Standard mrna and fibroblasts, Biology

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.

Posted Date: 2/19/2013 8:08:41 AM | Location : United States







Related Discussions:- Standard mrna and fibroblasts, Assignment Help, Ask Question on Standard mrna and fibroblasts, Get Answer, Expert's Help, Standard mrna and fibroblasts Discussions

Write discussion on Standard mrna and fibroblasts
Your posts are moderated
Related Questions

What is katal katal : the amount of  enzyme that  transforms  1 mol  of substrate into product in one second.

This constitutes about 2.4% of the body weight of man viz. Brain-1400 gram Spinal nerve-150 gram Spinal cord-30 gram Cranial nerve-10 gram C.S.F. is altogether not present in

Define Nutrient and Drug Interaction? Modern medicine has given us many useful drugs that not only prolong and save lives but in fact improve the quality of our lives. Have you

How was it proved in the case of Amoeba that the key to the life of a cell is Nucleus?

Q. What do you mean by Nervous System? Structural Organization:   CNS + PNS Functional Organization:   only concerned with PNS Sensory (Afferent) ->  CNS Motor (Effe

Explain about Posterior wall Posterior wall separates the antrum from the infra-temporal fossa and contains two important structures. Posterior superior alveolar nerve and

The secondary protein structure is produced by the manner its amino acids interact by intermolecular bond. These interactions make a spatial conformation of the polypeptide filamen

general character and classification of platyhelminthes

Q. Introduction of Quality Attributes of Food? Seasonal fruits and vegetables are grown in plenty, and are available in their respective seasons in abundance. While choosing th