Bioluminescence

In many biological studies it is essential to be able to translate into an observable and measurable signal the specific process under analysis. This is normally possible through the use of assay systems based on radioactivity, photon absorption or photon emission. Radioactivity and photon emission are less and less used nowadays, the first due to the restrictions and cares associated with the use of such hazardous materials, the second due to its low sensitivity.

Photon emission can occur by two different processes- either fluorescence or chemiluminescence. Both processes involve the emission of photons associated with transitions between energy states, but the way the excited state is generated varies. In fluorescence, the absorption of light (i.e. of photons) produces the excited state, while in chemiluminescence this is due to exothermic chemical reactions (see diagram- fluorescence is on the right, luminescence on the left).

Both of these processes have their advantages and disadvantages. Fluorescence (of which the most famous example is probably GFP) normally shows a much stronger signal, as the source of the excited signal are photons which can be introduced in the sample at very high rate. However, this jamming of photons tends to create a high background signal which influences the sensitivity of the assay. Chemiluminescence does create lower intensity signals, but as no photons need to be provided there is hardly any background signal. Whichever of the two systems is used will depend on the machinery required in each assay. If the efficiency of light collection is limited, then fluorescence is the best option, this being the reason why until recently fluorescence was the main assay system used. The development of more sensitive machinery, however, has made the background noise the most important problem to solve, therefore justifying the increased interest in chemiluminescence systems.

Bioluminescence is a form of chemiluminescence that naturally occurs in certain living organisms. The enzymes that catalyse this reaction are called luciferases and their substrates luciferins. Note, however, that these are very general terms as bioluminescence seems to have evolved several times independently. This is why the several existing luciferases have such different molecular structures. In fact, luciferases have been cloned from several different types of organisms, namely jellyfish (Aequorea), sea copepod (Gaussia princeps), corals (Tenilla), click beetle (Pyrophorus plagiophthalamus) and several bacterial species. The most successfully luciferase, however, is the widely used firefly luciferase, from the firefly Photinus pyralis. It is commonly used in its humanized variant (codons optimized for mammalian expression), requiring ATP and magnesium in the oxidation of its substrate luciferin, and yielding a yellow-green light with a maximum luminescence of 560 nm.

Firefly luciferase (FLuc) is normally used as a reporter gene, which may mean very different systems of reporting. The most obvious is, of course, the insertion of the luciferase gene in the same plasmid and under the same regulatory promoter as the gene of interest. In my current department (Gene Therapy department, NHLI), for example, firefly luciferase is the main reporter gene used in CFTR transfections. However, FLuc can report more than the expression levels of transfected genes. As it depends on ATP for its activity, it is ideal in assays of ATP concentration. It can also, for example, be an indicator of how well G-protein coupled receptors (GPCR) work. This is possible by using a cAMP response element (CRE) upstream of the luciferase gene. GPCR activation causes an increase in intracellular cAMP concentration. This increase leads to Protein Kinase A activation, which in turn phosphorylates CRE binding protein. The CRE binding protein will bind to the CRE upstream of the luciferase gene, leading to increased transcription. Overall, an increase in CPCR activity leads to increased luminescence signal. This type of report system, for example, is useful in the study of GPCR agonists. The complexity of the system, however, makes it prone to interference, which can lead to false positive results. To prevent this, a dual-reporter system can be used involving a second luciferase, Renilla Luciferase (RLuc), which is an internal control to detect aberrant data. A dual luciferase system can also be used to detect two different processes or expressed genes simultaneously (as FLuc and RLuc have different spectral maximuns, namely 560 and 480 nm).

As a reporter of transfected gene expression, FLuc shows a few limitations, namely the fact that it is an intracellular protein. This means that measuring of transfected gene expression requires, in in vitro cell culture the lysis of the transfected cells, and in in vivo studies the killing of the animal in order to access the transfected tissue. In patients under clinical trial, luciferase assays imply invasive obtainment of tissues (I am still trying to find out how this is done exactly). This is why the development of new luciferase, namely a secreted luciferase is important. Until now, around 4 types of secreted luciferases have been found, but the only one commercially available for cell supernatant assays is that produced by the copepod Gaussia princeps (see figure). Gaussia luciferase (GLuc) is naturally secreted from cells and used by this animal as a defence mechanism. As Gaussia princeps lives at a depth of between 350 and 1,000 m, the sudden production of light is a good distractive mechanism against dark-adapted predators. As a secreted luciferase, Gaussia is reported to give very good signals when medium supernatants are assayed. The important question right now (at least for me) seems to be if it can be assayed in something more… well, you will have to wait for my year-away talk next year for more information!

References

Fan F., Wood K. (2007). Bioluminescence Assays for High-Throughput screening, Assay and Drug Development Technologies, 5. pp 127-136

Markova S., Golz S., Frank L., Kalthof B., Vysotski E. (2004). Cloning and Expression of cDNA for a luciferase from the marine copepod Metridia longa, The Journal of Biological Chemistry, 5. pp 3212-3217

Roda A., Pasini P., Mirasoli M., Michelini E., Guardigli M. (2004). Biotechnological applications of bioluminescence and chemiluminescence, Trends in Biotechnology, 22. pp 295-303

Serganova I., Moroz E., Moroz M., Pillarsetty N., Blasberg R. (2006). Non-invasive molecular imaging and reporter genes, Central European Jornal of Biology, 1. pp 88-123

Tannous B., Kim D., Fernandez J., Weissleder R., Breakfield X. (2004). Codon-optimized Gaussia Luciferase cDNA for Mammalian Gene expression in culture and in vivo, Molecular Therapy, 11. pp 435-443

Wiles S., Ferguson K., Stefanidou M., Young D., Robertson B. (2005). Alternative Luciferase for monitoring bacterial cells under adverse conditions, Applied and Environmental Microbiology, 71. pp 3427-3432

Reaction or catalyst? Which started life?

If only the question was as simple as ‘which came first the chicken or the egg’ (answer: the egg). The question here is which came first - DNA or proteins. as we all know DNA stores the code to make proteins but proteins are needed to read this code and make more DNA. Obviously you can’t have one without the other. So the RNA world hypothesis was born. RNA can do both store information and catalyse reactions, perhaps even catalyse its own replication. I believe this is a beautiful theory. However beauty is only skin deep. There are real problems with this explanation of the origins of life apart from the obvious difficulties in testing it, which is true for any explanation of the origin of life. RNA is famous for being chemically unstable. It can break itself down quite easily (especially in alkaline conditions). Also where did the RNA come from? I don’t mean did it come from outer space (because radiation levels in space are so high RNA or cells could not have survived). I mean how the molecules were created on earth. Like it or not RNA is a complex molecule and no experiment trying to recreate the primordial soup has ever found RNA nucleotides. Or any really complex molecules, only the simplest of amino acids have been made.

This has lead to another explanation for the origins of life. Perhaps thinking of life as a bunch of replicators has blinded use to it. Living organisms can also be thought of as chemical factories. Genes and their products are simply there to control these reactions. Some believe it was chemical reactions that came before the proteins or RNA that catalyse the reaction. This is called the metabolism first theory. People who follow this theory have described what is needed for a chemical system to be the beginnings of life. 1) A boundary or form of membrane is needed to keep life and non-life away from each other. The 2^nd law of thermodynamics states the universe my decrease in order but life increases in order so inside the boundary entropy decreases but this generates heat that causes an increase in entropy outside it. 2) An energy source must have existed. Perhaps some sort of redox reaction to power the chemical reactions in the metabolism first model. Radiation may have been used. 3) The energy source must be linked to the other chemical reaction. For me this is difficult to see how this could happen without proteins there to help things along but perhaps if I knew more chemistry it would be clearer. We use ATP as our energy currency but how could redox reactions or radiation be linked to these ancient chemical reactions? 4) The chemical reactions must be able to change and evolve. If a cycle of reactions was created where A became B and B became C and C became D and D became A again we have something to expand upon. If we had a carbon input such as E we could take compounds off the cycle and expand it (see diagram). These reactions could be powered by a redox reaction of X to Y. Eventually complex molecules could be created 5) One final requirement for these reactions to have been the origins of life is need and that is to be able to replicate. It is hard to imagine how this is possible before a lipid membrane existed for it to divide into two. If possible this would have allowed for Darwinian evolution through the competition for recourses.

The RNA-first approach has some support for it. Minerals have been found that contain boron in ‘containers’ or ‘bowls’ in Death Valley which could help create the ribose sugar in RNA. If such pores with boron existed billions of years ago RNA could have been created. Laboratory experiments have shown some randomly generated RNA molecules can catalyse the addition of an ATP molecule to itself. This is tested by using an ATP molecule with a sulphur atom not an oxygen atom and using a column that pulls out the sulphur containing RNA molecules only. RNA can carryout many reactions such as making and breaking DNA and RNA links and amide bonds and even make links with sugars. So their is diversity in RNA’s ability to catalyse reactions but its limit appears to be speed. It is possible one reason proteins took over from RNA as life’s catalyst because proteins are faster as well as because protein is more stable. Metabolism first has the great weakness of not having much lab experiments to support it but only computer simulations.

I believe these two theories could work together. Perhaps it is only because I find the rna world aesthetically pleasing i want to save it but these reactions could eventually become so complex they make rna. Over time these built up and started to take control and then made proteins to do much of there job when dna then took over as the info store and rna was simply the messenger and helps out in only a few reactions today. This nicely explains why the formation of the peptide bond in the ribosome is still done by rna. We could go even further into theory and suggest there was a polymer before RNA that could act as catalyse and self replicator. PNA has been suggested. Instead of having the sugar-phosphate backbone like RNA and DNA it has peptides attached to bases forming a backbone. Sadly such a molecule does not exist in our cells today or leave fossils in the ground for us to examine so we cannot test if PNA was really the first molecules that lead to life. If PNA did exist it all became RNA and then DNA.

At the moment we have many ideas about what may have been involved with the start of life on earth but it is difficult to prove anything. What we can do is explore the potentials of these molecules or chemical systems. After all theories about the origins of life cannot be tested directly but the do make predictions and by testing these predictions we can hopefully learn a lot.

References

Albert et al. (2002) Molecular biology of the cell. 4th edition.

Shapiro (2007) A simpler origin for life. Scientific American 296: 24-31.