Question

2) A linear phage chromosome is labelled at both ends with 32P and digested with restriction enzymes. EcoRI produces fragments of sizes 2.9, 4.5. 6.2.7.4, and 8.0 kb. An autoradiogram developed from a Southern blot of this digest shows radioactivity associated with the 6.2 and 8.0 fragments. BamHI cleaves the same molecule into fragments of sizes 6.0, 10.1 and 12.9; the label is found to be associated with the 6.0 and 10.1 fragments. When EcoRI and BamHI are used together, fragments of sizes 1.0, 2.0, 2.9.3.5. 6.0, 6.2, and 7.4 kb are produced. (a) Draw a restriction-enzyme map of this molecule, showing relative positions and distances apart. (b) A radioactive probe made from a cloned phage gene X is added to Southern blots of single-enzyme digests that have used nonradioactive phage DNA. The autoradiograms show hybridization associated with the 4.5, 10.1 and 12.9 fragments. Draw in the approximate location of gene X on the restriction map. 3) A viral DNA fragment carrying a specific gene V is transfected (introduced into the cell by transformation) into a muscle-cell culture. Following incubation with 32P-labelled ribonucleotides, the virus-encoded RNA product is isolated at two time intervals. The radiolabelled viral RNA is treated as follows. First, it is hybridized to a specific cDNA previously constructed from viral gene V mature mRNA. Second, the hybrid is treated with Rnase. Finally, the hybrid is denatured and electrophoresed on a gel, which is then subjected to autoradiography. The following results suggest that the pathologic nature of the virus is time-related (the number of nucleotides is indicated on the bands observed): At 2 hours At 10 hours 1200 700 500 Protein V absent Protein V present (a) What is the size of the mature mRNA for gene V? 6) Draw a diagram of each hybrid and indicate what the illustrated bands represent? ©) Why is protein V not produced until after 2 hours?

Answer 1

3.a. The size of the mature mRNA is 12000 nucleotides.

Total - 29 kb 326 6.2kb | 2.9kb|2kb/3.5k0 | 7.4kb | 2Kb 6 kb Fig:- Treated with both EcoRI and BamH I are used together:- Showing relative position. and distance. 4.5 d. 12.9 Kb Fisa Gene-X 6.266 2.9 kb/1kb/3.5Kb/7.4kb/2k6 / 6Kb ] Showing the approximate location of gene x, which is present between 1 kb and 3. Skb. fragments. Because, the autoradiogram hybridization associated with 4.5kb; 10.1 kb; 12.9.

3.c. The protein v is not produced until 2 hours because the mature mRNA required more than 10 hours for its complete transcription.

-ODNA from matere MRNA-V RNA - 700 or soo nucleo sides. which is uncomplete - mature C.DNA - complete RNA mahered.