mock qualifying exam
Post on 13-Apr-2017
252 Views
Preview:
TRANSCRIPT
Identification of a daf-3 corepressor and mechanism of cofactor switch in high temperature induced dauer
formation
L1
L2
L3
Dauer
L4
Egg
Adult
C. Elegans Life Cycle
Adverse ConditionsFavorable Conditions
Lack of Food
Dauer Inducing Factors
HeatOvercrowding
TGF-B in Dauer Formation
daf-5
daf-1 daf-4
daf-3
Dauer promoting genes
daf-7
daf-14daf-8
Pheromone
TGF-β Pathway <26°C
daf-3
TGF-β Pathway >26°C
Dauer CONSTITUTIVEDauer DEFECTIVE
daf-5
No Effect
HYPOTHESIS
daf-5
daf-1 daf-4
daf-3
Dauer promoting genes
daf-7
daf-14daf-8
Pheromone
TGF-β Pathway >26°C
coREP
daf-3
Specific Aims
Identify daf-3 corepressor in high temperature induced dauer formation
Determine the mechanism of daf-3 cofactor exchange.
Specific Aim 1: Identify the daf-3 corepressor
RNAi mediated loss of known daf-3 binding proteins in WT
Non-lethal deletion mutants loss of known daf-3 bindg proteins
DAF-C PHENOTYPE AT 27°C
VSAdult Dauer
Specific Aim 1: Identify the daf-3 corepressor
Protein extract from 20C and 27°CALTERNATE APPROACHES
Purified with daf-3 antibody20°C 27°C
Specific Aim 1: Identify the daf-3 corepressorALTERNATE APPROACHES
Two Dimensional Gel
Identification of proteins by MALDI-MS
pH
Size
daf-5daf-3
Dauer promoting genes
coREP
• Temperature dependent cofactor expression
Real-time PCR of daf-5 and the corepressor at 20°C and 27°C
20°C 27°C
daf-5daf-3
Dauer promoting genes
coREP
• Temperature dependent cofactor expression
Ectopically express daf-5 and the daf-3 corepressor with HSP-GFP transgenics
20°C (+) Pheromone27°C (-) Pheromone
•Expect dauer promoting conditions•Expression of the corepressor should result in REDUCED dauer formation •Expect dauer repressing conditions•Expression of daf-5 should result in INCREASED dauer formation
daf-5daf-3
Dauer promoting genes
coREP
Competitive binding of the corepressor
•Corepressor-RFP/daf-5-CFP/daf-3-GFP young adults at 20°C and 27°C
RFP CFPGFP
20°C27°C
daf-5daf-3
Dauer promoting genes
coREP
Changes in daf-3 structures
20°C 27°CRNA Isolation
20°C 27°C
Changes in daf-3 structures: Northern Blot
Isoform XDaf-3 Null
+
Changes in daf-3 structures: Isoform Rescue
27°C
SUPPRESSION OF THE DAF-C PHENOTYPE
Specific Aims 2:Determine the mechanism of daf-3 cofactor exchange.
• Temperature dependent cofactor expression - real-time PCR of daf-5 and the corepressor at 20°C and 27°C
- Ectopically express daf-5 and the daf-3 the corepressor with HSP-GFP transgenics
• Competitive binding of the corepressor- corepressor-RFP/daf-5-CFP/daf-3-GFP young adults at 20°C and 27°C
• Changes in daf-3 structures- Examine relative amounts of daf-3 mRNA isoforms a-c at
both 20°C and 27°C via northern blot
- Rescue the daf-d phenotype at 27°C by expression of isoform’s cDNA in the daf-3 mgD90 mutant
top related