Lecture 21 Flashcards
Why do the primers used in PCR to analyse the effect of insulin on gene expression need to be close together
Because PCR can only work for between 10-20kbps
Describe the process of promoter analysis/promoter bashing
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.
What is the effect of activated PKB on glucose transport
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
What is the long-term effects of insulin exposure
Increased expression of liver enzymes that synthesise glycogen as well as enzymes involved in triacylglycerol synthesis in the adipocytes
Where is insulin produced
In the B-cells of the Islets of Langerhans in the pancreas
Explain the role of PI-3 Kinase in Ras-MAP-K independent signal transduction
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:
What is the benefit of using a heat stable DNA polymerase in PCR
Allows you to repeat the cycle (heating wont denature the polymerase but can still break the hydrogen bonding between the strands
How does FOXO act at low blood insulin levels
When there are low blood insulin levels, Foxo binds to an IRS near to PEPCK to activate its transcription
What is the name of the technique used to quantify gene expression from mRNA
Reverse Transcriptase PCR
How many PCR cycles are usually carried out
30 cycles – producing >1x109 fragments
The cysteine bonding that holds the insulin peptides together can only form outside the cell, why is this
These cysteine bonds can only form in an oxidative extracellular environment
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
PI-3 kinase pathway
Describe what happens following ligand binding to the insulin receptor
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)
What is the effects of activated PKB on glucose storage and how is this achieved
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
Give some examples of the downstream protein targets of activated PKB
GSK3, GLUT4, FOXO and Phosphoenolpyruvate Carboxykinase
Describe the structure of PI-3 Kinase
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
Describe the structure of the insulin receptor
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
What is the immediate effect of insulin signalling
Glucose uptake from the blood into muscle cells and adipocytes
Insulin’s effects on glucose synthesis are known as the faster response, T or F
F – it’s a slower response
What are the effects of PKB activity on glucose synthesis
FOXO is a transcription factor that activates expression of PEPCK, a glucose synthesis gene. PKB phosphorylates and inactivates FOXO and prevent glucose synthesis
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
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.
To look at gene expression specifically using PCR, it is essential that you must use high quality tissue. Why is this
You need to extract all of the mRNA transcribed by the tissue
Leprechaunism is a disease caused by mutations in the insulin pathway. Describe the symptoms of this condition
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
What is the effect of designing short primers that anneal to a specific gene or sequence of interest within a fragment
Primers trim down DNA sequence from the genomic dsDNA to only amplify the target sequence that lies between the two primers
The insulin receptor is an RTK, what is unusual about its structure
The insulin receptors is present as a dimer in its inactivated state
Explain the role of IRS in insulin signalling
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.
Explain the cleavage events that take place after insulin synthesis
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
IRS is known as a surrogate, what is meant by this
IRS binds to the signal docking site on the activated insulin receptor but then creates additional docking sites for downstream targets
