von Dr. Tiziana Masullo
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| [1.] Tim/Fragment 025 01 - Diskussion Zuletzt bearbeitet: 2014-10-25 14:31:47 Hindemith | BauernOpfer, Fragment, Gesichtet, Gurskaya 2003, SMWFragment, Schutzlevel sysop, Tim |
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| Untersuchte Arbeit: Seite: 25, Zeilen: 1 ff. (entire page) |
Quelle: Gurskaya 2003 Seite(n): 404, Zeilen: l.col. last lines |
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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 |
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). |
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Letzte Bearbeitung dieser Seite: durch Benutzer:Hindemith, Zeitstempel: 20141025143230