Keith V. Wood

Firefly luciferase gene: structure and expression in mammalian cells.

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5 end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5 sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5 deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants

The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants watered with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.

Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase

The gene for firefly luciferase (luc) can be used as a generalized genetic probe. A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here. Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction. Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay. This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.

Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns.

Poly (A)+ RNA was isolated from the lanterns of adult fireflies, Photinus pyralis. The Poly (A)+ RNA was translated in a cell-free translation mixture from rabbit reticulocytes and the synthesis of enzymatically active firefly luciferase was demonstrated. The translation products were immunoprecipitated with anti-luciferase and then subjected to SDS gel electrophoresis. It was shown that a newly synthesized polypeptide exhibited an identical electrophoretic mobility as the purified enzyme.

[1] Cloning firefly luciferase

Publisher Summary This chapter discusses determination of cloning firefly luciferase. Firefly luciferase catalyzes the ATP-dependent oxidative decarboxylation of luciferin (LH 2 ) resulting in the production of light. The most well characterized firefly luciferase is isolated from the common North American firefly Photinus pyralis . Isolating luciferase cDNA clones will allow the determination of the nucleotide sequence of this gene from which the amino acid sequence of the enzyme can be deduced. When the luciferase genes of other species of fireflies are cloned, comparison of their sequences can help pinpoint regions of the enzyme that are essential to its function and increase our understanding of bioluminescent reactions. The cloning of luciferase cDNA and the expression of the enzyme in an active form in Escherichia coli , and would provide an unlimited source of this enzyme. Cloned luciferase cDNA could be very useful for the monitoring of gene expression. The simple and sensitive assay for luciferase may allow the in vivo detection of this enzyme obviating the need to destroy the cells being monitored. As this is a single gene system, it is likely to be particularly useful in eukaryotic systems as an indicator of promoter activity in plant, animal, and lower eukaryote cells using both in vitro and in vivo tests.