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Lecture 5

Lecture 5. Reporter genes. Reporter Genes. A gene encoding an enzyme medium modification is added along with your gene nucleic acid sequences encoding easily assayed proteins

erasmus-goodman
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Lecture 5

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  1. Lecture 5 Reporter genes

  2. Reporter Genes • A gene encoding an enzyme medium modification is added along with your gene • nucleic acid sequences encoding easily assayed proteins • Reporter genes include -galactosidase (encoded by lacZ), -glucuronidase (encoded by uidA), chloramphenicol acetyltransferase, luciferase and green fluorescent protein (GFP) .

  3. Novel Reporter Genes • Luciferase - gene from fireflies with causes the glow - add a substrate to tissue that has been transformed and it lights up • Green Fluorescent Protein - from jellyfish - under lights and filter the transgenic plants will fluoresce • GUS - glucuronidase gene will convert added substrate to blue color.

  4. ß-glucuronidase (GUS) • GUS is probably the most widely used reporter gene in plants • low endogenous activity in plant • stable enzyme which hydrolyses wide range of ß-glucuronides • easily assayed for histochemical analysis, using X-gluc (5-bromo, 4-chloro, 3-indoyl ß–glucuronide). • After cleavage, oxidation of the indole derivative causes dimerisation and the production of an insoluble indigo dye

  5. GUS expression • The GUS gene creates blue coloration of transformed tissue when transformed cells or tissues are provided with the appropriate substrate.

  6. Luciferase (Luc) • enzyme from firefly (Photinus pyralis) • produces flashes of light in the presence of luciferin and ATP • detected in tissue extracts or even in the intact plant after watering with luciferin • allows non-destructive imaging of plants

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  8. Green fluorescent protein (GFP) • from the jelly-fish Aequoria victoria • intrinsically fluorescent • due to a chromophore in the protein by cyclisation and oxidation of the amino acids Ser-Tyr-Gly at positions 65-67 in the polypeptide • allows non-destructive imaging of plants and sub cellular localization of GFP by microscopy • several variants of GFP to give different colours – YFP (yellow), BFP (blue), CFP (cyan) • produced by alteration in the chromophore (Tyr66), or residues close to the chromophore in the 3-D protein structure

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