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

Treatment of diarrhoea, Treatment   Most cases of diarrhoea do not need...

Treatment   Most cases of diarrhoea do not need antibiotic therapy as the bacterial or parasitic organism are not isolated from most of the cases. However chemotherapeutics  ar

Zoology, what is the term used for study of sponges,protozoa,colentreta,mol...

what is the term used for study of sponges,protozoa,colentreta,molusca,fish,aves,mammal

Most common opportunistic diseases in aids, Q. Moniliasis is one of the mos...

Q. Moniliasis is one of the most common opportunistic diseases in AIDS. What is the etiological agent of moniliasis and what is the other name of the disease? Why is monilia also c

What is spermatogenesis in human biology, What is Spermatogenesis in human ...

What is Spermatogenesis in human biology? The male sex cells, or sperm, are produced in the oval-shaped testes, the primary sex organs of the male reproductive system, in a pro

Cardiac output - circulation, Cardiac Output - Circulation The cardiac...

Cardiac Output - Circulation The cardiac output can be determined in a number of ways. The most common method is the Fick's principle. A. Fick a German physiologist in 1870 de

Define about the centrifuge, Define about the Centrifuge? Centrifuge is...

Define about the Centrifuge? Centrifuge is an apparatus consisting essentially of a compartment spun about a central axis to separate contained materials of different specific

Define activation of the antagonist during movement, Explain the relationsh...

Explain the relationship between an agonist muscle and its antagonist as it relates to positional control, functional movement and neuromuscular activation. For example, how the tw

Deficiency diseases-zinc deficiency, Zinc deficiency Zinc is essential ...

Zinc deficiency Zinc is essential for several physiological functions in the body such as protein synthesis, carbohydrate and nucleic acid metabolism, and foetal growth. The de

What is sulphonylureas, Q. What is Sulphonylureas? Sulphonylureas: Tolb...

Q. What is Sulphonylureas? Sulphonylureas: Tolbutarnide is the mildest and its effect remains for 8 hours hence 2-3 doses need to be taken. Another drug chlorpropamide needs on

Define the dietary requirements of phosphorus, Define the Dietary Requireme...

Define the Dietary Requirements of phosphorus? We read in the previous section that the requirements of phosphorus are closely linked with those of calcium. The phosphorus req

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