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Unit III: Paper-10; MBT-II: M.Sc BT II yr

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  1. 1. Compiled & Prepared by,K.P.Senthil kumar.,M.Sc.,M.Phil.,ADAB
  2. 2. Tobacco plant?
  3. 3. Viral cells with luciferase
  4. 4.  Fireflies glow in summer night – “ LuciferaseActivity ” Aids molecular biologists interested inmammalian gene transcription Gut of firefly beetle and some marine organismsit is present Luciferase Activity not in any other eukaryotes Excellent Reporter gene in promoter analysis Derived from luc/lux gene of firefly - Photinuspyralis Enhance Research in Chemiluminescence andBioluminescence
  5. 5. Modified luciferase genes are constructed  Higher level of expression  Different assay reagents – Change the kinetics of light release  Use of small molecules to stable the enzyme  Isolation and expression in different organisms
  6. 6.  Luciferase gene – cDna  Peptide sequence at carboxy terminal  Targets the protein to peroxisome of the cell  Eliminated peroxisome targeting sequences  Elimination of sequences predicted to give Rna secondary structure  Inclusion of optimal translation initiation sequence  Swapping of prevalent insect codons for mammalian counterparts  Inclusion of polyadenylation sequences upstream and downstream from cDna of Luciferase gene
  7. 7. Luciferase has characteristic behavior  Substrate specificity  Light release kinetics  Allosteric modulation  Intracellular stability  Presence of Mg2+ is essential ATP + Luciferin + O2 AMP + Oxyluciferin + Ppi + light (560 nm)
  8. 8.  pGL series available from Promega Other luciferase genes isolated from diversemarine and bacterial organisms From sea pansy – Renilla reniformis Different substrate and different biochemicalproperties Dual reporter assay systems Optimum assay components differ fromluciferase from species to species
  9. 9. pGL series available from Promega1. pGL3-Control vector2. pGL3-Basic Vector3. pGL3-Promoter Vector4. pGL3-Enhancer Vector
  10. 10. Enhancer 2013-2249
  11. 11. Promoter 0048-0250
  12. 12. RVprimer3 is especially useful for identifyingpositions of nested deletions.Note: All three primers can be used for dsDNAsequencing, but onlyRVprimer4 and GLprimer2 also may be used forssDNA sequencing.
  13. 13.  Acetyl CoA : CAT, widely used as Reporter Indirect assay of transcriptional regulatory elements Transfected Mammalian cells Covalently modifies chloroamphenicol Transfer an acetyl group from acetyl CoA toprimary hydroxyl residue C3-chloroamphenicol Then to C1 and one more in C3 form 1,3-diacetylchloroamphenicol.
  14. 14.  CAT – Responsible for resistance toChloroamphenicol; An antibiotic Bind to peptidyl transferase center ofprokaryotic ribosomes CAT – Available in plasmids of Gram +ve andGram – ve organisms.  Trimers consists of identical subunits ;  Mw 25 kDa  Trimer- β pleated sheets extends from 1 subunit to next. Two substrates  Chloroamphenicol  Acetyl CoA
  15. 15.  Approach active site through tunnels Located in opposite sides of the molecule Active site located at subunit interface His residue – act as general base catalyst inacetylation reaction Expression of CAT in Mammalian cells: Plasmid pSV2CAT  SV40 promoter/enhancer  29 bp of Un Translated Sequence CAT coding sequence  8 bp of Dna 3’ to the UAA stop codon.
  16. 16. To assay putative promoters in mammalian cells  A derivative of pSV2CAT is constructed with pSV0CAT  Promoter of SV40 replaced with the one under test.No endogenous DNA in eukaryotes No competing activities Enzyme is stable Some assays measure in cell lysate Expensive and Time consuming Extracts prepared from C-14 labeledChloroamphenicol Modified product separated from unmodifieddrugs by TLC
  17. 17. Quantified by Autoradigraphy Sensitivity increased if condition of extract adjusted  10 mM EDTA  Heated at 60˚C  10 minutes incubation  Mixture extracted with ethyl acetate  Partition in organic phase Acetyl CoA remains in aqueous phase  liquid Scintilation counter Express CAT activity in product formed per mg cellextract per unit time.
  18. 18. Green Fluorescent Protein Bioluminescent jellyfish Aequorea victoria Emits characteristic Green Fluorescence Activity of two proteins  Calcium-binding photoprotein “aequorin” and its companion  Green Fluorescent Protein GFP most important protein in BC and Mob. Tool to understand and manipulate  Relation between protein structure and intracellular process  From bacteria to mice
  19. 19. 238 – residue peptide Mw – 26,888 Da Three Exons spread over 2.6 kb Protein is stable even at  Heat  Extreme pH  Chemical denaturants  Continues to emit fluorescence after fixation in formaldehyde. Fluorescence is rapidly quenched under reducingconditions. The emission spectrum peaks at 508 nm Distinct from chemiluminescence of aequorin i.e.,blue – peaks near 470 nm.
  20. 20. Structure β – barrel structure composed of 11 strands, encapsulates 1α-helix. Chromophore formed ; cyclization of residues  Ser-65  Tyr-66  Gly-67  contained in short helical structure  Buried inside β – barrel Insulation gives greater resistant to denaturants
  21. 21. Function: No separate biosynthetic pathway is requiredAutocatalytic intramolecular reaction to create thechromophore Post translation occurs  Cyclization  Oxidation of the trimer Ser-65, dehydro Tyr-66, Gly-67 Absorb light maximally at 390 nm However intact GFP emits green light  Peak at 508 nm  Shoulder at 540 nm
  22. 22.  It can be formed in wide range of cells thatnormally do not produce lightSensitive to  pH  Temperature  Ionic StrengthChromophore may exists in two formsTwo peaks of absorbance  390 nm – protonated tyrosyl hydroxyl groups  480 nm – deprotonated tyrosyl hydroxyl groups
  23. 23. GFP as Reporter:Ability to fluoresce in organisms other thanaequorea No other agents; such as  Abs  CofactorEnzyme – substrates required for its activity. GFP used as reporter in  Caenorhabditis elegans  Bacteria and Yeasts  Drosophila  Zebra fish; Plants ; Cultured mammalian cells  Transgenic mice
  24. 24. Potential difficulties in heterologous expression : Post translation modification requires > 1 hr  This delays the emission ability  Immediate readout is not possible Efficient expression in higher organisms Optimization of coding sequence required No definite prediction of fusion protein  i.e., Function protein to analyze and GFP  A variety of constructs generated Over expression of protein in some cells likeyeast
  25. 25.  Confocal microscopy, Careful choice offilters are required GFP exhibits significant spectral change  Protein concentration  Ionic strength  pH  Important to assay under standard condition
  26. 26.  The entire cDNA sequence encoding GFP hasbeen Mutagenized Synthesized in several different ways  various groups to alter  Signal produced by reporter has been increased considerably Low level transcription is addressed  Insertion of strong constitutive promoters  Cytomegalovirus  SV40
  27. 27. HIV  Long terminal repeats upstream of the GFP- coding regionExtensive structure driven ,site-directedmutagenesis. Variants each having altered fluorescenceexcitation and/or emission spectra
  28. 28.  Altered properties provide significantadvantage over wild type GFP  Mutation by substitution at Tyr-66 ; generates  Fluoresce yellow  Blue  Cyan  Mutation at Tyr-66 cause 20% reduced fluorescent output  Thr-65 cause 4 to 6 folds greater than wild type GFP.
  29. 29.  E.coli β Galactosidase – 465,412 Da Tetramer of 4 identical polypeptide subunits 1023 amino acids Encoded by first gene of the Lac operon – lac Z The individual polypeptide chain folds into 5sequential domains An extended section of 50 aminoacid residues atthe amino terminus This is α peptide β Galactosidase whose synthesis is induced bylactose and other galactosides
  30. 30. Catalyzes two enzymatic reactions  Hydrolysis of β-D-galactopyrinosides  Lactose into glucose and galactose  A transgalactosidation reaction  Lactose is converted to allolactose, true inducer of lac operon β Galactosidase interact with series of analogsof lactose  Glucose is replaced with other moieties  ONPG o-nitrophenyl-β-D-galactoside  X-gal  MUG 4-methyl umbelliferyl- β-D-galactoside  TPEG p-aminophenyl- β-D-thio-galactoside - INHIBITOR
  31. 31. PECULARITY: The aminoacid and carboxyl domains of theenzymes can be in different molecule  Two inactive fragments of the polypeptide chain  One lacking amino-terminal region (α acceptor)  Other lacking carboxy terminal region (α donor) Vector  Both able to associate in vivo and in vitro  To form Tetrameric active enzyme  This unusual form of Complementation is α- Complementation
  32. 32. Hydrolysis of ONPG Spectrophotometric Assay Cleaves β- galactosidase linkages Hydrolysis synthetic ONPG into o-nitrophenol Yellow in aqueous solutionAbsorbance at 420 nm
  33. 33. bacterial cells + permeabilized toluene or chloroform +buffer with high concentration of β mercaptoethanol + Incubation with ONPG ; Reaction terminated with Sodium carbonate OD 420 (o-nitrophenol + bacterial debris) Remove the debris (centrifugation) OD 420
  34. 34. Units of β galactosidase = 1000 OD 420 /t v OD 600 Miller units  t- time of the reaction in minutes  v-volume of the culture in ml used for assay  OD 600 Absorbance at 600 nm of bacterial cells just before enzyme assay  Induced culture contains almost 1000 units of β galactosidase  Uninduced <1 unit β galactosidase can also be expressed in mammaliancells
  35. 35. Reporter gene technology:  To define a gene with readily measurable phenotype  Can be distinguished easily over a background of endogenous proteins  Reporters selected :  Sensitivity  Dynamic range  Convenience  Reliability of their assay  Controlling the activity of genes by cis – reguation sequences (Response Elements)  Responsive to alterations in Gene regulations and Expression in host cells  Hormones and Growth Factors stimulate Target cells
  36. 36. 1.The cAMP response element (CRE) interacts with CREB (CRE-binding protein), which is regulated by cAMP2.Estrogen response element (ERE) are the recognition sites of estrogen receptor3.Glucocorticoid response element (GRE) and glucocorticoid receptorNote that hormones are not transcription factors, but many of their receptors
  37. 37. 4. Heat shock response element (HSE) is present in heat shock protein genes.In response to external stress (e.g. high temperature), the heat shock factor (HSF) will interact with HSE, stimulating expression of heat shock proteins.5. Serum response element (SRE) binds to serum response factor (SRF), which can be activated by many growth factors in serum. The Fos subunit of AP-1 is encoded by a gene containing SRE. Fos is known to play an important role in cell cycle progression.
  38. 38. Reference:Sambrook J. et al. Molecular Cloning: A LaboratoryManual. New York; Cold Spring Harbor 1989.[Book]