Already have an account? Get multiple benefits of using own account!
Login in your account..!
Remember me
Don't have an account? Create your account in less than a minutes,
Forgot password? how can I recover my password now!
Enter right registered email to receive password!
You have discovered a novel alternatively spliced gene (gene X) and you wish to study its regulation in an in vitro splicing assay. The alternatively spliced product retains intron 2 and is produced only in hepatocytes, whereas the standard mRNA is expressed in fibroblasts. The structure of the standard mRNA and the alternatively spliced form are shown below.
Conveniently, you have two cell lines available (one is derived from hepatocytes and the other from fibroblasts) that also show the same splicing patterns of this mRNA as that seen in tissue. You have already prepared a nuclear extract from each of these cell lines that you hope will provide the essential splicing proteins and snRNAs that are required for splicing your RNAs in an in vitro splicing reaction. You have already used PCR to amplify a genomic clone of the whole gene, which includes introns and exons. The primers are shown below with arrows. The 5' ends of the primers contain unique restriction enzyme sites to facilitate cloning in the later steps.
A. Briefly describe the major steps you would take to use the pcr product (500 bp) to generate a 32P-UTP labeled pre-mRNA run-off transcript (internal/uniformly labeled) that could be used in your in vitro splicing reactions. One or two sentences per step should be sufficient. Make sure you include details on the orientation of the PCR product in the construction.
B. You now prepare a freshly labeled gene X pre-mRNA from the construct you prepared from part a. You set up three in vitro splicing reactions in three different 0.5 ml tubes as described below. All have your labeled RNA but each contains a different protein extract or no extract. After incubation for 5 hours, which you have read should be long enough to get near complete splicing, you purify RNA from the reactions and run them on a denaturing PAGE followed by X-ray film exposure. The result is shown below. You suspect that the two darker bands (labeled a and b) may be the mature spliced mRNAs but you can't tell with certainty from this experiment. Using a technique we discussed in the lecture, suggest a follow-up experiment that could demonstrate that these two bands are in fact the correctly spliced mRNAs as depicted above. You can either use RNA purified from the gel shown below or you can go back and set up new reactions using your reagents. Explain briefly what you would do and what the experiment would show.
Q. Why is it not correct to assert that the DNA self- replicates? The DNA is not completely self-sufficient in its duplication process because the replication does not occur wi
Two atoms always represent the same element if they have. A) the same number of particles in the nucleus. B) The same number of electrons. C) The same number of shells. D) The same
Tick-Borne Encephalitis (TBE ) TBE occurs in Scandinavia, Western and Central Europe and countries of the former USSR, mainly in rural forested areas. Risk is greatest from Apri
Problem 1: How many different types of RNAs are present? What is the function of Promoter in Transcription? Mention different types of RNAs Discuss function of Promot
what is respiration
Q. What are the near point and the far point of the vision? The near point is the closest distance between the eye and an object that makes possible the formed image to be focu
Determine the Fluids and Electrolyte Requirement at high Altitude? In addition to cold induced diuresis, hyperventilation together with a dry environment at HA makes an individ
Sporozoans – Protozoan Sporozoans of the genus Plasmodium are responsible for causing a serious human disease, malaria. They are among, the best known parasites which live in
The movement of Na + and glucose from the lumen of the intestine across the epithelial cell to the blood sets up a dissimilarity in osmotic pressure across the cell. As
Design a controlled experiment to test the hypothesis that acid precipitate inhibits the growth of elodea, a common freshwater plant.
Get guaranteed satisfaction & time on delivery in every assignment order you paid with us! We ensure premium quality solution document along with free turntin report!
whatsapp: +1-415-670-9521
Phone: +1-415-670-9521
Email: [email protected]
All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd