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

Determine about the disease - brain tumor, Determine about the disease - br...

Determine about the disease - brain tumor Tumor, or neoplasm, is a mass of new tissue that persists and grows independently of its surrounding structures and has no physiologic

What is the cyclosis, Q What is the cyclosis? Cyclosis is a kind of int...

Q What is the cyclosis? Cyclosis is a kind of interior cell movement in which an oriented flow of circulating material is created and maintained in the cytoplasm by the action

Long-chain fatty acids, Fatty acid breakdown brings about the oxidation of ...

Fatty acid breakdown brings about the oxidation of long-chain fatty acids. The fatty acids are first transformed to their acyl CoA (coenzyme A) derivatives and then degraded by the

Surface run-off - causes of water pollution, Surface Run-off - Causes of Wa...

Surface Run-off - Causes of Water Pollution The surface run-off from cultivated lands where inorganic fertilisers, pesticides, insecticides and rich manure are applied and sol

What are the main structures of the human eye, What are the main structures...

What are the main structures of the human eye? The major structures of the human eye are the cornea, the iris, the pupil, the ciliary muscles, the crystalline lens and the ret

Describe organs of female reproductive system, Q. What are the organs that ...

Q. What are the organs that are part of the female reproductive system? The organs that constitute the female reproductive system are the ovaries, the uterus, the Fallopian tub

Use of beta-blockers-peri operative problems, Use of Beta-blockers :  ...

Use of Beta-blockers :  In some centres, patients are routinely put on beta-blockers after CABG. European coronary artery surgery study showed that only 25 per cent of patient

Which part of heart serves as an actuating signal, Which of the following s...

Which of the following serves as an actuating signal, or as part of an actuating signal, in a negative feedback system? A. Action potentials in sympathetic neurons that release

Explain what position of patient will during jvp examination, Explain what ...

Explain what Position of Patient will be During JVP Examination ? Position of Patient During JVP Examination: The patient is propped up to 45"on a back rest or pillow as in thi

Determine the nucleus transplantation technology, Why are the recombinant D...

Why are the recombinant DNA technology and the nucleus transplantation technology still dangerous? The recombinant DNA technology and the nucleus transplantation technology (cl

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