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

Ingestion of foreign bodies, Ingestion of Foreign Bodies: As we know  ...

Ingestion of Foreign Bodies: As we know  that small children are curious and  innocence children are notorious for inserting various object into their orifices like mouth, nos

Main moral problem about the cloning of human individuals, What is the main...

What is the main moral problem about the cloning of human individuals? As well biological perils, a very serious moral problem involves the nucleus transplantation technology c

About JACK FRUIT, How is the development of JACK FUIT fruit takes place fr...

How is the development of JACK FUIT fruit takes place from seed to fruit?

Joints, JOINTS - The structural arrangement of tissues which connect 2 ...

JOINTS - The structural arrangement of tissues which connect 2 or more bones together at their place of meeting is called a joint. Study of joint is arthrology. Joint make the

What do energy pyramids represent, Normal 0 false false fal...

Normal 0 false false false EN-IN X-NONE X-NONE MicrosoftInternetExplorer4

Define total body potassium (tbk), Define Total Body Potassium (TBK)? ...

Define Total Body Potassium (TBK)? Potassium in the body is an index of body's total cell mass. A gamma counter measures the amount of a type of potassium which is assumed t

Advantage & disadvantage of using fungi as source of protein, Define Advant...

Define Advantage & disadvantage of using fungi as source of protein? Advantages 1. Easy to harvest from culture medium. 2. Texture of the fungi improves the functional

Classifications of mechanical cleaning devices, Q. Classifications of mecha...

Q. Classifications of mechanical cleaning devices? In general, three classifications of mechanical cleaning devices are available for the dental office. They are:  ult

What is the parasite that causes giardiasis, Q. What is the parasite that c...

Q. What is the parasite that causes giardiasis? How is it transmitted and what are the typical manifestations of the disease? The Giardiasis is a protozoal infection caused by

How can the visual deficiencies known as myopia, How can the visual deficie...

How can the visual deficiencies known as myopia and hypermetropia be optically explained? Myopia is the visual condition in which the images are produced before (in front of)

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