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Our bodies are continuously makingNobel Lecture by Osamu Shimomura. Discovery of Green Fluorescent Protein. [Электронный ресурс]. – Режимдоступа:http://nslc.wustl.edu/courses/Bio1810/readings/Nobel%20shimomura_lecture.pdf (последняя дата обращения05.12.2017 г.)222Там же223Tsien Roger Microscopy: Fluorescent Proteins (Roger Tsien) [Электронный ресурс]. – Режим доступа:https://www.youtube.com/watch?v=qK9aYnkIr3w. (последняя дата обращения 10.09.2017 г.)221273new hemoglobin. When new hemoglobin is required, protein production is activated. The gene isread and the protein is manufactured224.b) Douglas Prasher envisioned that it would be possible to use biomolecular techniques to insert theGFP gene at the end of the hemoglobin gene, right before the stop codon. When the cell needed tomake hemoglobin, it would go to the hemoglobin gene, use the information encoded in the gene tomake it, but instead of stopping when the hemoglobin was made, this cell would carry on makingGFP until it reached the stop codon at the end of the GFP gene.
As a result, the cell would produce ahemoglobin molecule with a GFP attached to it, see below225.c) Besides attaching GFP to a protein and making it a fluorescent tag, Prasher also thought that GFPcould potentially be a very useful reporter molecule.
In order to activate protein production, DNApromoters are used. These are sequences of DNA next to genes that contain the information aboutwhere and when the gene should be read and make the protein (in other words be expressed). If GFPis linked to a specific promoter then it will be expressed in place of the protein, showing where andwhen the gene of interest is switched on226.d) Prasher's GFP work was funded by the National Cancer Institute.
In his grant he suggested that itshould be possible to take the GFP gene out of the jellyfish cell and attach it to cells so that theywould be labeled with a fluorescent tag. Prasher managed to find the gene for GFP in AequoreaVictoria and was able to express it in bacteria. In 1992, he published a paper in Gene; it reported thecloning of GFP and the sequence of the 238 amino acids in GFP, shown below.
Sadly it was only atwo-year grant and the funding ran out before he could express the GFP clone he had produced in amanner that would result in GFP227.Ex. 23. Read the text, underline key words and say in short what happened with ascientist, Douglas Prasher.Bittersweet prizeThe rules of the Nobel prize limit it to a maximum of three people. And, like many other importantdiscoveries, more scientists contributed to the development of GFP. This year the man who clonedand sequenced the GFP gene, and who first imagined its potential, Douglas Prasher, missed out onthis most prestigious of accolades.
What’s much worse is that Prasher’s career was cut short. Hefailed to find funding for his research and dropped out of science entirely. He is now working as abus driver.’That Doug could not be recognised certainly lends a bittersweet element to the prize,’ Chalfie says.’I think it must have been an agonising decision for the Nobel committee, and they could easily havegiven the prize to Doug instead of me.’228д) Упражнения, целью которых является осуществление операции идеализации:Ex.
24. Read two extracts from the lecture by a Nobel Laureate, Osamu Shimomura andmake up the scemes of the experiments:a) I immediately went back to the lab and tested the luminescence of light organs at variouspHs. I clearly saw luminescence at pH 7, 6 and 5, but not at pH 4. I ground the light organs in a pH 4buffer, and then filtered the mixture. The cell-free filtrate was nearly dark.
But it regainedPrasher D. [Электронный ресурс]. – Режим доступа: https://www.conncoll.edu/ccacad/zimmer/GFPww/prasher.html225 Там же226 Там же227 Там же228 Lewis Brindley A glowing green Nobel. Chemistry World. 29 October 2008. [Электронный ресурс]. – Режимдоступа: https://www.chemistryworld.com/feature/a-glowing-green-nobel/3004465.article224274luminescence when it was neutralized with sodium bicarbonate. The experiment showed that I couldextract the luminescence substance, at least in principle229.b) We first cut the molecule of GFP into small pieces of peptide by enzymic digestion. Weisolated and purified the peptide that contained the chromophore, and then analyzed the structure ofthe chromophore. I was surprised when I measured the absorption spectrum of the peptide. Thespectrum was nearly identical to that of a compound that I had synthesized in my study of Cypridinaluciferin 20 years earlier.
Based on the spectral resemblance and some other properties, I couldquickly identify the chromophore structure of GFP (Shimomura, 1979) 230.Ex. 25. Read the extracts from the lecture by a Nobel Laureate, Osamu Shimomura,find new information and sum it up.a) Osamu Shimomura discovered the green fluorescent protein GFP from the jellyfish Aequoreaaequorea in 1961 as a byproduct of the Ca-sensitive photoprotein aequorin (Shimomura et al., 1962;Johnson et al., 1962), and identified its chromophore in 1979 (Shimomura, 1979). GFP was abeautiful protein but it remained useless for the next 30 years after the discovery231.b) Now GFP and its homologues are indispensable in biomedical research, due to the fact that theseproteins self-contain a fluorescent chromophore in their peptide chains and they can be expressed inliving bodies.
The identification of the fluorescent chromophore, however, depended on the GFP thathad been accumulated for many years in our study of aequorin. Without the study of aequorin, thechromophore of GFP would have remained unknown and the flourishing of fluorescent proteinswould not have occurred232.c) Dr.
Shimomura decided to investigate how the jellyfish species Aequorea Victoria wouldspontaneously produce its green glow. He took over 1 million individuals from his field site inWashington State to do so! Through his research of the jellyfish he discovered that there were twoproteins involved, one is called aequorin that produced a blue glow, and another is called greenfluorescent protein (GFP), which would glow green when it was mixed with calcium233.е) Упражнения, целью которых является осуществление операции абстракции:Ex. 26.
Read the extracts from the lecture by a Nobel Laureate, Osamu Shimomura anddraw a conclusion by your own words:a) But a big surprise came the next moment. When I threw the extract into a sink, the inside of thesink lit up with a bright blue flash. The overflow of an aquarium was flowing into the sink, so Ifigured out that seawater had caused the luminescence.
Because the composition of seawater isknown, I easily found out that Ca2+ activated the luminescence. The discovery of Ca2+ as theactivator suggested that the luminescence material could be extracted utilizing the Ca-chelatorEDTA, and we devised an extraction method of the luminescent substance234.Nobel Lecture by Osamu Shimomura. Discovery of Green Fluorescent Protein.
[Электронный ресурс]. – Режимдоступа: http://nslc.wustl.edu/courses/Bio1810/readings/Nobel%20shimomura_lecture.pdf (последняя дата обращения05.12.2017 г.)230 Там же231 Там же232 Там же233 Там же234Nobel Lecture by Osamu Shimomura. Discovery of Green Fluorescent Protein. [Электронный ресурс]. – Режимдоступа: http://nslc.wustl.edu/courses/Bio1810/readings/Nobel%20shimomura_lecture.pdf (последняя дата обращения05.12.2017 г.)229275b) After five years of hard work, we determined the chemical structure of AF- 350 in 1972(Shimomura and Johnson, 1972). The result was surprising.
The structure of AF-350 contained theskeleton of a 2-aminopyrazine (Fig. 12) that was previously found in the oxidation products ofCypridina luciferin, although the side chains are different. This finding suggested a close relationshipbetween the luminescence systems of Aequorea and Cypridina. Based on that information, we wereable to determine the structure of the chromophore of aequorin to be coelenterazine 235.Ex. 27.
Read the extracts from the article and sum up the aim of investigation.a) Every summer, Shimomura would sail out into the bay of Friday Harbor, San Juan Island,Washington, when the sea became thick with jellyfish. During 1961, he extracted the faintly glowingrings from around 10,000 of the mouse-sized jellyfish and squashed them through a filter. Afterisolating a protein from the glowing ’squeezate’, Shimomura was surprised to find that it emittedblue light, not green.
This, it turned out, was aequorin - a protein that produces blue light in thepresence of calcium ions236.b) Shimomura realised that something else was converting this blue light into the green glow.Eventually he isolated a second protein, which was able to absorb both blue light and ultraviolet lightand then fluoresce green. This protein was referred to as green fluorescent protein (GFP) - it was asimple name that stuck.
Shimomura was finally able to characterise the chemical structure of theGFP chromophore in 1979 and lay the groundwork for future discoveries237.Ex. 27. Key:a) Shimomura isolated aequorin - a protein that produces blue light in the presence of calcium ions.b) Shimomura isolated the green fluorescent protein, characterised the chemical structure of the GFPchromophore in 1979 and laid the cornerstone for future discoveries.ж) Упражнения, целью которых является осуществление операции компрессии:Ex.30.
Compress the following sentences, using some of the following symbols:+ to add smth= to be equal to, in parallel with≠ not to be equal to≈ approximately, around, overΔ difference in smthǁ parallel thoghts∩ to combine smth, combinedwith? controversial,problematicnotclear,↑ smth increases↓ smth decreases↓↓↓ smth decrease dramatically (drastically)← left→ rightΣ in total, to sum upƎ smth exists! smth is important!!! smth is very important1. The population of Aequorea in the Friday Harbor area drastically decreased after 1990,thus making it practically impossible to prepare any new samples of natural aequorin or GFP 238.2.’We needed a lot of material, so our schedule required us to catch around 50,000 jellyfishТам жеТам же237 Там же238 Nobel Lecture by Osamu Shimomura.
Discovery of Green Fluorescent Protein. [Электронный ресурс]. – Режимдоступа: http://nslc.wustl.edu/courses/Bio1810/readings/Nobel%20shimomura_lecture.pdf (последняя дата обращения05.12.2017 г.)235236276every summer,’ says Shimomura. ’So in total we probably collected over 850,000.’2393. Shimomura worked in the field of chemiluminescence, Chalfie investigated aspects ofnerve cell development and function, and Tsien worked in the development of chemical probes.
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