies will express RNAi because they all have Gal 4 protein to drive its expression Legend + =wild type y-= yellow body w-=white eyes Cyo= curly wings
  • 9. Methods • Setting the Crosses: – 11 flies per vial (6 females, 5 males) • Allowed to mate for 2 to 3 days • Transfer parents to new vial • Parents in new vial left to lay eggs for 2 days (then parents transferred out) • Culturing: in vials with the standard cornmealagar-molasses-yeast medium at the bottom and a cotton swab at the top, 25°C • Pupae counting- at the end of all eclosion the number of pupae were counted and recorded
  • 10. Results: Eclosion: Cross Set: 10/01/2013 CROSS 1: Whole body Date: 10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21 Actin, RNAi 0 0 0 - 0 - - - 0 Actin, RNAi 0 0 0 - 0 - - - 0 Cyo, RNAi 21 21 23 - 4 - - - 1 Cyo, RNAi 17 19 25 - 17 - - - 3 CROSS 2: Nervous System Date: 10/13 10/14 10/15 10/16 10/17 10/18 10/19 10/20 10/21 C155 RNAi 0 0 0 - 0 - - - 0 C155 RNAi 0 0 0 - 0 - - - 0
  • 11. Cross 1: Actin-Gal 4 X RNAi • 154 empty pupae (154 flies to eclose) – All flies to eclose were curly= 154 curly flies eclosed – 1:1 ratio inheritance 154 knockdown flies SHOULD have eclosed • 7 pupae with uneclosed flies – Must have been caused by knockdown because pf the established 100% eclosion rate of curly flies • 7/154= 4.5% pupae lethal • 147/154= 95.4% larvae or embryonic lethal LEGEND 1: eclosed pupa 2: uneclosed pupa 3: larva http://www.killi.co.uk/graphics/fruitfly10.jpg
  • 12. Discussion • All flies with the fully body knockdown died before eclosion- significant lethality to knockdown, especially early in development – Pupa lethality rate- 4.5% – larva or embryonic lethality rate 95.4% • All flies with nervous system specific knockdown died before eclosion – More pupae had formed on the vials of nervous system knockdown – Less severe as full body • Charcot-Marie-Tooth disease – Inherited neurological disorder – Peripheral neuropathy • CMT caused by mutations in genes for proteins of myelin sheath
  • 13. Charcot Marie Tooth Disease • Recent study (McLaughlin HM et al.) associated point mutation in gene for alanyl-tRNA synthethase – P. Arg329His reduces enzyme activity • Several mutations in genes of aminoacyl-tRNA synthethase have been implicated in CMT • 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
  • 14. Next Step • Human “rescue” • Insert the human homolog into the flies simultaneously with knockdown of the protein • Tell whether the protein is conserved between flies and humans – Whether we can successfully express this human protein in flies
  • 15. • • • • • • • • • • Acknowledgements and References Dr. Grace Zhai, and her lab at University of Miami, especially Chris Bello Ms. Jennifer Gordinier References: 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 McLaughlin HM, Sakaguchi R, Giblin W; NISC Comparative Sequencing Program, Wilson TE, Biesecker L, Lupski JR, Talbot K, Vance JM, Züchner 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). Duffy B. Joseph, 2002 GAL4 System in Drosophila: A Fly Geneticist’s Swiss Army Knife, Department of Biology, Indiana University, Bloomington, Indiana National Institute of Neurological Disorders and Stroke: http://www.ninds.nih.gov/disorders/charcot_marie_tooth/detail_charcot_marie_tooth .htm Madame Curie BioScience Database, Lluis Ribas de Pouplana, Karin Musier-Forsyth and Paul Schimmel, Alanyl tRNA synthetase Raymond O. Flagg, Carolina Drosophila Manual, 2005 Carolina Biological Supply Company Motley WW, Seburn KL, Nawaz MH, Miers KE, Cheng J, et al. (2011) Charcot-MarieTooth–Linked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels. PLoS Genet 7(12): e1002399. doi:10.1371/journal.pgen.1002399