L19 - Insulin Signalling

Question 1

Leprechaunism is fatal after

Answer 1

Within the first two years of life

Question 2

Describe symptoms seen in leprechaunism

Answer 2

Elfin like facial appearance with protuberant ears and relatively large hands and feet
Decreased ammount of subcuatneous fat and muscle mass
Skin abnormal with increased hair growth

Question 3

Rabson-Mendenhall syndrome - life expect.

Answer 3

Survival into the second stage

Question 4

Describe symptoms of Rabson-mendenhall syndrome

Answer 4

Skin and teeth abdnormalities, hair over growth and pineal hyperplasia

Question 5

How long do people with type A insulin resistance survive

Answer 5

Into the middle age and beyond

Question 6

Where is inuslin produced

Answer 6

In the beta cells of the islets

Then secreted into the bloodstream

Question 7

What forms between _______ in insulin production

What environment does this require

Answer 7

Disulphide bridges form between cysteines

This requires an oxidative environment

Question 8

What is the immediate effect of insulin release

Answer 8

glucose uptake from the blood into muscle cells and adipocytes

Question 9

What is the effect of long term exposure to insulin

Answer 9

Increased expression of liver enzymes that synthesise glycogen and of adipocyte enzymes that synthesise triacyglycerols

Question 10

Insulin results in activation of

Answer 10

IRS

Question 11

Describe the synthesis of the two subunits of insulin

Answer 11

Aloha and beta subunits are synthesised as a single polypeptide which is then cleaved into two fragments

Question 12

Binding of insulin to the receptor results in

Answer 12

Autophosphorylation

Question 13

IRS stands for

What does it contain

What happens to this domain

Answer 13

Insulin receptor substrate

Phosphotyrosine binding domain (PTB)

This domain is highly phosphorylated by the insulin receptor

Question 14

IRS acts as a …

Answer 14

Docking sites for many other protein such as GRB2 to activate the RAS pathway

Question 15

What is PI-3 kinase made up of two subunits

Answer 15

P85

P110

Question 16

P85 contains a

Answer 16

SH2 domain

Question 17

P110 is a

Answer 17

Kinase

Question 18

Describe what occurs when Pi3 kinase binds to IRS

Answer 18

Pi3 kinase phosophrylates Pi-4,5-biphosphate and Pi-4-phosphate

Question 19

What happens when:

Pi-4-5-biphosphate is phroylated

Pi-4-phosphate is phosphorylated

Answer 19

Pi-3,4,5-triphosphate

Pi-3,4-biphosphate

Question 20

Formation of Pi-4,5-BP and Pi-4-P creates a binding site for

Answer 20

Protein kinase B

Question 21

Describe what haappens once protein kinase B is recruited to the membrane

What does this cause to happen

Answer 21

Phosphorylated by membrane associated kinases

Causes a confirmation change to become active and is then released and effects numerous proteins

Question 22

What is the effect of active PKB on

GLycogen synthase kinase

Glucose transporter

FOXO

Answer 22

Decrease

Increase

Decrease

Question 23

Phosphoenolpyruvate carboxykinase is what sort of enzyme

Answer 23

Glucose synthesis enzyme

Question 24

Describe on of the way in which we can identify genes that are activated by insulin signalling

Answer 24

Can track changes in gene expression using microarrays comparing to before and after insluin is added to the cells in culture

Question 25

Describe the action of FOXO when blood insulin levels are low

Answer 25

Foxo binds to IRES near to PEPCK to activate its transcription

Question 26

Describe what occurs with FOXO1 at high blood insulin levels

Answer 26

Phosphorylation of FOXO1 by protein kinase B which inactivates it

Question 27

What is PCR

Answer 27

Used to amplify the DNA

Question 28

PCR is used to

Answer 28

Clone DNA from minute samples and to quantify the levels of RNA

Question 29

For the first sequence describe how primers are designed

Answer 29

Based upon a known sequence - they must face each other and be on opposite strands thus defining the target sequence

Question 30

What type of DNA polymerase must be used with PCR - where is this obtained from

Answer 30

Heat stable DNA polymerase

From the thermus aquaticus bacteria (TAQ)

Question 31

How many ds strands produced after 1 orund of PCR

What about 2 cycles

What about 3 cycles

Answer 31

2
4
8

Question 32

Describe the porcess of PCR

Answer 32

Heat DNA - causes the separation of the strands
Primers anneal
DNA synthesis by polymerase from the primer template junction

Question 33

After 3 rounds of synthesis how many strands are there of the target sequence

Answer 33

8 (2 ds strands and 4 ss strands)

Question 34

Describe how PCr may be used to make genomic clones

Answer 34

Identify DNA to be cloned
Sep strands and add primers
PCr amplification

Question 35

Describe how PCR may be used to make cDNA clones

Answer 35

Isloate the mRNA
Add first primer and reverse transcriptase
DNTPs also required to be added
Separate strands - RNA/cDNA hybrid and add the second primer
Amplify with PCR

Question 36

Descirbe quantitative PCR

Answer 36

make cDNA from tissue
Perform PCR in the presence of a fluoresecent DNA dye
Measure the fluoresecnce after each PCR cycle

Question 37

what technique can be used an ana;yse the mechanism of trnascription regulation

Answer 37

Promoter bashing

Question 38

Describe how promoter bashing is performed

Answer 38

Make a transgene that uses a quantifiable reporter then make a series of deletions - test the activity of reporter to identify the critical regions of regulation

Question 39

Why is luciferase a good reporter for promoter bashing

Answer 39

An insulin receptor - produces a flourescent substrate which is quantifiable

Question 40

What can promoter bashing be used in conjunction with

Answer 40

EMSA and DNAseI protection