Question

Protein P is synthesized in relatively high amounts in the human pancreas. This protein has been isolated and purified, but its amino acid sequence has not been determined. We wish to clone the gene for protein P. (a) How can a probe be prepared to identify the gene for protein P? (b) If we have prepared a radioactive messenger RNA as our probe in part (a), how could we verify that it is the mRNA for protein P? (c) If we wish to make a gene library from the DNA of human pancreatic cells, we proceed by digesting the DNA with the restriction enzyme BamHI and then separating the fragments by agarose gel electrophoresis. The fragments are transferred (by Southern blotting) onto a nitrocellulose filter and flooded with the radioactive probe. Following incubation, the filter is washed to remove any probe that has not specifically bound to one or more DNA fragments. Autoradiography of the filter reveals two bands of sizes 300 and 700 bp. When experiment is repeated using restriction enzyme Psti, only one band of 1000 bp appears. If it is already known that pancreatic cells contain a single copy of the gene for protein P, which of these enzymes should be used to construct our gene library? (d) We can construct our gene library by cloning the Pst. fragments into either plasmid PBR322 or phage lambda. Which one should we choose and why? (e) Suppose we want to insert the Pst. fragments into the BamHI site within the gene for tetracycline resistance (tet) in the plasmid PBR322. If both of these restriction enzymes produce cohesive ends, how can this be done? (f) After transforming E. coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing a DNA insert)? (g) If a million tetracycline-sensitive, ampicillin-resistant clones are grown on nutrient agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?

Answer 1

Q.H. Genes are isolated from plasmid by :- as plasmid are naturally occurring in bacterial cell, most widely used for gene cloning, contains foreign DNA and enzyme DNA ligases.

To isolate plasmid Alkaline lysis is the method used, there is detergent present, which cleaves phospholipid bilayer, membrane.

Steps:- 1. Plasmid should be cut and , pasted in gene , use restriction enzyme dnto cuta , use dna ligase for joining.

2. Plasmid should be inserted in bacteria.

3. Growth of plasmid are their then make use of them to make proteins..

L.the selection or mechanism of Tetracycline, and ampicillin resistance which are encoded on pbr322 are very different.

The enzyme beta - lactamase encodes by Ampicillin resistance on pbr322, which is secreted by periplasm this is done by an act of inactivating or binding Ampicillin molecules.

In Tetracycline their is production of transmembrane efflux, by pumping in or pumping out Tetracycline molecules.

With the use of effective ampicillin , then the selection of transformants are fewer in Tetracycline plate then ampicillin.