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dcyphr | Green Fluorescent Protein as a Marker for Gene Expression.

Abstract

The jellyfish Aequorea victoria expresses green fluorescent protein (GFP). A complementary DNA can be expressed in prokaryotic or eukaryotic cells. Other substrates or cofactors are not necessary for this fluorescence. Thus, GFP can be used to monitor gene expression and protein localization in living organisms.

Introduction

In the jellyfish Aequorea victoria, the green fluorescent protein (GFP) emits a green light. When the photoprotein aequorin binds to calcium, GFP absorbs excitation energy from aequorin. GFP absorbs blue light and emits green light. The fluorescence is stable, and the fluorophore hardly breaks down. The fluorescence does not need any extra gene products. The fluorophore formation is not species-specific.

Aims

Chalfie et al. studies the properties of GFP. They examine whether some organisms can express GFP.

Methods

The researchers used PCR to amplify the fragment of GFP. They used the restriction enzymes NheI and EcoRI. They transformed organisms with ampicillin on the nutrient plates. Researchers used epifluorescence microscopy to see the GFP.

Results

Escherichia coli emitted green fluorescence with the expression of GFP. Researchers used an ultraviolet (UV) source to view the bacteria (Figure 1). The plates also contained isopropyl-β-D-thiogalactoside (IPTC), which induces the protein expression. GFP did not appear to have a toxic effect on cells. The excitation and emission spectra of the E. coli were identical to the spectra of the native jellyfish (Figure 2). Thus, the fluorescence does not need other jellyfish products. The researchers transformed GFP into the roundworm Caenorhabditis elegans (Figure 3). GFP was expressed in the neurons with the promoter for the mec-7 gene. The mec-7 gene codes for tubulin that is abundant in six touch receptor neurons. The GFP expression was like the fluorescence detected using the MEC-7 antibody. The touch neurons, including the terminal branches, grow throughout the embryonic stages. The neurons become functional in the fourth larval stage. The fluorescence of GFP in the neurons are consistent with previous data. Fluorescence is not detected in newly hatched or late first-stage larvae. Fluorescence is seen is some middle stage larvae. All the early fourth stage larvae and almost all the young adults emit fluorescence. GFP is stable when illuminated with 450 to 490 nm light in both E. coli and C. elegans. Some photobleaching occurs with lower wavelength lights.

Conclusion

GFP can be useful for monitoring gene expression and protein localization because its activity is not limited by other substrates. GFP does not seem to interfere with cell growth and function. So, GFP can be used as a marker for cell growth, such as in the C. elegans neuronal growth. GFP is small, so it will diffuse throughout the cytoplasm of cells. GFP fluorescence lasts even after killing cells with formaldehyde. Thus, GFP can be used to view fixed cells.