Cellular oncogenes and signalling

Question 1

What experiment proved that Ras bound to guanine (nucleotide)?

Answer 1

Radioactive displacement experiment
incubated immunoprecipitated Ras with radioactively labelled GDP- labels the guanine
saw the binding of guanine to Ras

Question 2

How did they find if other nucleotides bound to Ras?

Answer 2

By incubating immunoprecipitated Ras with non-radioactive nucleotides- measure the fall in radioactivity
aka the displacement of the radioactive GDP with other nucleotides

Question 3

Which other nucleotides were found to bind and not bind to Ras?

Answer 3

dGTP and GTP bind to Ras

GMP and cyclic GMP do not bind to Ras

Question 4

Which experiment proved that Ras was a GTPase?

Answer 4

they expressed Ras via recombinant bacteria
incubated the ras with radioactive GTP
looked via chromatography for the presence of GDP
in a time-dependent manner GDP appeared in the lane with Ras
not the case for mutant vial Ras (Ha-Ras)- no GDP was seen- this mutant does not act as an enzyme

Question 5

How good of a GTPase is Ras?

Answer 5

Not very good

Question 6

What experiment identified what stimulates Ras activity?

Answer 6

They added cell lysates from a cell line to the recombinant ras
the cytosol and ras lead to the appearance of GDP
purified a signal protein= GTPase activating protein (GAP)

Question 7

What is CDC25?

Answer 7

A gene in yeast which is dependent on the status of Ras for growth

Question 8

What was yeast used for in studying ras?

Answer 8

They knew that ras bound both GTP and GDP- didnt understand the mechanism behind this
they used a biochemical approach to see if anything could speed up the displacement of GDP from ras

Question 9

How was a protein found to help displace GDP from ras?

Answer 9

Brain cytosol was found to contain activity that facilitated the dissociation of GDP from ras
identified a protein- GEF- which was homologous to CDC25
GEF= guanine nucleotide exchange factor

Question 10

Which animal models were used to study how ras interacted with growth factors?

Answer 10

Drosophila

C.Elegans

Question 11

What is the mutant sevenless- what genes work up and downstream of sevenless?

Answer 11

Sevenless mutant= lacks the seventh cell in the ommatidium

bride of sevenless-> sevenless-> son of sevenless

Question 12

Which genes were found to be involved in vulval induction in c elegans?

Answer 12

Lin-3-> Let23-> SEM-5-> Let-60

Question 13

What did they find when they looked closer at the sequencing of the drosophila and c.elegans genes?

Answer 13

BOS= growth factor
S.less= EGF-R tyrosine kinase 
SOS=GEF (also the same as CDC25)
Lin23= growth factor 
Let23= EGF-R tyrosine kinase
SEM-5=?
Let-60=Ras

Question 14

How did they identify SEM-5?

Answer 14

Using an expression cloning strategy
library of mammalian genes expressed in bacteria- each gene product expressed in at least one bacterium
added a fluorophore to the phosphodomain of a receptor tyrosine kinase
incubated the probe with the bacteria- see if anything binds strongly to the RTKs

Question 15

What was SEM-5 found to be?

Answer 15

GRB2- growth factor receptor binding protein

Question 16

How do RTKS, GRB2, SOS and ras interact?

Answer 16

When a GF has bound to a RTK- autophosphorylation
GRB2 binds to the phosphorylated RTK and interacts with SOS
SOS interacts with ras and removes GDP so GTP can bind

Question 17

What does Ras signalling lead to in the cell?

Answer 17

Cell growth
gene expression
cell morphology
cell movement

Question 18

When will downstream effectors of Ras bind to ras?

Answer 18

When Ras is in its GTP activated form