1) The first voltage-gated sodium channel genes were isolated from the electric organ of the electric eel. Tetrodotoxin (TTX) is a potent neurotoxin that binds to sodium channels.
a) Describe how Noda and Numa isolated the first full length sodium channel cDNA sequence from electric eel from the knowledge that TTX binds specifically to the eel sodium channel.
b) Further explain how human sodium channel sequences were found once the eel sodium channel cDNA was identified.
2)Lily Jan’s laboratory isolated the first voltage-gated potassium channel gene, dubbed “Shaker” from the fruit fly. Mutant flies were created by irradiating fruit flies with gamma rays (radioactive cesium).
As a result of the radiation treatment, Shaker flies had uncontrollable leg shaking and wing scissoring behaviour under ether anaesthesia. Electrophysiological studies indicated that neurons in Shaker flies had reduced to no outward potassium currents, indicating abnormalities in a potassium channel gene.
No potassium channel gene had been identified by this time in 1987.
a) Explain how Lily Jan’s laboratory found the gene sequence of the Shaker mutant flies, knowing that irradiated flies had large deletions in the gene coding for the fruit fly Shaker potassium channel gene.
b) Further explain how the human Shaker potassium channel cDNA sequences were found once the fruit flyShaker potassium channel cDNA was identified.
3) Inward rectifying potassium channels were identified physiologically because they were potassium currents that were blocked by barium ions and was highly abundantly expressed in the adrenal medulla. The inward rectifying potassium channel and other expressible proteins from the adrenal medulla can be functionally assessed by injection of the mRNA coding for the inward rectifying potassium channels into immature frog eggs (Xenopus oocytes).
Explain how the inward rectifying potassium channel cDNA sequence was found in adrenal medulla cells using the expression cloning technique. Make sure to explain how cDNA libraries were constructed and screened to find the unique clone.
4) Two pore leak potassium channels from Arabidopsis plant was identified by complementation in yeast, utilizing CY162 yeast cell strain deficient in potassium transport.
The yeast cell strain CY1632 normally die in media lacking high concentrations of potassium ions. Explain how the leak potassium channel cDNA sequence was found in Arabidopsis plant using the CY162 yeast strain. Make sure to explain how cDNA libraries were constructed and screened to find the unique clone.
5) Long QT syndrome is a rate inherited heart condition, identified by an abnormal QT interval in an electrocardiogram. Mutations in HERG potassium channels were found to cause Long QT syndrome.
Mark Keating and colleagues found the human HERG cDNA sequence by positional cloning. They knew that Long QT syndrome was a heritable disease and the disease-causing gene was linked to a known polymorphic marker on chromosome
6) Explain how Mark Keating found the sequence of the human HERG cDNA sequence. Make sure to explain how cDNA libraries were constructed and screened to find the unique clone.
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