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 do you determine by Mantle? A thin sheetlike membranous extension of visceral mass of molluscs which forms two flaps of skin. Mantle secretes the shell on the dorsal side

Residue is defined medically as the solid contents that have reached the lower intestine. A low residue diet is composed of foods, which are easily digested and readily absorbed, r

Ethnobotany : This is the study of relationship between tribals and plants. Ethnobotany is from "ethnology" - study of culture or "botany" - study of plants. This is the scientific

Explain about the Fructnns as Food Ingredients? Oligosaccharides have several functional/technological attributes that give them a potential for incorporation into foods. They

Conduction in Myelinated Axons The myelin sheath provides insulation for the axon preventing movements of Na + and K + through the membrane. Therefore, if this myelin sheath

We now understand that mutations that cause the inhibition of apoptosis are found in tumours. Because proliferation itself is not induced by the inhibition of apoptosis, explain ho

What is the pineal gland? The pineal gland, also called as pineal body or epiphysis, is situated centrally in the head. It secretes the hormone melatonin, a hormone formed at n

What is the name of the DNA duplication process? What is the main enzyme that participates in it? The process of copying, or duplication, of the DNA molecule is called replica

Limitations of Five-Kingdom Classification As already pointed out, each system of classification has its own limitations. The two-kingdom system has outlived its usefulness as

Jaundice (Icterus) Jaundice is classified as pre-hepatic (hemolytic), hepatic and post-hepatic (obstructive) depending on origin of the problem, and is characterized by yellowi