Nervous System Function in Drosophila Randall D. Shortridge, Ph.D. 645-2363 ext 137 Cooke Hall, Room...
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Transcript of Nervous System Function in Drosophila Randall D. Shortridge, Ph.D. 645-2363 ext 137 Cooke Hall, Room...
Nervous System Function in Drosophila
Randall D. Shortridge, Ph.D.
645-2363 ext 137Cooke Hall, Room 336
Biological Sciences, SUNY-Buffalo
Project Collaborators
Randall D. Shortridge, Ph.D., Biological Sciences, SUNY-Buffalo; Molecular biology, transgenics, immunology, biochemistry, bioinformatics.
Satpal Singh, Ph.D., Pharmacology and Toxicology, SUNY-Buffalo; Electrophysiology, pharmacology, physiology, genetics.
Why study the nervous system?
Neuronal Disease • Diverse causes and common occurrence• Severely impacts the quality of life • Understanding causes can lead to treatments or cure
movie
Why use fruit flies to study the nervous system?
1. Relevance
2. Amenable to a variety of research approachesGenetics, behavioral analysis, transgenics, molecular biology, biochemistry, electrophisiology, immunology.
Studies can reveal complex processes in humans.
From: Genome Research, 2001, 11(6), 1114-25.
75% of Human disease genes listed in the Online Mendilian Inheritance in Man (OMM) database have counterparts in Drosophila melanogaster.
“In essence, we are nothing but a big fly,” Dr. Charles Zuker, Professor of Biology at UCSD – reported by ABC News Maggie Fox, March 23, 2003
Brundlefly in The Fly, 1986, starring Jeff Goldblum, Geena Davis
Drosophila Genetics
1. Drosophila is the best genetically-characterized
Metazoan.
2. Easy to induce mutations and isolate mutant lines.
3. Easy to transfer genes back into fly (transgenics).
4. Thousands of mutants available in stock centers.
5. Sophisticated mutant lines available for use in research studies (eg. enhancer-traps).
6. Allows a correlation of gene to function (functional genetics).
Genetic-Molecular approach (Drosophila) allows a direct correlation of specific genes to their in vivo function.
Organism (mutant phenotype)
Gene product
Functional lesion
Tissue
Forward Genetics; Functional Genetics
Bride of Frankenstein, Universal Studios, 1933
Cover of Science, March 24, 1995
Genetics and the making of FrankenFly
Edward B. Lewis Christiane Nüsslein-Volhard
Eric F. Wieschaus
California Institute of Technology Pasadena, CA, USA
Max-Planck-Institut für Entwicklungsbiologie Tübingen, Federal Republic of Germany
Princeton University Princeton, NJ, USA
b. 1918 b. 1942 b. 1947
The Nobel Prize in Physiology or Medicine 1995
"for their discoveries concerning the genetic control of early embryonic development"
1. Isolate Drosophila mutations that disrupt function of the nervous system (TS paralysis).
2. Identify the genes involved.
3. Determine the mechanism of gene expression in the nervous system.
The power of this approach is that it allows a direct correlation of specific gene products to
their in vivo function.
Basic Strategy
Equipment used in Temperature-Sensitive (TS) Paralytic Assays
Aquarium with heater
PencilWriting pad
Flies
Stopwatch
Worker
Temperature-Sensitive (TS) Paralytic Mutant Assays
TS mutants are identified by paralysis at 38oC. movie
Convention for naming TS paralytic mutants Drosophila TS mutants are named after individuals associated
with the 1991-1994 Buffalo Bills football team (eg. Norwood, Levy, Andre, Kelly, Thurmond).
Approximately 30 total TS alleles isolated
5 being actively studied at the present time.
Scott Norwood’s game-ending FG miss in Superbowl XXV Kelly was intercepted four times in Superbowl XXVI
Some TS paralytic mutants are bang-sensitive
Bang-sensitive assay: Vortex flies for 10 seconds then time paralytics for recovery. Recovery times vary among mutants from a few seconds to 20 minutes.
movie
Mutation Gene Identified? Ion
CurrentHeart Rates
Bang Sensitivity
levyYes (COX VIa
subunit) Ca2+ K+ 140% Yes
kelly Yes (novel) Ca2+ only 80% No
norwood Yes (novel) K+ only 100% No
andre 1 of 2 (novel) Ca2+ only 120% Yes
thurmond 1 of 11 (novel) Ca2+ only 122% No
Paralytic mutants under active study
DNA sequence analysis of levy1 identifies a mutation in the CG17280 gene
CG17280: 743 nucleotides, 2 exons, 1 intron, 109 amino acids, 47% identical to the human COX subunit VIa precursor
32 44WT A.A …S G G Y K V W K R L S F FcDNA ...TCTGGTGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTClevy cDNA ...TCTG--TGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTC A.A ....S V A T R C G S A C P S S 32 44
45 56WT A.A V A V P A V G L C M L N...cDNA GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC...levy cDNA GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC...A.A W P C P P W D C A C Stop 45 54
Exon-exon boundary
The levy1 mutation is predicted to cause a highly truncated form of the CG17280 (COX VIa) protein
WT
levy1
Exon 1
Exon 1
Exon 2
Intron
STOP
STOP
levy1 mutation alters splicing at intron-exon junction, resulting in a frame-shift defect.
The predicted transcript is detected by DNA sequencing after RT-PCR in levy1 mutant.
Rescue of levy mutant defect by gene therapy
1. Insert gene into transposable element.
2. Inject modified DNA into early embryo.
3. Isolate fly carrying DNA insert.
Transgenic Drosophila:
levy embryolevy mutant expressing CG17280
(transformant)
levy transformants are not TS paralytic
DNA constructinject
CG17280 (levy)
Levels of COX activity is reduced by the levy1 mutation, but restored in transformants
2.50
2.00
1.50
1.00
0.5
0
CO
X A
ctiv
ity (n
mol
/min
/g)
WT
(CS)
levy
TFc2
TFc1
levy
/ +
TF1
TF2
ATP is reduced in levy1 after paralysis at 38oC
120
100
80
60
40
20
0A
TP le
vel (
% C
S)
WT
(CS)
levy
TFc
TF
After 38oC, 5 min (paralytic)
120
100
80
60
40
20
0
ATP
leve
l (%
CS)
WT
(CS)
levy
TFc
TF
21oC 2 days old adults
ATP is reduced in levy1 mutants by age
120
100
80
60
40
20
0
ATP
leve
l (%
CS)
WT
(CS)
levy
TFc
TF
21oC 2 days old adults
120
100
80
60
40
20
0A
TP le
vel (
% C
S)
WT
(CS)
levy
TFc
TF
30 days old adults
Mitochondrial Respiration Machinery (Oxidative Phosphorylation)
levy is a part of Complex IV (Cyt C Oxidase)
Ox-Phos Figure from Alberts et al., MB of the Cell, 4e.
Gene contributions to assembling the mitochondrial COX complex
levy mutationFig from: R. Poyton, Nature Genetics 1998, 20:316
Cytochrome C Oxidase (COX) Deficiency
Fatal Infantile MyopathyHypotoniaWeaknessRespiratory distressRenal tubular defect: glycosuria, amino aciduria, etcMitochondrial myopathy with selective absence of subunits VII a/b of COX
Benign Infantile MyopathyHypotoniaWeaknessRespiratory insufficiencySpontaneous recovery by age 2-3Mitochondrial myopathy with selective absence of subunits VII a/b and II of COX
Leigh's Syndrome (Subacute Necrotizing Encephalomyelopathy)HypotoniaEpisodic vomiting and feeding problemsLoss of motor and verbal skillsHearing and visual lossSpasticityMuscle biopsy normal except for COX deficiency with absence of all subunits
Leigh’s DiseaseSynonyms:
• Leigh Necrotizing Encephalopathy• Leigh’s Syndrome • Necrotizing Encephalomyelopathy of Leigh’s • SNE • Subacute Necrotizing Encephalopathy
Closely Related Disorders:• Autism• Alzheimer's
Symptoms:• Classical Leigh’s has onset after 3 months of age, but adult onset possible.• 50% survival to 3 years; less than 20% by mid teens• Progressive neurodegeneration• Loss of motor skills is first noticeable trait• Lack of muscle tone (hypotonia); clumsiness; tremors; absence of reflexes; muscle spasms; seizures; visual disturbance; heart enlargement; partial paralysis, mental retardation.• Increased CO2 in blood; lactic acidosis
Causes:• COX deficiency• Lesion in non-COX Ox-Phos proteins
levy Mutant and Leigh’s Disease Parallels
1. Cytochrome C oxidase (COX) deficiency.
2. Muscular dysfunction (TS paralysis)
3. Brain (neuronal) degeneration.
4. Reduced life span.
5. Motor defects (paralysis; bang sensitivity; climbing behavior?; jumping ability?)
6. Visual dysfunction (?)
7. Ion channel regulatory defects (?)
Examples of Available Ph.D. or M.S Research Projects
1. What gene encodes the IKF (potassium) channel? Use RNAi to knockout candidate genes (8).
2. Will levy’s brother substitute for levy to rescue paralysis in the mutant? Construct hybrid genes and transfer back into mutant lines.
3. What are the expression profiles of levy and levy’s brother? Are they tissue-specific like in humans? RT-PCR, Northerns, epitope tags, transformants expressing reporter constructs.
4. Identify mutation in the thurmond gene. PCR amplify 10 candidate genes from mutant and compare sequence to wild-type.
5. Identify mutant in andre (2 candidate genes) or in 25 other TS-paralytic mutant lines. (same as above).
A multidisciplinary approach is used in analyzing the nervous system
1. Electrophysiology
2. Pharmacology
3. Genetics
4. Immunology
5. Molecular Biology
6. Transgenics
7. Physiology
8. Biochemistry
9. Bioinformatics