With Antibiotic

Survivors

Killed

FIGURE 25.01 Antibiotic Resistance as a Reporter Gene

Killed

Survivors

FIGURE 25.01 Antibiotic Resistance as a Reporter Gene

Antibiotic resistance genes are included on plasmids in order to determine whether the plasmids are present in a cell. When bacteria are transformed with plasmid DNA those that get a plasmid that carries an antibiotic resistance gene will survive when treated with the antibiotic whereas those cells that fail to get a plasmid will be killed.

that receive the plasmid become antibiotic resistant; those not getting the antibiotic resistance gene are killed. An antibiotic resistance gene carried on the same fragment of DNA can also report whether a transgene has integrated into another DNA molecule such as a chromosome or a virus.

Beta-galactosidase and alkaline phosphatase can be assayed using substrates that generate colored or fluorescent products.

Easily Assayable Enzymes as Reporters

The most widely used reporter gene for monitoring gene expression is the lacZ gene encoding b-galactosidase (Fig. 25.02). This enzyme normally splits lactose, a compound sugar found in milk, into the simpler sugars glucose and galactose. However, b-galactosidase will also split a wide range of compounds of galactose (i.e., galactosides) both natural and artificial (Fig. 25.02). The two most commonly used artificial galactosides are ONPG and X-gal. ONPG (o-nitrophenyl galactoside) is split into o-nitrophenol and galactose. The o-nitrophenol is yellow and soluble, so it is easy to measure quantitatively. X-gal (5-bromo-4-chloro-3-indolyl b-D-galactoside) is split into galactose plus the precursor to an indigo type dye. Oxygen in the air converts the precursor to an insoluble blue dye that precipitates out at the location of the lacZ gene.

Another widely used reporter gene is the phoA gene that encodes alkaline phosphatase. This enzyme cleaves phosphate groups from a broad range of substrates (Fig. 25.03). Like b-galactosidase, alkaline phosphatase will use a variety of artificial substrates:

1. o-Nitrophenyl phosphate is split, releasing yellow o-nitrophenol.

2. X-phos (5-bromo-4-chloro-3-indolyl phosphate) consists of an indigo dye precursor joined to phosphate. After the enzyme splits this, exposure to air converts the dye precursor to a blue dye, as in the case of X-gal.

3. 4-Methylumbelliferyl phosphate releases a fluorescent compound when the phosphate is removed.

The enzyme luciferase emits light when it reacts with its substrate, luciferin.

Light Emission by Luciferase as a Reporter System

A more sophisticated reporter gene encodes luciferase (Fig. 25.04). This enzyme emits light when provided with a substrate known as luciferin. Luciferase is found naturally in assorted luminous creatures from bacteria to deep-sea squid. The lux genes from bacteria and the luc genes from fireflies produce different brands of luciferase, but both work well as reporter genes. The luciferins used by the different types of luciferase are chemically different. Bacterial luciferase uses the reduced form of the cofactor FMN (flavin mononucleotide) as its luciferin. Oxygen and a long chain aldehyde (R-CHO) are also needed. Both the reduced FMN and the aldehyde are oxidized.

alkaline phosphatase Enzyme that cleaves phosphate groups from a broad range of substrates b-galactosidase Enzyme that splits lactose and other compounds of galactose galactoside Compound of galactose, such as lactose, ONPG or X-gal lacZ gene Gene encoding b-galactosidase;widely used as a reporter gene luc gene Gene encoding luciferase from eukaryotes luciferase Enzyme that emits light when provided with a substrate known as luciferin luciferin Chemical substrate used by luciferase to emit light lux gene Gene encoding luciferase from bacteria

4-methylumbelliferyl phosphate An artificial substrate that is cleaved by alkaline phosphatase, releasing a fluorescent molecule ONPG (o-nitrophenyl galactoside) Artificial substrate that is split by b-galactosidase, releasing yellow o-nitrophenol o-nitrophenyl phosphate Artificial substrate that is split by alkaline phosphatase, releasing yellow o-nitrophenol phoA gene Gene encoding alkaline phosphatase; widely used as a reporter gene

X-gal (5-bromo-4-chloro-3-indolyl b-D-galactoside) Artificial substrate that is split by b-galactosidase, releasing a blue dye X-phos 5-bromo-4-chloro-3-indolyl phosphate, an artificial substrate that is split by alkaline phosphatase, releasing a blue dye

Light Emission by Luciferase as a Reporter System 697

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