Already have an account? Get multiple benefits of using own account!
Login in your account..!
Remember me
Don't have an account? Create your account in less than a minutes,
Forgot password? how can I recover my password now!
Enter right registered email to receive password!
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.
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.
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.
Define observation or inference for barfoed test? 1. Reddish brown precipitate is seen on the sides and bottom of the tube. The precipitate of the sides and bottom indicates th
Q. What are the symptoms of Rheumatic Heart Disease? Symptoms generally appear after 1 to 6 weeks of the fever and sometimes the infection may have been too mild to have been r
Q Are there living beings without cells? Viruses are considered the only living beings that do not have cells. Viruses are constituted by genetic material RNA or DNA enwrapped
Q. How can the tubular-dorsal nervous system in chordates be compared to the nervous pattern present in invertebrates? In chordates the nervous system is dorsal and highly ceph
Q. Which kind of circulatory system do molluscs have? How can it be compared to the respiratory system of annelids? Molluscs, with the exception of cephalopods, have an open ci
Q. Show the Inventory of the plants? A flora provides an inventory of the plants of an area and is often restricted to the vascular plants. Floras contain description of plants
Q. What is the difference between rough and smooth endoplasmic reticulum? The endoplasmic reticulum is a delicate membranous structure contiguous to the nuclear membrane as wel
Examination of the Prosthesis It is important to examine the prosthesis thoroughly. This involves assessing mobility, checking screws, evaluating attachments and porcelain res
Define Unsafe Water and sanitation - public nutrition? Safe water and sanitation may seem tenuous in their link to food security but their impact is unquestionable. With 19% US
Zoonotic paramyxoviruses Three zoonotic paramyxoviruses (Hendra, Menangle, and Nipah viruses) emerged during 1994-2004 and all these viruses have a fruit bat reservoir. Humans are
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!
whatsapp: +1-415-670-9521
Phone: +1-415-670-9521
Email: [email protected]
All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd