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f^*Co*e -z' QP code:780600 F.y.B.Sc. Biotechnology Semester I Examination paper: Basic BiotechnoloryII Molecular Biology ModelAnswers Q.1 Do as directed (Any t5) (15 marks- one mark each) 1. F plasmid. 2. a. Insertional Inactivation of a marker gene, b. Visual screening methods c. Plaque phenotlpe 3. Streptococcus pneumonia - smooth and rough sffain 4. Transposition involves integration of a copy of a defined segment of a DNA from another locus, either in the same or different molecule of DNA. 5. Mutation- sudden change in the heritable material. 6' Mutagenesis- the process of change in genetic material naturally or on induction with a mutagen. 7. E. coli or Bacillus subtilis 8 Ethidium b'romide, proflavin (any one) 9 False 10. True 11. Multiple Cloning Site 12. Semiconservative 13. Primer 14. DNA Polymerase - 15. 3'- I 5' exonuclease activity 16. Leading 17. Primase 18. Cyclin dependent kinases (Cdks) 19. G$cosylase 20. Thymine dimers Q 2 a Explain how Meselson and Stahl proved semiconseryative mode of DNA replication (8 marks) watson and crick proposed the semiconservative model in which each progeny molecule retains one of the parental st Cs. Five years later, Matthew Meselson and Frank Stahl obtained experimental evidence that semiconservative replication was correct. Organism used - Escherichia coli. E'coli was grown for several generations in a minimal medium n which the only N source was l5NH4Cl. As a result, all the bacteria's cellular N-containing compound contained 15N instead ofN14. l5N labeled bacteria was transferred to a medium containing nitrogen in the normal 14N form and allowed to reproduce in the new conditions for several Then the generations. Throughout the period of growth in the r4N medium, sample of E.coli were take4 lysed to release the cellular contents and cellular DNA wasw analysed in cesium chloride density gradients. After one generation ----all the DNA had density intermediate between that of totally 15N DNA and totally 14N DNA. After 2 genaations, half the DNA wasof intermediate and half of the density of DNA containing entirely r4N. thus, the observation was exactly what semiconserva ive model predicted. If the conservative model of DNA replication had been correct, after one generation -- 2 bands of DNA" one in the heavy density position of the gradient and other in the light density position would be seen and in the subsequent generations the amount of DNA in the light density position would increase with eaci generation. But the fact that intermediate density DNA was found ruled out the conservative model. According to the dispersive model after one generation all DNA would be of intermediate density but after the 2"d generation, the DNA molecules would be found at one band located halfivay between the intermediate density and light density band positioned and with subsequent there would continue to be one band and it would become lighter in density with each generation. Such a slow shift in DNA density was not seen in Meselson therefore dispersive model ruled out. - stahl experiment and This is how Meselson and Stahl proved semiconservative mode of replication. \ "'n \r' :t i1f :.r ij '.t t.f ++ re ,* ill{,*gfi 6Ni{4t{tr Q 2 b differentiate between the reading and lagging strands formed during DNA replication (7 marks) To maintain the 5'- 3'polarity of DNA synthesis and one overall direction of the replication fork movement, the direction of DNA synthesis is different on the two template strands. Leading strand: As the replication fork migrates, synthesis of one v'v new Dlrcu\r (the 'vvv strand \ continuous. leading strand) is The template strand copied is in 3'_ 5, direction. The leading strand synthesized is in the same direction as that of the fork movement. Lagging strand: synthesis of lagging strand is discontinuous because the helix must untwist to expose a new segment of 3'- 5'template so that the new strand can be made in the 5' - 3, direction. Thus the DNA on the lagging strand is formed in fragments called okazaki fragments. The template strand copied is in 3'_ 5, direction. The lagging strand is spthesized in the opposite direction of the replication fork movement. OR Q 2c Elaborate on the different DNA polymerases in Eukaryotic DNA replication (8 marks) Replication Enzymes and proteins: Five different DNA polymertase have been identified in mammalian cells, designated as o (alpha), B @eta), y (gamma) and (epsilon). e The o, p, o and e polymerases af,e located in the nucleus, while the y polymerase is found in the mitochondria. DNA polymerase o, 6 are the two enzymes responsible for nuclear DNA replication. They function essentially like the E.cori DNA polymerase III. DNA polymerase p serves a DNA repair function, and DNA polymerase y catalyses mitochondrial DNA rep lic action. Both Nuclear replication polymerase a and 6 can use RNA primers for the initiation DNA synthesis; D can also use a DNA primer. The mitochondrial polymerase y uses an RNA polymer. uniquely among the DNA Polymerase, the polymerase c also has primase activity; a separate enzwe is used for the synthesizing a primer in DNA synthesis catalyzed.by the other DNA polymerase. It is not known whether or not these enzymes have 5, to 3, exonucrease activity. only 6, e and y have been shown to have proofreading (3, to 5, exonuclease) activity. of Q2 d With ^ neat labelled diagram illustrate the Holliiday model of recombination (7 marks) fi*lrs "iR.l? 6 -\]i i:-l ki $:. $;.' tex r':' +i. $: +t- sr s€ttica, plan$ t g teppsa )* lt€i,ms ;Jre orodtrcgd W;i*i Q 3 a Explain the term mutation and discuss the various tlpes of mutations (8 marks) Can be explained in form of a table Mutation- 2 Marks Tlpes (6 marks) - somatic, gerrn line, spontaneous, Induced, point, Gross, forward, Backward, lethal, nutritional etc. Q 3 b Discuss Nucleotide Excision repair (7 Marks) Can be explained only diagrammatically U*ABC endonuclease complex is a speciar class of endonucrease enzyme involved in DNA repair of prokaryotes. The name is derived from the root term ultraviolet radiation, since the level of most of these enzymes will lre elevated in the bacterial cells when the cells are exposed to UV light. The U*ABC complex of E. cori consists of four enz)nnes. They are named as urzrA, UwB, uwc and uvrD (uwD is also called as DNA hericase Irt). Among these uw erzymes, uvrB and uwc are the actual e,ndonucleases which cut the phosphodiester backbone of the DNA. UWABC complex is arso called as DNA excinuclease. A;n excinucrease is a special type of endo-nucrease enzyme which cut two phosplodiester bonds at a time simultaneously, which is different from conventional endonur:leases since usual endonucleases cut only one phosphodiester bond at a tims. NER mechanism also requires DNA pollnnerase I enzyme wtrich wil do the gap filling activity after the exinuclease removes the damaged basesr. NER also requires DNA ligase enzyme for the final nick joining. The sequential action of these enz)rynes such as uwA, uwEt, uwc, uwD, then DNA polymerase I and DNA ligase are essential for the orchestr.tion of NER machinery in the bacterial cells. NI'CLEOTIDE EXCISION REPAI* IN PROI(ARYOTES IIrrrABC ENDONITCLEASS [eXrNUCf-n.eslrl Er{ZyMEBY [fV radiatio.rr irrcltr ces DNA darnages {JrrrABC extrnrrclease find.s darnage. crrts DNA strand. L2 or 13 nucLeotid.es are rerrro.rre*I DIYA pol5rrnerasre f fil.ts in ttre resulting gap DNA ligase :sea,l.s ttre DlgA str:and tllu$tratlan: @ JotlaD Jarnastad/Tho F?or.a, Swed/sar AcadeqT. of ,scrcrnc* Q 3 c Explain the various types of point mutations (g Marks) Base pair substitution - Transition, transversion Base Pair addition/ deletion - Frame shift Resulting in phenotlpic changes - Missence, Nonsense, silent, and Neutral. Explanation in a table form should also be considered can be explained with or without example Q 3 d Discuss the mechanism of mutation induced by Base anarog (7 marks) Base analog - chemical mutagen, analogous to nitrogen bases of DNA example 5 BU- 5 Bromo uracil (2 Marks) Mechanism 5 marks ( with or without diagram) Normal form of 5 BU and rare form of 5 BU ,$t .rs|$ ^{'F&dh:Whffin fi#",*ffi ffiffiih Q4 A. How wourd you marks) use pBR 322 as a vector to introduce a gene of choice (g pBF- 322 is an anificial cloning plasmid vector, B Boliver, R _ Rodriguez. It has 4364 kbases' It has 2 selectable markers tetracycline and ampicillin resistant gene. Has unique single restriction site for 12 different - - restriction endonucleases within the marker genes' cut vector with PstI, place the insert in the amp gene and make it non functional' Bacteria with such a cloned vector cannot grow in presence of amp but can grow in presence of tetra. This is insertional inactivation. cells are first grown ln media containing tetra and a replica is made on a plate containing amp in the medium. which grow on master plate but fail to grow on the replica plate contain the "t]t: TT qeslred msert. 48' Explain Isoration of genomic DNA by a flow diagram (7 marks) Protocol of genomic DNA from any material can be considered bacterial DNA flow sheet is given as an example examiners need not restrict only to it. Expected is various solution used in extraction and isolation protocol of genomic DNA. Q4 c. Explain the different types of Restriction Endonucleases (g marks) Tlpe I - has 3 subunits - i. Restrictive subunit ii. Modification subunit iii. specificity subunit - specifies recognition site. Requir e Mg2+, _ S adenosyl methoinine and ATp as cofactors. The recognition site is 15bp long and cleavage site is l000bp away from the 5' end ofrecognition site. Tlpe II - require Mg2+ as cofactor. They have separate activities for reshiction and modification' They cut within the restriciion site at a specific place. The restriction site exhibits a twofold symmetry around a given point. The restriction sites palindromes. Enzymes cut to give staggered or blunt ends. Tpe III - two subunits, are one for recognition and modification and the other for cleavage. ATP is source of energy and Mg2+ as cofactor. They have symmetrical recognition sites and cut DNA at non palindromic sequences, 25_27 bp away from recognition site. Q4 D. Explain with an example plant vectors (7 Marla) Plant virus vectors are used for transfer of genes. The DNA insert linked with viral DNA replicates and expresses in the host cell along with viral genes. These are transmitted systemically in the plant but the DNA never integrates into the plant DNA' To act as vectors they should i. carry extra NA ii. have broad host range iii. be easily transmitted. Then write on any one - l.caulimoviruses - rinear open circle ds DNA . Spreads rapidry throughout the plant and found in high copy number. Replicates through .*"rr" transcription. Two genomic regions of the virus oRF I and oRF vlr are non essential and can be replaced by a foreign DNA. 2' Geminiviruses - ss DNA. Their genome consists of two elements - an origin of replication and replication protein, which are included in the vector. The coat protein sequence is replaced by the desired or reporter gene. They are used to insert foreign genes in protoplasts. 3' Tobacco mosaic viruses (TIW) linear RNA genome. The vector is constructed by ne into an intact viral genome which rapidly spreads through the with insert replicate only in protoplasts inoculated leaves or Q 5. Write short notes on any three (5 marks each) a S ' ' . 15 marks Early models of DNA Replication emiconse.ative, Disp ersive and conservative models conservative. Replicalion produces one helix made entirely of old DNA and one helix made entirely of newDNA semi-conservative. Replication produces two helices that contain one old and one new DNA strand. Dispersive b. Genetic Engineering in any one prokaryote _ Most common- E. coli Methods l' - Transformation entry of naked DNA through the cell membrane into the recipient E. cori cell. The cell has to be competent to take up the foreign DNA. Not all exogenous DNA survive in the recipient celr, they are wrnerable to 2. e specific transduction only specific regions recipient, either the gal or bio gene. The 3. 4' - conjugation transrer or genetic -",:1tJ"Tf:*':'l:j,t::i',',.* or Hfr) to the recipient cell (F-) through direct physical contact. Transposition - involves integration of a copy of a defured segment of a DNA from another locus, either in the same or different molecule of DNA. these mobile transposable elements carry one or more genes that provide added qualities to the cell that carries it, for eg, resistance to drugs, toxins; encode toxins. c Photoreactivation - Repair ofI-IV induced thymine dimers In prokaryotes Also called as Light repair Induction by visible light, Role of photolyase enzyme Splitting of bonds between the dimers Can be explained diagrammatically. d - W radiations as physical mutagens Non -ionizing radiations Induction ofthymine dimers Also C^C C^T Atrecting formation of covalent bonds between bases Forming gaps in DNA, no replication Bulging ofDNA A,$Nitn$(p)Fr e. pUC vector- Developed by Messing. has colEl origin gene, amp resistance gene and engineered version of lac z gene which contain *ut ipt, restriction sites within the first part of the coding region. This is calred the MCS - multiple .toning site. If targetDNA is inserted into any one of these sites the lac z gine is inactivated. Hence such a recombinant vector will form white colony on a suitable plate as compared to non recombinant vector. cells canying just the vector are capable of producing the hybrid beta galactosidase. so when grown in presence of IprG (inducer) and x-Gat ,,rb.,.ur", the enzyme acts on substrate to produce a blue product. cells carrying the vector with insert are not able to produce the hybrid beta galactosidase. Hence the substrate is not acted upon and the colonies remain white in colour. This plasmid vector is designed for blue white screening.