HOME
Table of Contents (all articles on this disk)
This Article: FIREFLY GENES LIGHT THE WAY
For this article:
      Educational Goals and Objectives  Reference Abstracts  Test Questions  References

FIREFLY GENES LIGHT THE WAY

Imagine if you could attach a tiny transmitter to genes, or even to molecules that make up genes, so that you could tell when the gene was involved in a biochemical reaction. Instead of conducting laborious assays, you would only need to receive the signal to know that the reaction or result you sought was, in fact, occurring. Actually, a technique much like this is being used in molecular biology labs throughout the world.

Luciferase is a naturally occurring enzyme that gives off light when it participates in the reaction it was designed to participate in. Sound unlikely? Then watch a lightening bug in action. Luciferase is the key to its bioluminescence. What is bioluminescence? Luciferase, and other enzymes like it, are known as oxygenases1. They require a substrate (luciferin) in the presence of oxygen to transform a molecule by raising its electrons to higher energy ("excited") states. When those electrons eventually fall back down to their normal ("rest") state, they give off light. Lightening bugs, also called fireflies (but which are actually beetles; Photinus pyralis) hold no monopoly on bioluminescence. The phenomenon can be found in microscopic bacteria, fungi, dinoflagellates and marine animals such as arthropods (such as crabs or shrimp), mollusks (such as clams), echinoderms (starfish) and cordates (a primitive worm-like animal). In fact, the flashes of light that can be seen at night as waves crash upon the beach are caused by thousands of tiny dinoflagellates. On dry land, various annelid worms and insects luminesce.

Because of the large quantities of luciferase required for research, it would be impractical to try to harvest it from a limited supply of beetles. Instead, the luciferase gene has been inserted into various strains of bacteria and algae where it can then grown and harvested in enormous quantities. This is referred to as recombinant luciferase. Promega Corp., of Madison WI, holds the exclusive license to produce recombinant luciferase.

In the laboratory, luciferase is commonly used as a reporter gene to analyze the actions of other genes, proteins or peptides that function as promoters and enhancers. Although luciferin is poorly taken up by cells, a synthetic form of luciferase known as caged luciferase, readily crosses cell membranes and can be used to measure delicate intracellular processes such as ATP conversion or internal gene expression. Japanese researchers have created transgenic trout2 by implanting firefly genes ("luciferase" genes) into fertilized fish roe (eggs). Proof of the successful gene transfer was the fact that the fish fry emitted light. Corn plants, bioengineered to contain genes for an insecticide, also were made to "glow" when luciferase genes were attached as "reporter" to the foreign insecticide genes, this providing the researcher with a clear signal that the gene transfer worked.

Instrumentation: the more sensitive the light detection equipment used, the more information will be retrieved from the reaction being studied. Light detectors used are microplate readers, standard luminometers, scintillation counters and video cameras. A luciferase flash decays quickly; about half of the light is emitted within the first 15 seconds. One extremely sensitive system, designed to be used with luciferase, contains a video component capable of capturing the act of transcription in individual cells. This system has been used to study transcription of circadian clock genes in transgenic plants and living fruit flies (Drosphilia). Circadian rhythms, commonly known as biorhythms, are controlled by clock genes. In blue-green algae, biological clock activity can be tracked visually by linking firefly luciferase to promoter genes that control light/dark cycles. By linking the luciferase gene to a promoter gene that is only active during photosynthesis (which normally requires sunlight) and recording when the organism lights up, you have a visual record of the activity of the cells biological clock. From such studies it was learned that the circadian clock controls the actions of hundreds of genes. Circadian clocks are found in a variety of mammals (e.g. mice and hamsters) as well as insects, algae and higher plants. Experiments using luciferase have provided a method of studying time and positional aspects of gene regulation in living, intact organisms. From such data we are beginning to draw conclusions as to how the human biological clock works. Some day this understanding may permit us to design therapeutics to address sleep disorders and the problems that afflict jet lagged travelers and night workers.

Luciferase is also being used in the field of medicine for diagnostic purposes. In the case of HIV (human immunodeficiency virus) infections, the Tat protein activates transcription of HIV by acting on the LTR (long terminal repeat) region. When the luciferase gene was fused to LTR in a carefully designed cell line, a flash of light is given off whenever HIV begins its attack, signalling the start of an infection. In another study, luciferase has been used to detect the presence of drug-resistant strains of TB. A culture of TB organisms was first exposed to an anti-TB drug, and then to a phage (an tiny bacteria-like organism that infects bacteria) carrying the luciferase gene. If the TB strain was drug sensitive, then there would be no light. A drug-resistant strain would give off light, thereby quickly alerting the physician that a change in therapy is required. Normal procedures for measuring the sensitivity of a strain of TB to drugs takes several weeks. The procedure using luciferin gave results in several days.

Another important use of luciferase is in the area of quality assurance ("QA") testing for industrial microbiologists. It is estimated that 2.5 billion microbial tests were done world wide in 1994 and that this number will increase to 3.2 billion by the year 2000. What is needed is a fast, effective and low cost system to detect low-level biological contamination. Such a kit is currently being marketed. Taking advantage of the fact that all living organisms (and only living organisms) contain quantities of the high energy molecule ATP, the kit combines ATP reagents with luciferase, and uses the bioluminescent property of luciferase to visually indicate the presence of ATP, signalling biological contamination. Kits are currently available for personal care product, foods (tests for milk quality and milk and fruit juice sterility), and water. The main QA application for this type of ATP bioluminescence is the surface testing in food processing plants to determine the existence of films of microbes that are capable of causing corrosion of pipework or contamination of products. The bioluminescence test, which can be read in ten minutes, is cost-effective and convenient compared with the conventional process involving sample collection and culture, which could take up to five days.

In the field of environmental testing, bioluminescence has also found a place. In one such system, using freeze-dried bioluminescent marine bacteria (Photobacterium phosphoreum), bacterial metabolism is impaired if contaminants are present, resulting in a dimming of bioluminescence. This system can be used for toxicity testing in research and development (R & D) product development, process control, raw materials testing, biomass protection in waste treatment plants, effluent monitoring, and analyzing sediment, hazardous waste and runoff water quality. Designed to be completed in under thirty minutes, some states now require the test to be performed.

Further refinements of luciferase-based test kits are expected. By inducing mutations in the luciferase gene, variations in response to temperatures has been obtained. And, four luciferase isozymes (related enzymes) have been obtained by cloning of click beetle (Photinus plagiophthnalmus) luciferase. The four colors are green, yellow-green, yellow, and orange. This development should provide the ability to monitor at least four processes simultaneously.


1 Aequorin (from jelly fish) is another naturally occurring bioluminescent material that is also being produced commercially through recombinant DNA technology.
2 Fish containing genes from another species; animal or plant.



HOME
Table of Contents (all articles on this disk)
This Article: FIREFLY GENES LIGHT THE WAY
For this article:
      Educational Goals and Objectives  Reference Abstracts  Test Questions  References