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

• Reporter genes include -galactosidase (encoded by lacZ), -glucuronidase (encoded by uidA), chloramphenicol acetyltransferase, luciferase and green fluorescent protein (GFP) .

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.

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

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

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

)

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