Douglas C. Prasher | |
---|---|
Born | August 1951 |
Nationality | American |
Alma mater | Ohio State University |
Scientific career | |
Fields | Molecular biology |
Douglas C. Prasher (born August 1951) is an American molecular biologist. He is known for his work to clone and sequence the genes for the photoprotein aequorin [1] and green fluorescent protein (GFP) [2] and for his proposal to use GFP as a tracer molecule. [3] He communicated his pioneering work to Martin Chalfie and Roger Y. Tsien, but by 1991 he was unable to obtain further research funding, and left academia. Eventually, he had to abandon science. Chalfie and Tsien were awarded the 2008 Nobel Prize in Chemistry for work that they publicly acknowledged was substantially based on Prasher's work; through their efforts and those of others, he returned to scientific research in June 2010.
Prasher received his Ph.D. in biochemistry from the Ohio State University in 1979. From 1979 to 1983, he worked in genetics and biochemistry research at the University of Georgia, where he identified the gene sequence for aequorin. [1] [4] He then joined the Biology Department of the Woods Hole Oceanographic Institution, Woods Hole, Massachusetts where he studied bioluminescence. In 1988, he received a two-year, $200,000 grant from the American Cancer Society to clone the gene for green fluorescent protein (GFP), the protein that gives the jellyfish its glow. Prasher succeeded in this project, and later shared his findings with Martin Chalfie and Roger Tsien after each scientist had communicated with him. [5] [6]
Reports that Prasher had difficulty in achieving fluorescence of GFP in other species in recombinant studies [7] are inaccurate, as Prasher had successfully worked with the Chalfie group to show recombinant expression in the bacterium E. coli and the nematode C. elegans , [8] and later in the plant Arabidopsis thaliana . [9] By the time Prasher's ACS funding ended, he had isolated a partial, but almost complete gfp cDNA, with 965 bases out of the 1,050 bases of the corresponding mRNA. [2] It would require construction of another cDNA library during the following (non-funded) year for Prasher to isolate a full-length cDNA clone, although this partial cDNA clone was subsequently used and found to be sufficient for successful heterologous expression in E. coli, C. elegans [8] and A. thaliana. [9] By this time, Prasher could not afford to devote limited resources to expression studies in E. coli. [10] It wasn't until the Nobel Prize announcement that it became clear how unfortunate this had been. Chalfie and Tsien went on to their successful expression studies. GFP has subsequently found application as a biochemical tracer in areas such as fluorescent studies of gene expression.
Prasher had applied to the National Institutes of Health for funding but had been turned down, and by the time he was undergoing review for promotion from assistant to untenured associate, he had decided to leave academia.[ citation needed ] Subsequently, Prasher worked for the Animal & Plant Health Inspection Service, U.S. Department of Agriculture at its Otis Plant Protection Center in Cape Cod, Massachusetts as a population geneticist, and was later transferred to the Plant Germplasm Quarantine & Biotechnology Laboratory in Beltsville, Maryland. After a mild heart attack, he went to work for NASA subcontractor AZ Technology in Huntsville, Alabama, working on an existing project to develop hand-held devices to monitor cabin environment and to perform human diagnostics during long-term spaceflight. [11] A year and a half later, he lost his job when NASA reorganized and canceled the project. [11] [12]
On 8 October 2008, the 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura, Chalfie, and Tsien for their work on GFP. [13] Prasher was not included among the Nobel laureates, as only three individuals can share in a single Nobel Prize. Chalfie said of Prasher's contribution: "(Prasher's) work was critical and essential for the work we did in our lab. They could've easily given the prize to Douglas and the other two and left me out." [14] Tsien also agreed that they couldn't have done it without Prasher and "Doug Prasher had a very important role." [15]
In an October 9, 2008 phone interview with National Public Radio (NPR) and October 14, 2008 TV interview with Inside Edition , Prasher reported that he was unable to find a job in science, his life savings had run out, and he was working as a courtesy shuttle bus driver for a Toyota dealership in Huntsville at $8.50 an hour. [6] [11] [14] [15] [16] In the NPR broadcast, one of his former colleagues called Prasher's current situation a "staggering waste of talent". [6] Prasher stated his wish to resume a career in science but not particularly with jellyfish. [12] He also expressed his pleasure at learning of the Nobel Prize awarded to Shimomura, Chalfie, and Tsien: "I'm really happy for them. I was really surprised that particular topic carried that much weight." [12]
Chalfie and Tsien invited Prasher and his wife, Virginia Eckenrode, to attend the Nobel Prize ceremony, as their guests and at their expense. [17] All three of the 2008 Chemistry laureates thanked Prasher in their speeches. [18]
In June 2010, Prasher was finally able to return to science, working for Streamline Automation in Huntsville until December 2011, and then from 2012 to 2015 in Tsien's lab at the University of California in San Diego. [19] [20]
The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.
Aequorea victoria, also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America.
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism express its fluorescence under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates. pGLO is made by Bio-Rad Laboratories.
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea victoria. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occurs together with the green fluorescent protein to produce green light by resonant energy transfer, while aequorin by itself generates blue light.
The GFP-cDNA project documents the localisation of proteins to subcellular compartments of the eukaryotic cell applying fluorescence microscopy. Experimental data are complemented with bioinformatic analyses and published online in a database. A search function allows the finding of proteins containing features or motifs of particular interest. The project is a collaboration of the research groups of Rainer Pepperkok at the European Molecular Biology Laboratory (EMBL) and Stefan Wiemann at the German Cancer Research Centre (DKFZ).
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish Aequorea victoria. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP is a useful tool in cell and molecular biology because the excitation and emission peaks of YFP are distinguishable from GFP which allows for the study of multiple processes/proteins within the same experiment.
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.
Roger Yonchien Tsien was an American biochemist. He was a professor of chemistry and biochemistry at the University of California, San Diego and was awarded the Nobel Prize in Chemistry in 2008 for his discovery and development of the green fluorescent protein, in collaboration with organic chemist Osamu Shimomura and neurobiologist Martin Chalfie. Tsien was also a pioneer of calcium imaging.
Aequorea forskalea is a species of hydrozoan in the family Aequoreidae. Discovered in 1810 by Péron and Lesueur, A. forskalea was initially found in coastal to offshore waters of the Mediterranean Sea. This species is commonly referred to as the many-ribbed jellyfish. The species is often mixed up with some other members of the genus due to some similarities including the capability of bioluminescence.
Renilla-luciferin 2-monooxygenase, Renilla luciferase, or RLuc, is a bioluminescent enzyme found in Renilla reniformis, belonging to a group of coelenterazine luciferases. Of this group of enzymes, the luciferase from Renilla reniformis has been the most extensively studied, and due to its bioluminescence requiring only molecular oxygen, has a wide range of applications, with uses as a reporter gene probe in cell culture, in vivo imaging, and various other areas of biological research. Recently, chimeras of RLuc have been developed and demonstrated to be the brightest luminescent proteins to date, and have proved effective in both noninvasive single-cell and whole body imaging.
Bioreporters are intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment. Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene. The promoter gene is turned on (transcribed) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. As a result, turning on the promoter gene also turns on the reporter gene, leading to the production of reporter proteins that output a detectable signal. The presence of a signal indicates that the bioreporter has sensed a particular agent in its environment.
Osamu Shimomura was a Japanese organic chemist and marine biologist, and professor emeritus at Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Boston University School of Medicine. He was awarded the Nobel Prize in Chemistry in 2008 for the discovery and development of green fluorescent protein (GFP) with two American scientists: Martin Chalfie of Columbia University and Roger Tsien of the University of California-San Diego.
Martin Lee Chalfie is an American scientist. He is University Professor at Columbia University. He shared the 2008 Nobel Prize in Chemistry along with Osamu Shimomura and Roger Y. Tsien "for the discovery and development of the green fluorescent protein, GFP". He holds a PhD in neurobiology from Harvard University.
Paul Brehm is a researcher at the Vollum Institute at Oregon Health and Science University. It was during a seminar by Brehm that Martin Chalfie became inspired to work on Green fluorescent protein for which Chalfie shared the Nobel Prize in Chemistry in 2008.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). As an RFP, mCherry was derived from DsRed of Discosoma sea anemones, unlike green fluorescent proteins (GFPs) which are often derived from Aequorea victoria jellyfish. Fluorescent proteins are used to tag components in cells so that they can be studied using fluorescence spectroscopy and fluorescence microscopy. mCherry absorbs light between 540 and 590 nm and emits light in the range of 550-650 nm. mCherry belongs to the group of fluorescent protein chromophores used as instruments to visualize genes and analyze their functions in experiments. Genome editing has been improved greatly through the precise insertion of these fluorescent protein tags into the genetic material of many diverse organisms. Most comparisons between the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that affect protein performance in cells or organisms. It is hard to perfectly simulate cellular environments in vitro, and the difference in environment could have an effect on the brightness and photostability.
Tulle Inger Hazelrigg is an American biologist who is Professor of Cell Biology at Columbia University. Her research considers the propagation and differentiation of germ cells. Hazelrigg was the first to attach green fluorescent protein to other proteins, which changed the way biological research could be conducted.