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

Explain acid butt - carbohydrate utilization pattern test, Acid butt - carb...

Acid butt - carbohydrate utilization pattern test ? Acid butt (yellow) and acid slant (yellow) with or without gas production – Glucose along with lactose and/or sucrose, is deg

Algae, What is the basis of classification of algae

What is the basis of classification of algae

Simple proteins, SIMPL E PROTEINS The proteins are made of amino acids...

SIMPL E PROTEINS The proteins are made of amino acids only. Additional chemicals are absent. These are of two types -    (i) Fibrous        (ii) Globular ( i )

Classification of zoonoses disease , Classification of zoonoses More t...

Classification of zoonoses More than 300 zoonoses of diverse etiologies are recognized. Thus, a very large number of zoonoses call for classification, especially for teaching

Schematic representation of curser position of pulse wave, A lot of hemodyn...

A lot of hemodynamic information including maximum and mean velocity, maximum and mean pressure gradients and pressure half time can be obtained from Doppler trace that helps in

Response to cold - responses of plants to stress, Response to Cold - Respon...

Response to Cold - Responses of Plants to Stress Let us first see what happen to a plant when it is exposed to a cold climate with temperatures below 0°C. If the temperature i

What is the difference among systole and diastole, What is the difference a...

What is the difference among systole and diastole Systole and diastole are the two stages into which the cardiac cycle is separated. Systole is the stage when the contraction o

What is the difference between rna and dna, Q. Concerning the nitrogen- con...

Q. Concerning the nitrogen- containing bases that participate in nucleotides, what is the difference between RNA and DNA? In DNA nucleotides can be formed of thymine (T), adeni

Food processing and preservation, various changes that occur during concent...

various changes that occur during concentration of food

Define the purposes of counselling, Q. Define the Purposes of Counselling? ...

Q. Define the Purposes of Counselling? 1. Supporting individuals to take charge of their own life by: Providing information; Facilitating emotional adjustments; and

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