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Pune University - Molecular Biology - II, April 2010

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Total No. of Questions : 5] [Total No. of Pages : 3 P1016 [3725] - 402 M.Sc. MICROBIOLOGY MB - 802 : Molecular Biology - II (2008 Pattern) Time : 3 Hours] [Max. Marks : 80 Instructions to the candidates: 1) All questions are compulsory. 2) All questions carry equal marks. 3) Draw neat labeled diagrams wherever necessary. 4) Use of scientific calculator and log table is allowed. 5) Assume suitable data, if necessary. Q1) Explain any two of the following : a) Enlist different vectors and their role in RDT. b) Explain role of aminoacyl t-RNA in translation. c) [16] Explain non coding RNAs and their role. Q2) With reference to transcription and translation in eukaryotes, describe any four from the following : a) Structure of typical eukaryotic promoter. b) Assembly of basal apparatus and RNA pol. II. c) Post translational modifications. d) Protein splicing. e) [16] Active centers of ribosomes. P.T.O. Q3) Describe the principle, working and applications of any two of the following : [16] a) Northern and Western hybridization technique. b) RFLP. c) PCR. Q4) Comment on a ny two of the following : a) Site directed mutagenesis for protein engineering. b) Genome mapping and sequencing. c) DNA footprinting. [16] Q5) Your first task as a newly arrived post doc in the laboratory of Dr. Ursh is to analyze the structure of the shellase gene from the Bulgarian spotted grosbeak. Luckily, your professor, Dr. Klassik, has left you a small, but very pure preparation of mRNA of this protein. The mRNA is 9.5 kb in length. As the starting material for your study, you prepare whole genomic DNA from the spotted grosbeak. Using the R.E. BamHI, EcoRI, Hind III, and Sall, in all combinations, you digest the DNA and subject it to electrophoresis on an agarose gel. You than do the southern transfer from the gel to nitrocellulose paper. As a probe you take half of the Dr. Klassiks mRNA preparation and incubate it with 32P-ATP and polynucleotide kinase to radioactively label 5 end. You then hybridize the probe to the DNA on the nitrocellulose After washing off unhybridized probe, you autoradiograph the filter. Table lists the sizes of bands seen after overnight exposure. You know from Klassiks former work that there are about 20 copies of the shellase gene per haploid genome. a) Draw the diagram of the restriction map of the gene. [4] b) What, if any, are the unusual features of the gene structure? [4] c) The boss is still not satisfied, and wants a better autoradiogram for publication. You leave another sheet of film on the filter-this time for 3 days. You are aghast at the results! Table shows your data. How would you explain the extra bands? d) [4] Can you say how large this piece of DNA is? Why or Why not? [4] [3725]-402 2 Table : Sizes of fragments identified by southern transfer in digests of Grosbeak genomic DNAa. Enzyme Fragments (kb) Overnight Three-day exposure exposure EcoRI 10 10,7* EcoRI + HindIII 6,4 7*, 6,4 EcoRI + BamHI 9,1 9,6*,1 EcoRI + Sal I 6,4 7*,6,4 HindIII 10 17*, 10 5(dark) 7*, 5(dark) HindIII + Sal I 8,2 15*, 8,2 Sal I 10 17*, 10 Sal I + BamHI 7,3 7,3 BamHI 10 10, 7* 5,4,1 6*,5,4,1 EcoRI + HindIII + Sal I 4(dark),2 7*,4(dark),2 EcoRI + BamHI + Sal I 6,3,1 6,3,1 4,3,2,1 6*,4,3,2,1 HindIII + BamHI EcoRI + HindIII + BamHI EcoRI + BamHI + HindIII + Sal I i) Restriction digests were probed with purified 32p labeled shellase mRNA. ii) Astriks indicate faint bands, less than 10% as intense as the main bands. vvvv [3725]-402 3

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Additional Info : M.Sc. MICROBIOLOGY, MB - 802 : Molecular Biology - II (2008 Pattern), Pune University
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