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

Define malnutrition - effects on milk and effects on mothers, Define Malnut...

Define Malnutrition - Effects on Milk and Effects on Mothers? Milk is the sole source of nourishment for many infants for upto 6 months or a year or even more. Therefore, the r

Stable atoms have no vacancies in their outer shell, Select all that are tr...

Select all that are true/correct: Stable atoms have no vacancies in their outer shell. Hydrogen is more stable than carbon Helium is more stable than carbon Helium is more stable t

Chapter-2, multiple choise question

multiple choise question

Define the prevention of iron deficiency anaemia, Define the Prevention of ...

Define the Prevention of iron deficiency anaemia? As in the case of vitamin A deficiency, correction and prevention of dietary inadequacy of iron are important sustainable meth

What is immune memory, How can an organism that once underwent contact with...

How can an organism that once underwent contact with an antigen be immunized against future infections by the same agent? This phenomenon is known as immune memory . When an a

Proteins made by the same total number of amino acid, Are there different p...

Are there different proteins made by the same total number of amino acids? Different proteins with the similar total number of amino acids may exist. In such cases the separati

Endocrine glands - heart, HEAR T - The cells called cardiocytes of atr...

HEAR T - The cells called cardiocytes of atria of the heart secrete peptide hormone called atrial natriuretic factor (ANF) in response of an increased return of the deoxygen

Write the blood sugar in normal range and range in diabetes, Q. Write the b...

Q. Write the blood sugar in normal range and range in diabetes? Normal           Diabetes mellitus >126 mg / dl >200 mg / dl

SYMBIOTIC THEORY, WHAT IS SYMBIOTIC THEORY. PLEASE EXPLAIN THIS TO ME IN A ...

WHAT IS SYMBIOTIC THEORY. PLEASE EXPLAIN THIS TO ME IN A VERY SIMPLE WAY.

Explain aortic valvuloplasty, Q. Explain Aortic Valvuloplasty? Valvar a...

Q. Explain Aortic Valvuloplasty? Valvar aortic stenosis accounts for 4-6 per cent of CHD. LV Outflow Tract obstruction eventually leads to LV dysfunction and congestive heart f

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