Generation And Screening Of cDNA Libraries
Generation And Screening Of cDNA Libraries
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.
Step1- Purification of sodium channels: Given the fact that tetrodotoxin binds specifically to the sodium channels, they were purified by affinity chromatography. Tetrodotoxin immobilized on sepharose beads was used as an affinity resin. When the cell lysate containing sodium channels from the electric organ of electric eel was passed over this affinity matrix, sodium channels were bound specifically with the matrix. Subsequently, the channels were eluted using increasing concentration of tetrodotoxin. An aliquot of purified protein was injected into the rabbit to obtain antisera for sodium channels and another aliquot was subjected to trypsin digestion. The peptides obtained post trypsin digestion were separated by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). The sequence of the peptides was determined by Edman’s degradation. Radiolabeled (P32) synthetic probes were synthesized based on peptide sequences.
Step 2- Generation and screening of cDNA libraries: Total mRNA was isolated from electric organ of electric eel using affinity column containing polyT. The isolated total mRNA was reverse transcribed using reverse transcriptase to form cDNA. The synthesized cDNA ligated with expression vector was packaged in the bacteriophage which infected E. coli and produced plaques. The cDNAs from plaques were transferred on nitrocellulose membrane and radiolabeled oligonucleotides were used as probe to fish out cDNA coding for sodium channel. cDNA clones which hybridized with radiolabeled probes were further investigated by western blotting using antisera for sodium channels. The cDNAs positive for both radioactivity and immunogenicity with antisera were further validated by injecting selected cDNA in Xenopus oocytes and monitoring the current using patch clamp.
b) Further explain how human sodium channel sequences were found once the eel sodium channel cDNA was identified.
Human and electric eel sodium channels are homologous proteins and hence they share sequence similarity. Once cDNA sequence of sodium channel of eel is known, cDNA sequence of human sodium channel was elucidated as follows. cDNA of sodium channel of eel was digested with restriction enzyme. The cDNA fragments were separated using agarose gel electrophoresis and each fragment was radiolabeled. Once the cDNA library of human genes was made, the radiolabeled cDNA fragments of sodium channel from electric eel were used as probes. The cDNA clones from humans that were positive for radioactivity with highest number of radiolabeled cDNA fragments of sodium channel were further checked for their activity using patch clamp.
2) 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.
The total mRNA from adrenal medulla was isolated using polyT as an affinity tag. mRNA contains poly A tail at the 3’end that enables it to bind to the affinity column containing polyT. The purified mRNA was reverse transcribed using reverse transcriptase to form RNA-DNA duplex (RNA corresponds to mRNA and DNA is the strand complementary to mRNA which was synthesized by reverse transcriptase). Formation of DNA-RNA duplex was succeeded by the addition of dCTP mediated by terminal transferase on 3’ end of both the RNA and DNA. RNA-DNA duplex was digested with RNase H leaving single stranded DNA. Subsequently, a primer containing stretch of G (acts as a primer) and DNA polymerase were added to synthesize complementary DNA strand which resulted in double stranded cDNA. The cDNA was ligated with expression vector. The cDNAs thus synthesized were fractioned based on the size on agarose gel (round 1 of cDNA fractionation).
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.
Step1- Purification of sodium channels: Given the fact that tetrodotoxin binds specifically to the sodium channels, they were purified by affinity chromatography. Tetrodotoxin immobilized on sepharose beads was used as an affinity resin. When the cell lysate containing sodium channels from the electric organ of electric eel was passed over this affinity matrix, sodium channels were bound specifically with the matrix. Subsequently, the channels were eluted using increasing concentration of tetrodotoxin. An aliquot of purified protein was injected into the rabbit to obtain antisera for sodium channels and another aliquot was subjected to trypsin digestion. The peptides obtained post trypsin digestion were separated by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). The sequence of the peptides was determined by Edman’s degradation. Radiolabeled (P32) synthetic probes were synthesized based on peptide sequences.
Step 2- Generation and screening of cDNA libraries: Total mRNA was isolated from electric organ of electric eel using affinity column containing polyT. The isolated total mRNA was reverse transcribed using reverse transcriptase to form cDNA. The synthesized cDNA ligated with expression vector was packaged in the bacteriophage which infected E. coli and produced plaques. The cDNAs from plaques were transferred on nitrocellulose membrane and radiolabeled oligonucleotides were used as probe to fish out cDNA coding for sodium channel. cDNA clones which hybridized with radiolabeled probes were further investigated by western blotting using antisera for sodium channels. The cDNAs positive for both radioactivity and immunogenicity with antisera were further validated by injecting selected cDNA in Xenopus oocytes and monitoring the current using patch clamp.
b) Further explain how human sodium channel sequences were found once the eel sodium channel cDNA was identified.
Human and electric eel sodium channels are homologous proteins and hence they share sequence similarity. Once cDNA sequence of sodium channel of eel is known, cDNA sequence of human sodium channel was elucidated as follows. cDNA of sodium channel of eel was digested with restriction enzyme. The cDNA fragments were separated using agarose gel electrophoresis and each fragment was radiolabeled. Once the cDNA library of human genes was made, the radiolabeled cDNA fragments of sodium channel from electric eel were used as probes. The cDNA clones from humans that were positive for radioactivity with highest number of radiolabeled cDNA fragments of sodium channel were further checked for their activity using patch clamp.
2) 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.
The total mRNA from adrenal medulla was isolated using polyT as an affinity tag. mRNA contains poly A tail at the 3’end that enables it to bind to the affinity column containing polyT. The purified mRNA was reverse transcribed using reverse transcriptase to form RNA-DNA duplex (RNA corresponds to mRNA and DNA is the strand complementary to mRNA which was synthesized by reverse transcriptase). Formation of DNA-RNA duplex was succeeded by the addition of dCTP mediated by terminal transferase on 3’ end of both the RNA and DNA. RNA-DNA duplex was digested with RNase H leaving single stranded DNA. Subsequently, a primer containing stretch of G (acts as a primer) and DNA polymerase were added to synthesize complementary DNA strand which resulted in double stranded cDNA. The cDNA was ligated with expression vector. The cDNAs thus synthesized were fractioned based on the size on agarose gel (round 1 of cDNA fractionation).
Each separated fraction of cDNA (round 1 of cDNA fractionation) was injected in Xenopus oocytes and patch clamp studies were performed in presence and absence of barium ions. The cDNA fraction for e.g. 5.5 kb-6.5 kb resulted in electric current during the patch clamp studies. This CDNA fraction was further fractioned and patch clamp studies were performed (both in presence and absence of barium ions) with each cDNA obtained from round 2 of fractionation.
The process of cDNA fractionation and subsequent patch clamp studies were continued till single cDNA of rectifying potassium channel was obtained.
3) 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.
The total mRNA from Arabidopsis was isolated using polyT as an affinity tag. mRNA contains poly A tail at the 3’end that enables it to bind to the affinity column containing polyT. The purified mRNA was reverse transcribed using reverse transcriptase to form RNA-DNA duplex (RNA corresponds to mRNA and DNA is the strand complementary to mRNA which was synthesized by reverse transcriptase).
3) 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.
The total mRNA from Arabidopsis was isolated using polyT as an affinity tag. mRNA contains poly A tail at the 3’end that enables it to bind to the affinity column containing polyT. The purified mRNA was reverse transcribed using reverse transcriptase to form RNA-DNA duplex (RNA corresponds to mRNA and DNA is the strand complementary to mRNA which was synthesized by reverse transcriptase).
Formation of DNA-RNA duplex was followed by addition of dCTP mediated by terminal transferase on 3’ end of both the RNA and DNA. RNA-DNA duplex was digested with RNase H leaving single stranded DNA. Subsequently, a primer containing stretch of G (acts as a primer) and DNA polymerase were added to synthesize complementary DNA strand which resulted in double stranded cDNA. The cDNA was ligated with expression vector and Transformed in the CY162, which are known to be deficient in potassium transport. The transformed CY162 were grown in a media lacking potassium ions. The CY162 containing only pore leak potassium channels cDNA will be able to grow in potassium deficient media while the CY162 containing cDNA clones of other proteins will require high potassium to grow. The cDNA was isolated from CY162 that continued to grow on a potassium deficient media which was subsequently sequenced to determine the complete cDNA sequence of pore leak potassium channels.
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