Lecture 21

Question 1

Why do the primers used in PCR to analyse the effect of insulin on gene expression need to be close together

Answer 1

Because PCR can only work for between 10-20kbps

Question 2

Describe the process of promoter analysis/promoter bashing

Answer 2

Promoter bashing is used to identify the actual DNA sequences that act as promoters and enhancers. You start with the promoter of interest and much of the sequence adjacent it. A transgene is then made that uses a quantifiable reporter gene such as luciferase, GFP, B-gal or HRP. Then make a series of deletions in the promoter and flanking sequences to test the responsiveness to insulin to find which sequences are critical in the activity after ligand binding Look at the sequences that were deleted that lead to decreased levels of activity to identify the sequences acting as promoters.

Question 3

What is the effect of activated PKB on glucose transport

Answer 3

GLUT4 is already synthesised in the cells but is present in vesicles beneath the membrane. Insulin binding and subsequent activation of PI-3 Kinase leads to the increase translocation and insertion of GLUT4 into the membrane hence increase sugar uptake from the blood

Question 4

What is the long-term effects of insulin exposure

Answer 4

Increased expression of liver enzymes that synthesise glycogen as well as enzymes involved in triacylglycerol synthesis in the adipocytes

Question 5

Where is insulin produced

Answer 5

In the B-cells of the Islets of Langerhans in the pancreas

Question 6

Explain the role of PI-3 Kinase in Ras-MAP-K independent signal transduction

Answer 6

Upon binding to IRS, PI-3 kinase phosphorylates phosphoinositol-4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate and PI 3,4-biphosphate, respectively. This creates a docking site for protein kinase B which, once recruited to the membrane, is phosphorylated by membrane associated kinases such as 3-phosphoinositide-dependent kinase (PKD1). PKB then goes through a conformational change to become active and is released to effect numerous protein:

Question 7

What is the benefit of using a heat stable DNA polymerase in PCR

Answer 7

Allows you to repeat the cycle (heating wont denature the polymerase but can still break the hydrogen bonding between the strands

Question 8

How does FOXO act at low blood insulin levels

Answer 8

When there are low blood insulin levels, Foxo binds to an IRS near to PEPCK to activate its transcription

Question 9

What is the name of the technique used to quantify gene expression from mRNA

Answer 9

Reverse Transcriptase PCR

Question 10

How many PCR cycles are usually carried out

Answer 10

30 cycles – producing >1x109 fragments

Question 11

The cysteine bonding that holds the insulin peptides together can only form outside the cell, why is this

Answer 11

These cysteine bonds can only form in an oxidative extracellular environment

Question 12

What is the name of the other main branch of signal transduction from RTKs such as the insulin receptor other than the Ras/MAP-K pathway

Answer 12

PI-3 kinase pathway

Question 13

Describe what happens following ligand binding to the insulin receptor

Answer 13

Ligand binding brings the intracellular kinase domain together and results in autophosphorylation of the receptor. Autophosphorlyation creates a single docking site for the insulin receptor substrate (IRS)

Question 14

What is the effects of activated PKB on glucose storage and how is this achieved

Answer 14

PKB phosphorylates GSK3 to inactivate it this allows glycogen synthase to be active and to promote the storage of sugars. Normally, GSK3 phosphorylates glycogen synthase to inactivate it and block the storage of sugars

Question 15

Give some examples of the downstream protein targets of activated PKB

Answer 15

GSK3, GLUT4, FOXO and Phosphoenolpyruvate Carboxykinase

Question 16

Describe the structure of PI-3 Kinase

Answer 16

PI-3 Kinase is made up of two subunits; the P85 subunit which contains an SH2 domain and the P110 subunit which contains the kinase activity

Question 17

Describe the structure of the insulin receptor

Answer 17

The insulin receptor is also synthesised as a full length protein that is then cleaved into ? and ? subunits that are held together by disulphide bridges

Question 18

What is the immediate effect of insulin signalling

Answer 18

Glucose uptake from the blood into muscle cells and adipocytes

Question 19

Insulin’s effects on glucose synthesis are known as the faster response, T or F

Answer 19

F – it’s a slower response

Question 20

What are the effects of PKB activity on glucose synthesis

Answer 20

FOXO is a transcription factor that activates expression of PEPCK, a glucose synthesis gene. PKB phosphorylates and inactivates FOXO and prevent glucose synthesis

Question 21

Microarrays are one way of measuring changes in gene expression as a result of insulin signalling, what is the other main method of measuring these changes and how does it work

Answer 21

Quantitative PCR - This allows you to quantify the levels of RNA and hence levels of gene expression. A dsDNA template is first denatured by heating to break the hydrogen bonding between complimentary base pairs. This can either be genomic dsDNA or produced from mRNA using reverse transcriptase. Primers are designed to bind to a known target gene and are introduced to the sample with DNA polymerase and dNTPs. Successive rounds of DNA replication are carried out leading to an exponential increase in the amount of DNA. This can be quantitively analysed by the incorporation of a fluorescent DNA dye into the reaction mixture which will allow you to determine the amount of DNA present after each PCR cycle. From this you can determine the amount of DNA present initially.

Question 22

To look at gene expression specifically using PCR, it is essential that you must use high quality tissue. Why is this

Answer 22

You need to extract all of the mRNA transcribed by the tissue

Question 23

Leprechaunism is a disease caused by mutations in the insulin pathway. Describe the symptoms of this condition

Answer 23

Usually fatal within the first 2 years of life. Patients have an elfin-like facial appearance with protuberant ears and relatively large hands and feet. Also there is a decreased amount of subcutaneous fat and muscle mass is seen, and the skin is abnormal with increased hair growth

Question 24

What is the effect of designing short primers that anneal to a specific gene or sequence of interest within a fragment

Answer 24

Primers trim down DNA sequence from the genomic dsDNA to only amplify the target sequence that lies between the two primers

Question 25

The insulin receptor is an RTK, what is unusual about its structure

Answer 25

The insulin receptors is present as a dimer in its inactivated state

Question 26

Explain the role of IRS in insulin signalling

Answer 26

The insulin receptor substrate contains a phosphotyrosine binding domain (PBD) similar to SH2 that binds to the phosphorylated site on the activated insulin receptor. IRS then acts as a docking site for other proteins such as GRB2 leading to the activation of the Ras pathway.

Question 27

Explain the cleavage events that take place after insulin synthesis

Answer 27

The full-length insulin protein is heavily modified after synthesis. These multiple cleavage steps leave the final product with only amino and carboxyl terminal peptides. These fragments are held together by cysteine bonds that can only form outside the cell

Question 28

IRS is known as a surrogate, what is meant by this

Answer 28

IRS binds to the signal docking site on the activated insulin receptor but then creates additional docking sites for downstream targets