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Autor     Jörg Wiedenmann, Carsten Elke, Klaus-Dieter Spindler, and Werner Funke
Titel    Cracks in the b-can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria)
Zeitschrift    PNAS
Jahr    2000
Seiten    14091–14096
URL    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC18876/
Fußnoten    yes
Fragmente    5


Fragmente der Quelle:
[1.] Tim/Fragment 005 05 - Diskussion
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Mainly green and orange fluorescence from unidentified pigments was described for various no bioluminescent cnidarians (Catala, 1959; Kawaguti, 1944, 1966; Schlichter et al., 1986, 1988; Mazel, 1995, 1997; Doubilet, 1997). The green and orange fluorescent pigments were found in the color morphs of the Mediterranean sea anemone A. sulcata revealed quite similar properties as GFP from A. victoria. Many authors have found that the tentacles of this species exhibit a bright green and orange fluorescence and a nonfluorescent reddish color in the tips of its tentacles and the exclusive expression of GFPs in the ectoderm of the tentacles (Wiedenmann et al., 2000, Leutenegger et al., 2007) determined that AsGFP contributed ~ 5% to the total soluble cellular protein of non-bleached individuals. Mainly green and orange fluorescence from unidentified pigments was described for various nonbioluminescent cnidarians (13–20). The green and orange fluorescent pigments we found in the color morphs of the Mediterranean sea anemone Anemonia sulcata revealed quite similar properties as GFP from A. victoria. [...] Most recently, this view was confirmed by the cloning of six fluorescent proteins from nonbioluminescent Anthozoa homologous to the GFP from A. victoria (22).

Our interest was focused on the morph var. rufescens of A. sulcata (23). The tentacles of this morph exhibit a bright green and orange fluorescence and a nonfluorescent reddish color in the tips of its tentacles.


14. Kawaguti, S. (1944) Palao. Trop. Biol. Stn. Stud. 2, 617–674.

15. Kawaguti, S. (1966) Biol. J. Okayama Univ. 2, 11–21.

16. Schlichter, D., Fricke, H. W. & Weber, W. (1986) Mar. Biol. 91, 403–407.

17. Schlichter, D., Fricke, H. W. & Weber, W. (1988) Endocyt. C. Res. 5, 83–94.

18. Mazel, C. H. (1995) Mar. Ecol. Prog. Ser. 120, 185–191.

19. Mazel, C. H. (1997) Ocean Optics XIII SPIE 2963, 240–245.

20. Doubilet, P. (1997) Nat. Geogr. 192, 32–43.

22. 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.

23. Andres, A. (1883) in Accademia dei lincei, ed. Atti, R. (Rendicont, Rome), pp. 211–674.

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The source is mentioned, but the extent of the copying (which includes 7 references to the literature) doen't become clear to the reader.

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[2.] Tim/Fragment 008 21 - Diskussion
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Localization.

Because of their content of protein pigments, the tentacles of A. sulcata var. rufescens exhibit three hues under daylight conditions.

The upper side is green, the underside is orange, and the tips of tentacles show a vivid reddish color (fig. 3A).

The green and the orange pigments exhibit a bright fluorescence under irradiation with UV light at 366 nm (fig. 3B). The reddish protein of the tips is nonfluorescent. The protein pigments all are located in the ectoderm of the tentacles (fig. 3C).

Localization. Because of their content of protein pigments, the tentacles of A. sulcata var. rufescens exhibit three hues under daylight conditions. The upper side is green, the underside is orange, and the tips of tentacles show a vivid reddish color (Fig. 1A). The green and the orange pigments exhibit a bright fluorescence under irradiation with UV light at 366 nm (Fig. 1B). In a few specimens, two opposite spots at the mouth and the verrucae of the column also fluoresce in orange. The reddish protein of the tips is nonfluorescent. The protein pigments all are located in the ectoderm of the tentacles (Fig. 1C).
Anmerkungen

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[3.] Tim/Fragment 009 01 - Diskussion
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Fig. 3. Pigments of A. sulcata var. rufescens. (A) Localization of GFPs in the upper side, orange fluorescent protein in the underside, and reddish protein in the tips of tentacles under daylight (scale bar: 1 cm) and UV (366 nm) (B). (C) Kryo-section (20 mm) of a tentacle fixed in seawatery4% paraformaldehyde irradiated with UV (365 nm) shows the ectodermal localization of the fluorescent proteins. The orange fluorescent protein located in the underside of the tentacle shows a yellow-shifted emission. This shift most likely is induced by the fixation. The red fluorescence of the entoderm is produced by chlorophyll of zooxanthellae (scale bar: 0.5 mm). (Wiedenmann et al., 2000).

Spectral properties.

The spectral properties are summarized in table 1 (appendixes) (Wiedenmann et al., 2000). The green fluorescence of partially purified protein solutions is characterized by an excitation spectrum with two maxima at 480 nm and 511 nm and a shoulder around 400 nm. A region of high excitation is found in the UV spectrum.

The emission spectrum shows two maxima at 499 nm and 522 nm. The ratio between the emission maxima varies in dependence of the excitation wavelength. This points to the existence of two different forms of GFP in the tissue with excitation/emission maxima at 480 nm/499 nm and 511 nm/522 nm.

The orange fluorescence distinguishes itself by three excitation maxima at 278 nm, 337 nm, and 574 nm and a single emission peak at 595 nm.

In the visible region the maximum of absorption of the nonfluorescent red protein is at 562 nm. According to the nomenclature introduced by Matz et al. (1999) the fluorescent proteins from A. sulcata var. rufescens were named asFP499, asFP522, and asFP595. The nonfluorescent red protein from A. sulcata var. rufescens was named asCP562. In this context, CP stands for colored protein and the number identifies the major absorption maximum.

Spectral properties. The spectral properties are summarized in Table 1. The green fluorescence of partially purified protein solutions is characterized by an excitation spectrum with two maxima at 480 nm and 511 nm and a shoulder around 400 nm. A region of high excitation is found in the UV spectrum. The emission spectrum shows two maxima at 499 nm and 522 nm (Fig. 2A). The ratio between the emission maxima varies in dependence of the excitation wavelength. Such a variance also is found in samples derived from different specimens (data not shown). This points to the existence of two different forms of GFP in the tissue with excitation/emission maxima at 480 nm/499 nm and 511 nm/522 nm. The orange fluorescence distinguishes itself by three excitation maxima at 278 nm, 337 nm, and 574 nm and a single emission peak at 595 nm (Fig. 2A). In the visible region the maximum of absorption of the nonfluorescent red protein is at 562 nm (Fig. 2C).

According to the nomenclature introduced by Matz et al. (22) the fluorescent proteins from A. sulcata var. rufescens were named asFP499, asFP522, and asFP595. The nonfluorescent red protein from A. sulcata var. rufescens was named asCP562. In this context, CP stands for colored protein and the number identifies the major absorption maximum.

Tim 009a source.png

Fig. 1. Pigments of A. sulcata var. rufescens. (A) Localization of GFPs in the upper side, orange fluorescent protein in the underside, and reddish protein in the tips of tentacles under daylight (scale bar: 1 cm) and UV (366 nm) (B). (C) Kryo-section (20 mm) of a tentacle fixed in seawatery4% paraformaldehyde irradiated with UV (365 nm) shows the ectodermal localization of the fluorescent proteins. The orange fluorescent protein located in the underside of the tentacle shows a yellow-shifted emission. This shift most likely is induced by the fixation. The red fluorescence of the entoderm is produced by chlorophyll of zooxanthellae (scale bar: 0.5 mm).

Anmerkungen

Source is mentioned twice: once to reference the image and its caption and once for table 1. It does not become clear to the reader, however, that the entire page is taken from the source.

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(SleepyHollow02), Hindemith

[4.] Tim/Fragment 010 01 - Diskussion
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Molecular masses.

Wiedenmann et al. (2000) purified all proteins to near homogeneity. Both AsGFP 499/522 could not be separated by the purification steps. The molecular masses, determined by SDS-PAGE, are 26.2 kDA for the mixed fraction of AsGFP499/522 and 19.1 kDa for both asFP595 and asCP562. In gel filtration experiments under physiological conditions all protein pigments show apparent molecular masses of 66 kDa. For the GFPs this indicates a natural occurrence as dimers or trimers. Under denaturing conditions these oligomers can be partially split into monomers with apparent molecular masses of 23 kDa. Molecular masses for the AsGFP499 and asCP562 cloned were 25.4 kDa and 16.5 kDa.

Stability.

All colored proteins show a remarkable stability after treatment with heat, detergent, chaotrop, reducing agent, and pH extremes (Table 2. appendixes). Thermostability of asFP595 fluorescence is clearly increased compared with that of AsGFP499/522. In contrast, fluorescence of AsGFP499 is more stable when the solutions are exposed to 1% SDS, 8 M urea, and pH 11. Fluorescence of AsGFP499/522 and asFP595 is also stable when the proteins are treated with 4% paraformaldehyde (Wiedenmann et al., 2000).

Reversible denaturation.

Wiedenmann et al. (2000) heat-denatured the partially purified extracts of the proteins asFP595 and asCP562 in the presence of 2% SDS and 10% β-mercaptoethanol. After, proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane by semidry blotting. On the membrane, the band corresponding to asFP595 appears red under daylight conditions and exhibits orange fluorescence with an emission maximum at 595 nm under UV light. The fluorescence is stable and can even be detected after passage of the renatured protein through the membrane because of extended blotting. Renaturation with full restoration of fluorescence in the presence of β-mercaptoethanol has been described for heat-denatured GFP (Surpin & Ward, 1989). Surprisingly, asCP562, which is nonfluorescent in vivo, behaves in the same manner and shows identical fluorescence as renatured asFP595.

Molecular masses [...] All proteins were purified to near homogeneity. Both asFP499 and asFP522 could not be separated by the purification steps. The molecular masses, determined by

[page 14093]

SDS/PAGE are 26.2 kDA for the mixed fraction of asFP499/ asFP522 and 19.1 kDa for both asFP595 and asCP562 (Fig. 3 A and B). In gel filtration experiments under physiological conditions all protein pigments show apparent molecular masses of 66 kDa (Fig. 3.1 and 3.3). For the GFPs this indicates a natural occurrence as dimers or trimers (Fig. 3.3). Under denaturing conditions these oligomers can be partially split into monomers with apparent molecular masses of 23 kDa (Fig. 3.4). Stable dimers also are reported for GFP from Renilla and some other pennatularians (12, 27).

Stability. All colored proteins show a remarkable stability after treatment with heat, detergent, chaotrop, reducing agent, and pH extremes (Table 2). Thermostability of asFP595 fluorescence is clearly increased compared with that of asFP499/asFP522. In contrast, fluorescence of asFP499 is more stable when the solutions are exposed to 1% SDS, 8 M urea, and pH 11. Fluorescence of asFP499/asFP522 and asFP595 is also stable when the proteins are treated with 4% paraformaldehyde. Overall, the stability of the pigments from A. sulcata is comparable to that of the GFPs from Aequorea and Renilla (27–30).

Reversible denaturation. Partially purified extracts of the proteins asFP595 and asCP562 were heat-denatured in the presence of 2% SDS and 10% β-mercaptoethanol. Proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane by semidry blotting. On the membrane, the band corresponding to asFP595 appears red under daylight conditions and exhibits orange fluorescence with an emission maximum at 595 nm under UV light (Fig. 4 A and C). The fluorescence is stable and can even be detected after passage of the renatured protein through the membrane because of extended blotting. Renaturation with full restoration of fluorescence in the presence of β-mercaptoethanol has been described for heat-denatured GFP (31). Surprisingly, asCP562, which is nonfluorescent in vivo, behaves in the same manner and shows identical fluorescence as renatured asFP595 (Fig. 4 A and C).


31. Surpin, M. A. & Ward, W. W. (1989) Photochem. Photobiol. 49, 65.

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The authors (Wiedenmann et al., 2000) noted that the two green fluorescent pigments can be distinguished by their spectral properties in the tentacles of A. sulcata var. rufescens although they could not be separated. This indicates that the two proteins have identical molecular weights or that they reflect two states of the same protein. The second hypothesis is supported by the construction of a cDNA library. In fact they assumed the existence of a second stable conformation of AsGFP499, with red-shifted fluorescence, which can be formed only in the expressing cells of the sea anemone.

Also the orange fluorescent protein asFP595 and the no [sic] fluorescent red protein asCP562 most likely represent two states of the same protein. Both proteins have the same molecular weight. A striking argument for their relationship is the finding that after a denaturation-renaturation process both asFP595 and asCP562 exhibit orange fluorescence with an emission maximum that matches that of asFP595. Therefore, the primary structure of asCP562 also must carry all features necessary for orange fluorescence. The putative semi-β-can structure of asCP562 can give a possible explanation of these phenomena. For GFP fluorescence the β-can structure is essential. They proposed the formation of a β-can-like structure in a multimerization process. This type of β-can consists of at least two molecules of asCP562. Their hypothesis was supported by the finding that both asCP562 and asFP595 show apparent molecular masses of 66 kDa. This molecular mass corresponds exactly to a tetramer consisting of four molecules asCP562 (4*16.5 kDa) (Table 1). Under the conditions necessary for renaturation of asFP595 a second fluorescent band can be observed if the samples are denatured without the presence of β-mercaptoethanol (fig. 4B). The second band migrates at twice the molecular mass of asFP595. This result confirms the hypothesis that asFP595 consists of at least two asCP562 monomers.

Unlike GFP dimers, in asFP595 disulfide bonds seem to be involved in dimer interactions as a complete splitting is only possible under reducing conditions. However, to obtain the fluorescent state, it seems likely that a β-can-like structure is formed in which at least two molecules of asCP562 are linked with a disulfide bond.

Two green fluorescent pigments can be distinguished by their spectral properties in the tentacles of A. sulcata var. rufescens although they could not be separated (Fig. 2A). This indicates that the two proteins have identical molecular weights or that they reflect two states of the same protein. The second hypothesis is supported by the construction of a cDNA library. It resulted in the cloning of several hundred GFPs of the asFP499 type. As the content of asFP499 and asFP522 in the tissue of tentacles is comparably high, one would expect that this ratio should be detectable in the library as well. We therefore assume the existence of a second stable conformation of asFP499, with red-shifted fluorescence, which can be formed only in the expressing cells of the sea anemone.

Also the orange fluorescent protein asFP595 and the non-fluorescent red protein asCP562 most likely represent two states of the same protein. Both proteins have the same molecular weight. A striking argument for their relationship is the finding that after a denaturation-renaturation process both asFP595 and asCP562 exhibit orange fluorescence with an emission maximum that matches that of asFP595. Therefore, the primary structure of asCP562 also must carry all features necessary for orange fluorescence. The putative semi-β-can structure of asCP562 can give a possible explanation of these phenomena. For GFP fluorescence the β-can structure is essential. [...] We propose the formation of a β-can-like structure in a multimerization process. This type of β-can consists of at least two molecules of asCP562. Together they could form a β-can with at least 12 β-strands surrounding at least two stretches homologous to the fluorophore region of GFP. Our hypothesis is supported by the finding that both asCP562 and asFP595 show apparent molecular masses of 66 kDa (Fig. 3.1 and 3.2). This molecular mass corresponds exactly to a tetramer consisting of four molecules asCP562 (4 x 16.5 kDa) (Table 1). Under the conditions necessary for renaturation of asFP595 a second fluorescent band can be observed if the samples are denatured without the presence of β-mercaptoethanol (Fig. 4B) The second band migrates at twice the molecular mass of asFP595. This result confirms the hypothesis that asFP595 consists of at least two asCP562 monomers. Unlike GFP dimers, in asFP595 disulfide bonds seem to be involved in dimer interactions as a complete splitting is only possible under reducing conditions. [...] However, to obtain the fluorescent state, it seems likely that a β-can-like structure is formed in which at least two molecules of asCP562 are linked with a disulfide bond.

Anmerkungen

The source is given at the beginning of the page, but without clear indication that is meant to reference any more than maybe the first paragraph of the page.

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