We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

/pdf/gus-fusions-beta-glucuronidase-as-a-sensitive-and-versatile-4b3n5r6gfw.pdf

GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.

https://www.pnas.org/content/pnas/89/12/5547.full.pdf

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

DeJi~eitio, r Ge~e lrlt.~irm Much of tile attention and interest in modern molecular biology is fi~cussed on the regulation of gene expression. Factors influencing or mediating such regulation are often best studied using gene Alsions. Gene fusions can be defined its DNA constructions (perfi3rmed ill vitro or i~e Hvo) that result in the coding sequences from one gene (r@o,ter) being transcribed and/or translated under the direction of the controlling sequences of another gene (cmltrr Gene fusions can be of two general types, with many wtriatiuns within types. Transcriptional fusions are defined as fusions in which all protein coding sequences are derived from the reporter, with none from the cmm,//e~. Thus, although the m R N A produced may consist of sequences from both control/o and re/;o~ter, the protein synthesized will be encoded only by the reporter. Translational fusions, in contrast, are defined as those in which the polypeptide produced is the result of coding information provided by both copraoiler and reporter.

Assaying chimeric genes in plants: The GUS gene fusion system

RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

https://science.sciencemag.org/content/sci/247/4949/1465.full.pdf

Direct gene transfer into mouse muscle in vivo.

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.

Firefly luciferase gene: structure and expression in mammalian cells.

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.

https://science.sciencemag.org/content/sci/234/4778/856.full.pdf

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

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.

https://www.pnas.org/content/pnas/82/23/7870.full.pdf

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

Publisher Summary This chapter discusses purification and properties of firefly luciferase. Firefly luciferase in the presence of luciferin (d-LH2), adenosine triphosphate (ATP)-Mg, and molecular oxygen catalyzes the production of light. The color of the emitted light shows a peak at around 560 nm. This emission can be affected by temperature, pH, and metal ions. At low pH or in the presence of lead, mercury, or other heavy metals, the emission peak is shifted to red, showing an emission around 615 nm. Thus, when this system is used for the assay of ATP, it is critical to run internal controls to make sure that the light color has not been shifted. Because of the greater sensitivity of phototubes in blue in contrast to the red, a shift to red will give an apparent lower level of ATP. A dark-adapted eye can readily discern the difference in color between the control and one emitting at longer wavelengths. In crude firefly extracts and some luciferin preparations, dehydroluciferin is usually present. It is a potent inhibitor of the light reaction. The chapter describes the method of collecting and drying fireflies in order to prepare an appropriate acetone powder, the preparations and properties of a crude and partially purified luciferase and finally the preparation of crystalline firefly luciferase.

Marlene DeLuca

Papers

Firefly luciferase gene: structure and expression in mammalian cells.

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

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

[1] Purification and properties of firefly luciferase

Kinetics of the firefly luciferase catalyzed reactions