Standard mrna and fibroblasts, Biology

Assignment Help:

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.

1078_RNA.png

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.

2448_DNA.png

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.


Related Discussions:- Standard mrna and fibroblasts

Why is it not correct to assert that the dna, Q. Why is it not correct to a...

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

Why two atoms always represent the same element, Two atoms always represent...

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

Explain tick-borne encephalitis, Tick-Borne Encephalitis (TBE ) TBE occu...

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

Function of promoter in transcription, Problem 1: How many different ty...

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 are the near point and the far point of the vision, Q. What are the ne...

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 fluids and electrolyte need at high altitude, Determine the Fluid...

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 – Protozoan Sporozoans of the genus...

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

Glucose rehydration therapy, The movement  of Na + and glucose  from the l...

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, Design a controlled experiment to test the ...

Design a controlled experiment to test the hypothesis that acid precipitate inhibits the growth of elodea, a common freshwater plant.

Write Your Message!

Captcha
Free Assignment Quote

Assured A++ Grade

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!

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd