Lecture 5
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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

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Lecture 5

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Lecture 5

Lecture 5

Reporter genes


Reporter genes

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) .


Novel reporter genes

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.


Glucuronidase gus

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


Gus expression

GUS expression

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


Luciferase luc

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


Lecture 5

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Green fluorescent protein gfp

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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