What is the DNA sequence of the luciferase gene?
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.
What does RNA polymerase do with the luciferase gene?
PCR is used to generate a DNA template with the desired elements. mRNA encoding a luciferase reporter gene is synthesized in vitro using a T7 RNA polymerase-based system. A Firefly luciferase (Fluc) mRNA is co-transfected with a Renilla luciferase (Rluc) mRNA into uninfected or VACV-infected cells.
What is the role of luciferase?
Luciferases are proteins with enzymatic activity that, in the presence of ATP, oxygen, and the appropriate substrate (typically luciferin), catalyze the oxidation of the substrate in a reaction that results in the emission of a photon.
What is the firefly gene?
The firefly luciferase gene (luc) from Photinus pyralis is a new tool for visualizing gene expression in plants. Luciferase (LUC) catalyzes the ATP-dependent oxidative decarboxylation of luciferin, a reaction that produces light.
Where is luciferin from?
Luciferin got its name from the Latin word lucifer (meaning “light-bearing”), which is also a source of the word that is sometimes used as a name of the devil.
When was luciferase first used?
1885
While DuBois discovered the reaction between luciferin and luciferase in 1885, it was not until the late 1940s when the luciferase protein was first extracted and purified firefly lanterns by Drs. Green and McElroy. Using this process, they isolated the enzyme and determined its conformational structure.
How is luciferase produced?
Luciferases can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate.
Where is luciferase produced?
insect fireflies
Luciferase is a light-producing enzyme naturally found in insect fireflies and in luminous marine and terrestrial microorganisms.
How do I activate luciferase?
A Luciferase Reporter Assay. When this protein activates transcription, the cell will produce luciferase enzyme. After the addition of a lysis buffer and a substrate, a luminometer quantifies the luciferase activity. If your protein activates the expression of the target gene, the amount of signal produced increases.
How is luciferase used to test microRNA effect?
The luciferase reporter gene assay has recently been adapted to test the effect of microRNAs. In this chapter, we use a previously identified miR-138 targeting site in the 3′-untranslated region (3′-UTR) of the RhoC mRNA as an example to describe a quick method for testing the interaction of microRNA and mRNA.
How is luciferase used in bioluminescence in Firefly?
Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly. The scientist produces a construct in which the regulatory region of a target gene is fused with the DNA coding sequence for luciferase (Figure 14.25). A separate DNA construct encodes the protein hypothesized to affect transcription.
How is luciferase control RNA used in translation reactions?
Luciferase Control RNA is a unique functional control for in vitro translation reactions. Luciferase Control RNA is an uncapped in vitro-transcribed RNA containing a 30-base poly (A) tail that produces functional luciferase when translated.
What can luciferase reporter gene assay be used for?
The most common application of luciferase reporter gene assay is to examine the regulation of transcriptional activities by promoters and transcription factors. Recently, this assay has also been adapted for testing the effect of miRNA-mediated, post-transcriptional regulation on target genes.