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Angaben zur Quelle [Bearbeiten]

Autor     Nadya G. GURSKAYA, Arkady F. FRADKOV, Natalia I. POUNKOVA, Dmitry B. STAROVEROV, Maria E. BULINA, Yurii G. YANUSHEVICH, Yulii A. LABAS, Sergey LUKYANOV and Konstantin A. LUKYANOV
Titel    A colourless green fluorescent protein homologue from the non-fluorescent

hydromedusa Aequorea coerulescens and its fluorescent mutants

Zeitschrift    Biochem. J.
Jahr    2003
Seiten    403–408
URL    http://www.inbi.ras.ru/competition/2003/articles/labas1.pdf

Literaturverz.   

yes
Fußnoten    yes
Fragmente    2


Fragmente der Quelle:
[1.] Tim/Fragment 001 02 - Diskussion
Zuletzt bearbeitet: 2014-12-05 06:47:39 Graf Isolan
BauernOpfer, Fragment, Gesichtet, Gurskaya 2003, SMWFragment, Schutzlevel sysop, Tim

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Green Fluorescent Protein (GFP) from the hydromedusa Aequorea victoria is intensively used in biomedical sciences (Tsien, 1998).

In nature, GFP is a component of the A. victoria bioluminescent system. Like many other marine organisms, this jellyfish can produce bright flashes in response to external stimulation. GFP is a secondary emitter that transforms blue light (460 nm), emitted by the Ca2+- dependent photoprotein aequorin, into green light (508 nm) (Johnson et al., 1962). Although GFPs similar to Aequorea GFP are found in other bioluminescent coelenterates (Chalfie, 1995), the biological significance of blue light transformation is not clear, particularly since the majority of bioluminescent animals do not possess this mechanism. The discovery of GFP-like proteins in the non-bioluminescent Anthozoa species indicates these proteins are not necessarily linked to bioluminescence (Matz et al., 1999). In fact, these organisms can produce bright flashes through GFP, as an electromagnetic radiation source with specific wavelength in response to external stimulation (Gurskaya et al., 2003).


Tsien, R., 1998. Annu. Rev. Biochem. 67: 509-544.

Johnson, F. H., Shimomura, O., Saiga, Y., Gershman, L. C., Reynolds, G. T., and Waters, J. R., 1962. J. Cell. Comp. Physiol. 60, 85-104

Chalfie, M., 1995. Green fluorescent protein. Photochem. Photobiol. 62, 651–656

Matz M.V., Fradkov A.F., Labas Y.A., Savitsky A.P., Zaraisky A.G., Markelov M.L., Lukyanov S.A., 1999. Nat. Biotechnol., 17:969-973.

Green fluorescent protein (GFP) from the hydromedusa Aequorea victoria is intensively used in biomedical sciences [1]. In nature, GFP is a component of the A. victoria bioluminescent system. Like many other marine organisms, this jellyfish can produce bright flashes in response to external stimulation. GFP is a secondary emitter that transforms blue light (460 nm), emitted by the Ca2+- dependent photoprotein aequorin, into green light (508 nm) [2]. Although GFPs similar to Aequorea GFP are found in other bioluminescent coelenterates (for review see [3]), the biological significance of blue light transformation is not clear, particularly since the majority of bioluminescent animals do not possess this mechanism. The discovery of GFP-like proteins in the non-bioluminescent Anthozoa species indicates these proteins are not necessarily linked to bioluminescence [4].

1 Tsien, R. Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544

2 Johnson, F. H., Shimomura, O., Saiga, Y., Gershman, L. C., Reynolds, G. T. and Waters, J. R. (1962) Quantum efficiency of Cypridina luminescence, with a note on that of Aequorea. J. Cell. Comp. Physiol. 60, 85–104

3 Chalfie, M. (1995) Green fluorescent protein. Photochem. Photobiol. 62, 651–656

4 Matz,M.V.,Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L. and Lukyanov, S. A. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973

Anmerkungen

The source is given at the end of the paragraph.

It is remarkable that the only sentence the reader sensibly would expect to come from the source cannot be found in it, while the rest of the documented passage is taken from it verbatim .

Sichter
(SleepyHollow02), Hindemith

[2.] Tim/Fragment 025 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 14:31:47 Hindemith
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[Although A. coerulecens and A. victoria look similar, some of their] features are clearly different. The most obvious difference is that A. victoria carries only 1 tentacle in the radial channel, whereas A. coerulescens possesses 3–6 tentacles between each pair of adjacent radial channels.

In contrast to A. victoria, A. coerulescens medusae displayed blue, not green, luminescence. Gurskaya et al. (2003) described for the wild type acGFP no detectable fluorescence under either UV light or using a fluorescence microscope. However, using monoclonal antibodies against GFP they found that a protein extract from A. coerulescens contained a GFP-like protein that had the 92% identity at the amino acid level with AvGFP. It was detected in the umbrella border.

All known key GFP residues are conserved in AcGFP, including the chromophore forming Ser65, Tyr66 and Gly67 residues, the evolutionarily invariant Arg96 and Glu222, and His148, Phe165, Ile167 and Thr203 which are all spatially close to the chromophore.

Gurskaya et al. (2003) used a random mutagenesis to generate fluorescent mutants of wt GFP of this species. The substituting E222G appeared to be the key event in creating a fluorescent form. Moreover five amino-acid substitutions compared with the wild-type, specifically V11I, F64L, K101E, T206A and E222G determined a very bright mutant.

Although A. coerulecens and A. victoria (other names for the latter are A. forscalea and A. aequorea) look similar, some of their features are clearly different. The most obvious difference is that A. victoria carries only 1 tentacle per radial channel, whereas A. coerulescens possesses 4–6 tentacles between each pair of adjacent radial channels [15].

In contrast to A. victoria, A. coerulescens medusae displayed blue, not green, luminescence. We cloned the aequorin-like photoprotein apparently responsible for the bioluminescence observed in A. coerulescens specimens (GenBank® accession number AY236998). This molecule shared 84% identity at the amino acid level with A. victoria aequorin, and had an emission maximum at 455 nm. No detectable fluorescence was observed in A. coerulescens under either UV light or using a fluorescence microscope. However, using monoclonal antibodies against GFP we found that a protein extract from A. coerulescens contained a GFP-like protein (see Figure 3A).

[...]

[...] All known key GFP residues are conserved in acGFPL, including the chromophore-forming Ser65, Tyr66 and Gly67 residues, the evolutionarily invariant Arg96 and Glu222, and His148, Phe165, Ile167 and Thr203 which are all spatially close to the chromophore. [...]

[...]

Random mutagenesis was used to improve protein folding and to generate fluorescent mutants of acGFPL. After the first round of mutagenesis several green fluorescent colonies of different brightness were found. The three brightest clones (named G1, Z1 and Z2) were further characterized and the mutants were found to contain the same substitution, E222G. Additional V11I/K101E and N19D mutations were found in G1 and Z2 respectively. Thus substituting Glu222 appeared to be the key event in creating a fluorescent form of acGFPL.

[...]

Two additional rounds of random mutagenesis on the G1 clone (only G1 was able to mature at 37 ◦C, whereas Z1 and Z2 required incubation at low temperature to develop fluorescence) generated a very bright mutant, named aceGFP, which matured fast at 37 ◦C. This mutant contained five amino-acid substitutions compared with the wild-type acGFPL, specifically V11I, F64L, K101E, T206A and E222G (Figure 1).


15 Kramp, P. L. (1968) The hydromedusae of the Pacific and Indian Oceans. Dana Rept. 72, 201–202

Anmerkungen

The source is given and from the context it becomes clear that some results of Gurskaya et al. (2003) are reported.

However, it is not clear that this reporting is done using the words of Gurskaya et al. (2003) and there are also passages taken from Gurskaya et al. (2003) that the reader would not attribute to Gurskaya et al. (2003) (first and third paragraph).

Sichter
(SleepyHollow02), Hindemith

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