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 benefit does the sea anemone get in the sea anemone-hermit crab facultative mutualism? Give an alternative term for this part of mutualism. Name the nitrogenous wast

Selective Destruction - Wildlife The selective destruction of one species of an existing fauna can produce equally unfortunate results. The perfect demonstration of unexpected

ACRODYNAM IC S Division of air in lungs. Measured by spirometer. 1. Tidal Air (T.A.): Quantity of air taken in or given out in a breath 500 cc. 2. Inspiratory Reserv

CHARACTERISTIC S OF MAN - Humans posses erect posture and bipedal locomotion. Humans possess large sized brain. Humans are less dependent on smell. Humans possess

What are cotyledons? Cotyledons, or seed leaves, are structures formed by the embryo of angiosperms to absorb nutrients from the endosperm and to keep and transfer these nutrie

What is the initial concentration of Beta-adrenergic receptor in blood?

discuss the merits nd demerits of five kingdom classification.

EGG S OF FROG This egg is round & its diameter is of 1-2 mm. It is mesolecithal & has distinct animal pole & vegital pole. The nucleus is found in the centre of the anim

Heat Loss Temperature regulation is extremely uneconomical if it depends only on variations in metabolism. Therefore, mechanisms for losing excess heat have been developed by

What are the two big groups into which cells are classified? Cells can be divided as eukaryotic or prokaryotic. Prokaryotic cell is the cell that without a delimited nucleus