Reacts With Oxygen In

INDIGO TYPE DYE dark blue and insoluble

FIGURE 25.02 Substrates Used by ß-Galactosidase

INDIGO TYPE DYE dark blue and insoluble

The enzyme, p-galactosidase, normally cleaves lactose into two monosaccharides, glucose and galactose. p-galactosidase also cleaves two artificial substrates, ONPG and X-Gal, releasing a group that forms a visible dye. ONPG releases a bright yellow substance called o-nitrophenol, whereas X-gal releases an unstable group that reacts with oxygen to form a blue indigo dye.

CH2OH

ch2oh ch2oh ch2oh ch2oh ch2oh

FIGURE 25.03 Substrates Used by Alkaline Phosphatase

Alkaline phosphatase removes phosphate groups from various substrates. When the phosphate group is removed from o-nitrophenyl phosphate, a yellow dye is released. When the phosphate is removed from X-phos, further reaction with oxygen produces an insoluble blue dye as for X-gal. Additionally, alkaline phosphatase releases a fluorescent molecule when the phosphate is removed from 4-methylumbelliferyl phosphate.

Luciferin

FIGURE 25.04 Luciferase Degrades Luciferin and Emits Light

Luciferase is an enzyme that alters the structure of luciferin. When the structure is altered, a pulse of light is emitted, which is detected by a photodetector. The luciferin shown in this figure is FMN (flavin mononucleotide), which is used by bacterial luciferases.

FIGURE 25.04 Luciferase Degrades Luciferin and Emits Light

Luciferase is an enzyme that alters the structure of luciferin. When the structure is altered, a pulse of light is emitted, which is detected by a photodetector. The luciferin shown in this figure is FMN (flavin mononucleotide), which is used by bacterial luciferases.

Green fluorescent protein does not need a substrate or a cofactor. It emits green light after illumination with longwave UV.

GFP can be used to follow gene expression or to localize proteins inside the cell.

Different groups of eukaryotes make several chemically distinct luciferins that are used solely for light emission. Firefly luciferase requires ATP as well as oxygen and firefly luciferin.

luciferin + O2 + ATP ^ oxidized luciferin + CO2 + H2O + AMP + diphosphate + hu

If DNA carrying a gene for luciferase is incorporated into a target cell, it will emit light only when the appropriate luciferin is added. Although high-level expression of luciferase can be seen with the naked eye, usually the amount of light is small and must be detected with a sensitive electronic apparatus such as a luminometer or a scintillation counter.

Green Fluorescent Protein as Reporter

The products of most reporter genes are enzymes that must be assayed in some manner. Unlike the products of most reporter genes, green fluorescent protein (GFP) is not an enzyme, and it does not need a non-protein cofactor for it to fluoresce. GFP is a stable and non-toxic protein from jellyfish that can be visualized by its inherent green fluorescence. Consequently, GFP can be directly observed in living tissue without the need for adding any reagents. Nearly 2,000 years ago, the Roman author Pliny noted that the slime from certain jellyfish would generate enough light when rubbed on his walking stick to help guide his steps in the dark.

The original GFP came from the jellyfish Aequorea victoria. Wild type GFP is excited by long wavelength UV light (excitation maximum 395 nm) and emits at 510 nm in the green. A variety of genetically engineered variants of GFP are also in use. Many of these are available as the Living Colors™ series from Clontech Corporation. Some of these were chosen for showing higher fluorescence and/or emitting at a different wavelength. Thus enhanced GFP (EGFP), enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) can be detected simultaneously using an argon-ion laser plus a detector with appropriate filters. A recent addition is a "Red GFP" (DsRed2—really a shade of orange). Other modifications include adapting GFP for high-level expression in different organisms by altering the codon usage (i.e., by changing bases in the redundant third codon position). Humanized versions of GFP exist that are adapted for use in cultured human cell lines.

Fusions of regulatory regions and promoters to the gfp gene have been used to monitor the expression of many genes, especially in living animals. The nematode, Caenorhabditis, and the zebrafish are both transparent and so GFP can be used to follow differential gene expression in different internal tissues of living animals. Trans-genic mice, rabbits, monkeys and several plants have been engineered that have the gfp gene inserted into the host genome (Fig. 25.05).

In addition to monitoring gene expression, GFP is widely used to localize proteins within the cell (Fig. 25.06). Gene fusions are constructed that yield a hybrid protein. These are normally designed so that the GFP protein is attached at the C-terminal end of the protein under investigation. The fluorescence due to GFP will reveal the sub-cellular location of the target protein. For example, a fusion of Red GFP (DsRed) to the targeting sequence from subunit VIII of cytochrome c oxidase is located in the mitochondrial inner membrane. Fusions between actin or tubulin and GFP are used to study cell architecture.

Gene Fusions

Reporter genes can be used to track the physical location of a segment of DNA or to monitor gene expression. In particular reporter genes are often incorporated into gene fusions where they are used to follow the level of expression of the target gene. Many green fluorescent protein (GFP) A protein, originally from a jellyfish, whose green fluorescence makes it useful as a reporter molecule

FIGURE 25.05 Transgenic Organisms with Green Fluorescent Protein

The gene for GFP has been integrated into the genome of animals, plants and fungi. After exposure to long wavelength UV the organisms emit green light.

A) Transgenic mice with GFP among normal mice from the same litter. The gfp gene was injected into fertilized egg cells to create these mice. GFP is produced in all cells and tissues except the hair. Credit: Eye of Science, Photo Researchers, Inc.

B) Phase contrast and

C) Fluorescence emission of germlings of the fungus Aspergillus nidulans. Original GFP was used to label the mitochondria and a red GFP variant (DsRed) for the nucleus. From: Toews et al., Current Genetics 45 (2004):383-389.

Gene fusions are used to monitor genes whose products are difficult to assay. Reporter genes are fused to the regulatory region of the target gene.

genes have products that are complicated or tedious to assay by direct measurement or may even be unknown. To avoid this, the original gene product is replaced by fusing its regulatory region to the structural region of a reporter gene. The target gene is cut between its regulatory region and coding region. The same is done with the reporter gene. Then the regulatory region of the gene under investigation is joined to the coding region of the reporter gene (Fig. 25.07). This hybrid structure is a gene fusion.

The gene fusion will be controlled the same way the original target gene was controlled, but instead of making the original gene product, it makes the enzyme belonging to the reporter gene. The cells carrying the fusion can be grown under an immense number of different conditions and assayed for the reporter enzyme. This approach, especially using lacZ and b-galactosidase, is widely used in surveying gene regulation (Fig. 25.08). In addition, gene fusions may be used to test for possible effects of regulatory genes. A mutation that inactivates a regulatory gene is introduced into the cell gene fusion Structure in which parts of two genes are joined together, in particular when the regulatory region of one gene is joined to the coding region of a reporter gene

Promoter

Promoter

Fusion protein

FIGURE 25.06 GFP for Protein Localization

GFP can be used to reveal where a protein is localized within the cell. The first step is to fuse the GFP gene in frame with all or part of the structural gene that encodes the protein of interest. The fused construct is then expressed in a host cell. The cells are excited with long wavelength UV light and visualized under the microscope. If the protein is normally located in the membrane, as in this example, the cell membrane will fluoresce green under the microscope.

Protein takes up correct

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