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

Db is a standard abbreviation, DB is a standard abbreviation used for the q...

DB is a standard abbreviation used for the quantitative expression of: 1. the density of bacteria in a medium 2. a particular pollutant 3. the dominant Bacillus in a cultu

Demography, Demography : This is the study of populations. In the other wor...

Demography : This is the study of populations. In the other words demography is the statistical study of human population. This may be a very general science which can be applied t

Explain physical properties of an oil, Explain Physical properties of an oi...

Explain Physical properties of an oil Physical properties of an oil or fat are of critical importance in determining its functional characteristics or use in food products. One

Illustrate briefly about vitamin B6, Vitamin B 6 (Pyridoxine hydrochloride...

Vitamin B 6 (Pyridoxine hydrochloride) Pyridoxine hydrochloride is a white, crystalline powder, practically odourless. The dry substance is sufficiently stable in air. With pr

Blood pressure - blood vessels, Normal 0 false false false ...

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

What are some prophylactic measures against hiv infection, What are some pr...

What are some prophylactic measures against HIV infection? The major prophylactic measures against HIV infection are: the use of condoms in sexual relations, not to share syrin

What is molecular weight, What is molecular weight? Molecular Weight ...

What is molecular weight? Molecular Weight :  The molecular weight of a molecule refers to the sum of the atomic weights of all the atoms making up the molecule. The gram mol

Explain the properties of gum karaya, Explain the Properties gum karaya ...

Explain the Properties gum karaya Gum karaya is water-swellable rather than water-soluble and absorbs water very rapidly to form viscous colloidal dispersions at low concentra

Explain in brief about cytokines, Cytokines activate inflammatory cells lik...

Cytokines activate inflammatory cells like neutrophils and eosnophils.IL-5 is important in activation of eosnophils, IL-4 enhances IgE production by B-cells. IL-4, Il-5, IL-6, TNF-

How common are coiled-coil motifs to each other, 1. Name four experimental ...

1. Name four experimental techniques to determine protein-protein interactions other than X-ray & NMR techniques. 2. How common are Coiled-coil motifs? Where are they found? Giv

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