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Flashcards in Lecture 4 Deck (14)
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1
Q

What is Ras?

A
  • Ras is a guanine nucleotide binding protein
  • Ras is a GTPase
  • GTPase function is compromised in oncogenic forms of ras
2
Q

Gross et al (1984):

A
  • Expressed and purified recombinant ras in bacteria
  • Analysed bound nucleotides by chromatography
  • Incubated with radioactive GTP radioactive in the guanine bit not phosphate
  • C-Ras (Wt) and Ha-ras (viral mutant)
  • Chromatogram
  • Expose to film
  • In presence of ras GDP is present – telling us its enz that hydrolyses GTP
  • But Ha- Ras isn’t a GTPase – viral ras don’t work as enz – capable of binding but not hydrolyzing
    • can calculate how many molecules per second it hydrolyses
3
Q

Erikson

A

Immunoprecipitated Ras and incubated with radioactive guanine nucleotides

4
Q

Ed Skolnick

A
  • Radioactive displacement experiment
  • Bound to ras
  • Incubated with non radioactive nucleotide
  • If you’ve got something that binds specifically you could bind a non radioactive substances that displaces the radioactive substance
  • Compete away radioactive with different nucleotides to see if competitive with GDP
  • Incubate with no radioactive GDP – compete it oit with cold
  • CGMP doesn’t compete so ras doesn’t bind cGMP or GMP
    Competitive assay
5
Q

McCormack et al (1988)

A

Identification of a GAP

  • Presence of something theres an increase in activity more GDp when cytosol added
  • Used assay as a biochemical purification – protein GAP identified stim GTPAse activity of Ras and other molecules
6
Q

Macara and wolfson-

A
  • Look to see if you can displace GDP from Ras more quickly
  • Can incubate for hours but GDP doesn’t come of Ras easily
  • Brain cytosol contains an activity that facilitates dissociation of GDP
  • Conc dependent more in more easily GDP off Ras
  • First GEF identified was a functional homologue of CDC25
7
Q

What animal model was used to identify if ras works upstream, downstream or in parallel ?

A

Drosophila- looking at the ommatidia

  • 7cells
  • sevenless mutant has 7th cell missing
  • looked at son and Bride of sevenless
8
Q

What strategy

led to the identification of SEM-5

A

An expression cloning strategy

    • Using bacteria to express mammalian genes in each bacterial colony
  • Each gene product should be expressed in each
  • Individual colonies express 1 polypeptide
  • Chemically attach fluorophore to phosphorylated domains
  • Transfer to bacterial plot – wash nitrocellulose and wash in solution with fluorophore and see if anything binds it tightly
  • Spot where protein expressed tightly should be able to map back to what colony it came from
9
Q

GRB2 is highly homologous to what ?

A

To SEM 5

10
Q

Describe the model for RTL activation of Ras

A
- see diagram 
Involves:
GRB2 
SH2 
SOS
11
Q

GRB2 can only bind after what has occurred to RTK

A

Phosphorylated

12
Q

SOS

A

is a GEF teases out GDP from Ras allowing it to bind GTP

13
Q

What type of system is RAs?

A

Two switch system- on and off state

Eventually GTP will hydrolyse and switches itself off which occurs more quickly if GAP present

14
Q

What type of system is RAs?

A

Two switch system- on and off state
Eventually GTP will hydrolyse and switches itself off which occurs more quickly if GAP present
- RasGTP adopts an altered conformation
which binds and activates downstream effectors
on one specific surface all protein effectors seem to bind one side only when in GTP bound form