Incorporation of the Firefly Luciferase Gene into Plant Cells
- D. W. Ow
- S. H. Howell
Reporter, or indicator genes such as lacZ (beta-galactosidase) and CAT (chloramphenicol acetyltransferase), encode for enzymes that allow convenient and sensitive biochemical detection of gene activity. For this reason, they have been used extensively for analysis of gene regulation. Recently, luciferases, enzymes that catalyze light emission, have been successfully used to measure gene expression in bacteria, mammalian cells and plant cells (Engebrecht et al. 1985; Ow et al. 1986, 1987; de Wet et al. 1987). In principle, a luciferase gene (or gene operon) is the biological equivalent of an indicator light. By placing it under the control of a biological switch, such as when fused behind a gene promoter, light emission becomes a reflection of promoter activity. Not only does it indicate when and where the biological switch is turned on, but the intensity of light emission measures transcriptional strength. Aside from monitoring gene promoter activity, a biological indicator light can also be put to many other uses. For example, it can serve as a screen-able marker in the transfer of DNA into eukaryotic cells, or as a convenient tag for studies of transmission and population genetics. This chapter reviews our recent work on the development of the firefly luciferase gene as a reporter of gene expression in plants.
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Science: Firefly factor makes colourful genetic marker
BACTERIA such as Escherichia coli will glow in the dark if supplied
with the gene for the firefly enzyme luciferase and a supply of its natural
substrate, luciferin. Now Keith Wood and his colleagues at the University
of California in San Diego, who were first to isolate the firefly gene,
have created four different clones of E. coli that glow with four different
colours. The new clones could prove very useful for studying the way genes
work within living cells (Science, vol 244, p 700).
All bioluminescent insects use the same system of luciferase acting
on luciferin, but different species emit different colours. Luciferase isolated
from a particular species always glows with that species’ characteristic
colour, regardless of the source of the luciferin, so the wavelength of
the light is a property of the enzyme, not of the substrate. To examine
this phenomenon in more detail, Wood and his colleagues turned to the click
beetle, Pyrophorus plagiophthalamus.
Click beetles have two lights, one on the top of the head and one at
the front end of the abdomen, and are unusual because different individuals
emit a wide range of colours. The headlamp is greenish, but varies from
green at 548 nanometres to yellow-green at 565nm. The abdominal light has
a longer wavelength, but again varies from beetle to beetle, with colours
between green (547nm) and orange (594nm).
Wood collected the beetles in Jamaica and froze them in liquid nitrogen.
Back in the laboratory, the team isolated messenger RNA from the abdominal
light organ and prepared complementary DNA copies of the RNA. Putting the
DNA into the bacteria and washing luciferin over the colonies revealed a
total of 11 clones of E. coli that had taken up click beetle DNA and were
The clones emitted one of four wavelengths: green 546, yellow-green
560, yellow 578, and orange 593nm. (Beetles in the wild emit other wavelengths
within this range, so it is possible that there are other luciferase genes
besides these four.) Wood sequenced the luciferase genes from four different
colours and discovered that they all code for an enzyme 543 amino acids
long. The exact sequence, however, varied slightly, by between 1 and 5 per
cent among the four different genes. So it does not take many changes to
the amino acid sequence of the luciferase to make it glow with a different
Forty-eight per cent of luciferase from click beetles is the same as
firefly luciferase, but the differences are spread throughout the whole
enzyme. This suggests that there are no particular regions that have to
be maintained to enable the enzymes to work.
Molecular biologists have already used the gene for firefly luciferase
to tell them whether other genes in the cell are working. If they put the
firefly gene downstream of another gene, any light emitted will reveal that
the other gene is operational. With four different colours at their disposal,
they will be able to follow up to four different genes in one cell.