In , Demorest Davenport at the University of California at Santa Barbara in Santa Barbara, California, and Joseph Nicol at Plymouth Marine Laboratory in Plymouth, England, used photoelectric recording and histological methods to confirm Harvey's descriptions, and they identified the green fluorescent materials in the marginal canal of the umbrella.
In the same year, Osamu Shimomura became a research assistant at Nagoya University in Nagoya, Japan, and he crystallized the luciferin, a light-emitting compound found in the sea-firefly Vargula hilgendorfii. Shimomura published his results in One of Harvey's students, Frank H. Johnson, studied bioluminescence at Princeton University. Johnson followed Shimomura's work and invited him to work in the US, and in Shimomura received a Fulbright Travel Grant and started working with Johnson.
After catching about 10, jellyfish, Shimomura took the extracts of the jellyfish and preserved it in dry-ice to bring it back to Princeton in September of At Princeton, Shimomura and his colleagues started to purify the bioluminescent substance, and they found that it was a protein, which they called aequorin. When they purified aequorin, they also discovered traces of another protein, which showed green fluorescence.
Shimomura's team published the findings in "Exraction, Purification, and Properties of Aequorin" in The paper was about aequorin, but it also described a green protein, which exhibited green fluorescence under sunlight. John W. Hasting and James G. Morin, who later researched aequorin, termed the protein as green fluorescent protein in Shimomura focused on aequorin, purified the protein, crystallized it, and elucidated its underlying structure.
From to , many researchers studied various aspects of GFP, including the use of Nuclear Magnetic Resonance to study the amino acids of the protein, the use of X-rays to study its crystal, and the evolution of GFP. In the early s, molecular biologist Douglas Prasher, at the Marine Biology Laboratory, used GFP to design probes, a technology involving fragments of DNA to detect the presence of nucleotide sequences.
When he applied for funding from the US National Institute of Health in Bethesda, Maryland, the reviewer argued that Prasher's research lacked contributions to society. As Prasher could not secure funding to support his research any further, he left the Marine Biology Laboratory to work for the US Department of Agriculture in Massachusetts.
After Prasher's publication in , many scientists tried to transfer and express the Gfp gene in organisms other than jellyfish using DNA recombinant technology, and Martin Chalfie was the first who succeeded. Chalfie's team obtained the cDNA of the gene Gfp from Prasher and inserted only the coding sequence of Gfp gene first in the bacterium Escherichia Coli , and then in C. Chalfie and his team found that Gfp gene produced GFP without added enzymes or substrates in both organisms.
The detection of GFP needed only ultraviolet light. Thereafter, many biologists introduced GFP into their experiments to study gene expression. Coli in Many scientists tried to mutate the Gfp gene to make the resultant protein react to wider wavelengths and emanate different colors. Comfort A. The pigmentation of Molluscan shells.
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Is the flower fluorescence relevant in biocommunication? Fluorescent prey traps in carnivorous plants. Plant Biol ;— Download references. The authors thank Francesca Strano for lovely discussions on an early version of the project. We would like to thank Eva Jimenez-Guri for her critical reading of the manuscript and Christopher Bowkett for English proofreading. You can also search for this author in PubMed Google Scholar. All the authors edited the manuscript and approved the final version.
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Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. The construction of monomeric DsRed variants has proven to be a difficult task.
More than 30 amino acid alterations to the structure were required for the creation of the first-generation monomeric DsRed protein termed RFP1. However, this derivative exhibits significantly reduced fluorescence emission compared to the native protein and photobleaches very quickly, rendering it much less useful then monomeric green and yellow fluorescent proteins.
Mutagenesis research efforts, including novel techniques such as somatic hypermutation, are continuing in the search for yellow, orange, red, and deep red fluorescent protein variants that further reduce the tendency of these potentially efficacious biological probes to self-associate while simultaneously pushing emission maxima towards longer wavelengths.
Improved monomeric fluorescent proteins are being developed that have increased extinction coefficients, quantum yields, and photostability, although no single variant has yet been optimized by all criteria. In addition, the expression problems with obligate tetrameric red fluorescent proteins are being overcome by the efforts to generate monomeric variants, which have yielded derivatives that are more compatible with biological function.
Perhaps the most spectacular development on this front has been the introduction of a new harvest of fluorescent proteins derived from monomeric red fluorescent protein through directed mutagenesis targeting the Q66 and Y67 residues. Named for fruits that reflect colors similar to the fluorescence emission spectral profile see Table 1 and Figure 5 , this cadre of monomeric fluorescent proteins exhibits maxima at wavelengths ranging from to nanometers. Further extension of this class through iterative somatic hypermutation yielded fluorescent proteins with emission wavelengths up to nanometers.
These new proteins essentially fill the gap between the most red-shifted jellyfish fluorescent proteins such as Venus , and the coral reef red fluorescent proteins. Although several of these new fluorescent proteins lack the brightness and stability necessary for many imaging experiments, their existence is encouraging as it suggests the eventuality of bright, stable, monomeric fluorescent proteins across the entire visible spectrum.
One of the most interesting developments in fluorescent protein research has been the application of these probes as molecular or optical highlighters see Table 2 , which change color or emission intensity as the result of external photon stimulation or the passage of time. As an example, a single point mutation to the native jellyfish peptide creates a photoactivatable version of green fluorescent protein known as PA-GFP that enables photoconversion of the excitation peak from ultraviolet to blue by illumination with light in the nanometer range.
Unconverted PA-GFP has an excitation peak similar in profile to that of the wild type protein approximately to nanometers. After photoconversion, the excitation peak at nanometers increases approximately fold. This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell.
Illustrated in Figure 6 a is a transfected living mammalian cell containing PA-GFP in the cytoplasm being imaged with nanometer argon-ion laser excitation before Figure 6 a and after Figure 6 d photoconversion with a nanometer blue diode laser.
Other fluorescent proteins can also be employed as optical highlighters. Three-photon excitation at less than nanometers of DsRed fluorescent protein is capable of converting the normally red fluorescence to green. This effect is likely due to selective photobleaching of the red chromophores in DsRed, resulting in observable fluorescence from the green state.
The Timer variant of DsRed gradually turns from bright green nanometer emission to bright red nanometer emission over the course of several hours. The relative ratio of green to red fluorescence can then be used to gather temporal data for gene expression investigations.
A photoswitchable optical highlighter, termed PS-CFP , derived by mutagenesis of a green fluorescent protein variant, has been observed to transition from cyan to green fluorescence upon illumination at nanometers note photoconversion of the central cell in Figures 6 b and 6 e. Expressed as a monomer, this probe is potentially useful in photobleaching, photoconversion and photoactivation investigations.
Additional mutagenesis of this or related fluorescent proteins has the potential to yield more useful variants in this wavelength region. Optical highlighters have also been developed in fluorescent proteins cloned from coral and anemone species. Kaede , a fluorescent protein isolated from stony coral, photoconverts from green to red in the presence of ultraviolet light. Unlike PA-GFP, the conversion of fluorescence in Kaede occurs by absorption of light that is spectrally distinct from its illumination.
Unfortunately, this protein is an obligate tetramer, making it less suitable fur use as an epitope tag than PA-GFP. Another tetrameric stony coral Lobophyllia hemprichii fluorescent protein variant, termed EosFP see Table 2 , emits bright green fluorescence that changes to orange-red when illuminated with ultraviolet light at approximately nanometers.
In this case, the spectral shift is produced by a photo-induced modification involving a break in the peptide backbone adjacent to the chromophore. Further mutagenesis of the "wild type" EosFP protein yielded monomeric derivatives, which may be useful in constructing fusion proteins. A third non- Aequorea optical highlighter, the Kindling fluorescent protein KFP1 has been developed from a non-fluorescent chromoprotein isolated in Anemonia sulcata , and is now commercially available Evrogen.
Kindling fluorescent protein does not exhibit emission until illuminated with green light. Low-intensity light results in a transient red fluorescence that decays over a few minutes see the mitochondria in Figure 6 c. Illumination with blue light quenches the kindled fluorescence immediately, allowing tight control over fluorescent labeling. In contrast, high-intensity illumination results in irreversible kindling and allows for stable highlighting similar to PA-GFP Figure 6 f.
The ability to precisely control fluorescence is particular useful when tracking particle movement in a crowded environment. For example, this approach has been successfully used to track the fate of neural plate cells in developing Xenopus embryos and the movement of individual mitochondria in PC12 cells. As the development of optical highlighters continues, fluorescent proteins useful for optical marking should evolve towards brighter, monomeric variants that can be easily photoconverted and display a wide spectrum of emission colors.
Coupled with these advances, microscopes equipped to smoothly orchestrate between illumination modes for fluorescence observation and regional marking will become commonplace in cell biology laboratories.
Ultimately, these innovations have the potential to make significant achievements in the spatial and temporal dynamics of signal transduction systems. Fluorescent proteins are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These ubiquitous probes have been extremely useful as reporters for gene expression studies in cultured cells and tissues, as well as living animals.
In live cells, fluorescent proteins are most commonly employed to track the localization and dynamics of proteins, organelles, and other cellular compartments. A variety of techniques have been developed to construct fluorescent protein fusion products and enhance their expression in mammalian and other systems. The primary vehicles for introducing fluorescent protein chimeric gene sequences into cells are genetically engineered bacterial plasmids and viral vectors.
Fluorescent protein gene fusion products can be introduced into mammalian and other cells using the appropriate vector usually a plasmid or virus either transiently or stably. In transient, or temporary, gene transfer experiments often referred to as transient transfection , plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes, but can be expressed in the cytoplasm for a short period of time.
Expression of gene fusion products, easily monitored by the observation of fluorescence emission using a filter set compatible with the fluorescent protein, usually takes place over a period of several hours after transfection and continues for 72 to 96 hours after introduction of plasmid DNA into mammalian cells.
In many cases, the plasmid DNA can be incorporated into the genome in a permanent state to form stably transformed cell lines. The choice of transient or stable transfection depends upon the target objectives of the investigation. The basic plasmid vector configuration useful in fluorescent protein gene transfer experiments has several requisite components. The plasmid must contain prokaryotic nucleotide sequences coding for a bacterial replication origin for DNA and an antibiotic resistance gene.
These elements, often termed shuttle sequences, allow propagation and selection of the plasmid within a bacterial host to generate sufficient quantities of the vector for mammalian transfections. In addition, the plasmid must contain one or more eukaryotic genetic elements that control the initiation of messenger RNA transcription, a mammalian polyadenylation signal, an intron optional , and a gene for co-selection in mammalian cells.
Transcription elements are necessary for the mammalian host to express the gene fusion product of interest, and the selection gene is usually an antibiotic that bestows resistance to cells containing the plasmid.
These general features vary according to plasmid design, and many vectors have a wide spectrum of additional components suited for particular applications. Illustrated in Figure 7 is the restriction enzyme and genetic map of a commercially available BD Biosciences Clontech bacterial plasmid derivative containing the coding sequence for enhanced yellow fluorescent protein fused to the endoplasmic reticulum targeting sequence of calreticulin a resident protein.
Expression of this gene product in susceptible mammalian cells yields a chimeric peptide containing EYFP localized to the endoplasmic reticulum membrane network, designed specifically for fluorescent labeling of this organelle.
The host vector is a derivative of the pUC high copy number approximately plasmid containing the bacterial replication origin, which makes it suitable for reproduction in specialized E. The kanamycin antibiotic gene is readily expressed in bacteria and confers resistance to serve as a selectable marker. Additional features of the EYFP vector presented above are a human cytomegalovirus CMV promoter to drive gene expression in transfected human and other mammalian cell lines, and an f1 bacteriophage replication origin for single-stranded DNA production.
The vector backbone also contains a simian virus 40 SV40 replication origin, which is active in mammalian cells that express the SV40 T-antigen. Six unique restriction enzyme sites see Figure 7 are present on the plasmid backbone, which increases the versatility of this plasmid. Successful mammalian transfection experiments rely on the use of high quality plasmid or viral DNA vectors that are relatively free of bacterial endotoxins.
In the native state, circular plasmid DNA molecules exhibit a tertiary supercoiled conformation that twists the double helix around itself several times.
For many years, the method of choice for supercoiled plasmid and virus DNA purification was cesium chloride density gradient centrifugation in the presence of an intercalation agent such as ethidium bromide or propidium iodide.
This technique, which is expensive in terms of both equipment and materials, segregates the supercoiled plasmid DNA from linear chromosomal and nicked circular DNA according to buoyant density, enabling the collection of high purity plasmid DNA. Recently, simplified ion-exchange column chromatography methods commonly termed a mini-prep have largely supplanted the cumbersome and time-consuming centrifugation protocol to yield large quantities of endotoxin-free plasmid DNA in a relatively short period of time.
Specialized bacterial mutants, termed competent cells, have been developed for convenient and relatively cheap amplification of plasmid vectors. The bacteria contain a palette of mutations that render them particularly susceptible to plasmid replication, and have been chemically permeabilized for transfer of the DNA across the membrane and cell wall in a procedure known as transformation.
After transformation, the bacteria are grown to logarithmic phase in the presence of the selection antibiotic dictated by the plasmid. The bacterial culture is concentrated by centrifugation and disrupted by lysis with an alkaline detergent solution containing enzymes to degrade contaminating RNA. The lysate is then filtered and placed on the ion-exchange column. Alcohol isopropanol precipitation concentrates the eluted plasmid DNA, which is collected by centrifugation, washed, and redissolved in buffer.
The purified plasmid DNA is ready for duty in transfection experiments. Mammalian cells used for transfection must be in excellent physiological condition and growing in logarithmic phase during the procedure.
A wide spectrum of transfection reagents has been commercially developed to optimize uptake of plasmid DNA by cultured cells. These techniques range from simple calcium phosphate precipitation to sequestering the plasmid DNA in lipid vesicles that fuse to the cell membrane and deliver the contents to the cytoplasm as illustrated in Figure 8. Collectively termed lipofection , the lipid-based technology has met with widespread acceptance due to its effectiveness in a large number of popular cell lines, and it is now the method of choice for most transfection experiments.
Although transient transfections usually result in the loss of plasmid gene product over a relatively short period of time several days , stably transfected cell lines continue to produce the guest proteins on a continuous long-term basis ranging from months to years. Stable cell lines can be selected using antibiotic markers present in the plasmid backbone see Figure 7.
One of the most popular antibiotics for selection of stable transfectants in mammalian cell lines is the protein synthesis-inhibiting drug G, but the required dose varies widely according to each cell line. Other common antibiotics, including hydromycin-B and puromycin , have also been developed for stable cell selection, as have genetic markers.
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