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Typus
KomplettPlagiat
Bearbeiter
Hindemith, Graf Isolan
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Untersuchte Arbeit:
Seite: 24, Zeilen: 1ff (entire page)
Quelle: Embark 2004
Seite(n): 30, 31, 32, Zeilen: 30: 1ff; 31: 22ff; 32: 1ff
3.3 Xenopus laevis oocytes

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA.

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (112). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte. For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles.

The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl- conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV. Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (113). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered. Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of the sodium-phosphate transporter NaPi, mediated by the PKA and PKC pathway.


112. Costa,PF, Emilio,MG, Fernandes,PL, Ferreira,HG, Ferreira,KG: Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp. J.Physiol 413:199-211, 1989

113. Tzounopoulos,T, Maylie,J, Adelman,JP: Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes. Biophys.J. 69:904-908, 1995

1.8 Xenopus laevis oocytes and electrophysiological recording

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA (Gurdon et al., 1971).

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (Dumont, 1972). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system in which to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte (reviewed in Wagner et al., 2000).

For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. [...] The main ion conductance in Xenopus oocytes is a Ca2+ -dependent Cl-conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, (Dascal, 1987).

[page 31]

Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (Tzounopoulos et al., 1995). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered (Wagner et al., 2000).

[page 32]

Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA and PKC pathway.

Anmerkungen

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Parts of this documented passage can also be found in an earlier source: Sj/Dublette/Fragment 024 01

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(Hindemith) LieschenMueller

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