Molekularbiologische Untersuchungen über ein neues Cytoskelett-verwandtes Protein

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2001
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Steppan, Sonja
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Molecular Characterization of a Novel Cytoskeleton-related Protein
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Abstract
This work describes the initial molecular characterization of a novel protein, p170, from the african claw frog Xenopus laevis. The cDNA encoding p170 was identified in a random manner when an expression library from X.laevis ovary was screened with an affinity purified antibody originally designed to recognize the replication factor cdc6. The original p170 cDNA fragment was completed by RACE ('rapid amplification of cDNA ends'), and the full length cDNA (4441bp) was found to encode a protein of 1335 aa related to certain cytoskeleton associated proteins. p170 has an extraordinary amino acid composition with a high content of leucine, glutamic acid and glutamine, but low content of proline and glycine. Secondary structure analysis of the p170 protein reveals that the protein has a high potential to form coiled coils. To allow a biochemical characterization of the p170 protein, two fragments were subcloned and expressed in E.coli to generate p170-specific antibodies by the immunization of rabbits. With these antibodies, immunofluorescence microscopy and biochemical cell fractionation experiments show that the p170 protein is preferentially localized in the cytoplasm of cultured Xenopus cells. The p170 protein is highly expressed in eggs and the p170 amount decreases during early development. Computer searches have shown that p170 is conserved in vertebrates, and homologues in human, mouse, cow, pig and zebrafish could be identified by expressed sequence tags (ESTs). On the basis of published sequence information the genomic organization of the complete human and mouse p170 genes could be determined The human p170 gene is located on Chromosome 1 and consists of 25 exons over a length of at least 107 kb. Further experiments are in progress to test the function of the p170 protein in cytoskeleton assemby.
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In dieser Dissertation wird die molekulargenetische und biochemische Charakterisierung eines hier neu entdeckten Proteins in Xenopus laevis dargestellt. Dieses Protein ist zu Cytoskelett-assoziatierten Proteinen verwandt und wird vorläufig als p170 bezeichnet.
Die p170 cDNA wurde aus einer X.l. Ovarien Expressionsbibliothek isoliert und mit der RACE Technik vervollständigt. Die gesamte p170 cDNA beträgt 4441 bp und codiert ein Protein mit 1335 Aminosäuren. p170 verfügt über eine außergewöhnliche Aminosäurezusammensetzung mit einem hohen Anteil an Leucin, Glutaminsäure und Glutamin und einem geringen Anteil an Prolin und Glycin. Sekundärstrukturanalysen sagen ein alpha-helicales, hydrophiles Protein mit großer Neigung zur Ausbildung von Coiled-Coil Domänen voraus.
Zur Charakterisierung von p170 wurden Teilfragment bakteriell exprimiert und zur Herstellung von Antikörpern verwendet. Lokalisationsstudien durch Immunfluoreszenz-Mikroskopie und Zellfraktionierungen haben gezeigt, daß p170 im Cytoplasma vorliegt. Das p170 Expressionsmuster wurde in Immunoblots auf Extrakten verschiedener Gewebe und Embryonalstadien untersucht. Dabei zeigte sich, daß p170 im Ei angereichert vorliegt und daß es zu einem Abbau des Proteins in der frühen Embryonalentwicklung kommt. Datenbankvergleiche der p170 Proteinsequenz haben ergeben, daß p170 in der Vertebraten Evolution konserviert vorliegt. Entsprechende Homologe in Schwein, Rind und Zebrafisch konnten durch EST-Klone identifiziert werden. Die cDNA- und Aminosäure-Sequenzen der p170 Mensch und Maus Homologe konnten durch die Analyse von öffentlich zugänglichen Sequenzdaten assembliert werden. Das p170 Gen ist auf Chromosom 1 lokalisiert und die Genstruktur des humanen p170 stellt sich als ein mindestens 107 kb großer Sequenzabschnitt mit 25 Exons dar. Die hier präsentierten Ergebnisse stellen einen wichtigen Ausgangspunkt dar, um die weiteren Eigenschaften und die Funktion von p170 in der eukaryontischen Zelle zu erforschen.
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570 Biosciences, Biology
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Xenopus laevis,Cytoskelett,coiled coil,alpha helical,african claw frog Xenopus laevis,Cytoskeleton,coiled coil,alpha helical
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ISO 690STEPPAN, Sonja, 2001. Molekularbiologische Untersuchungen über ein neues Cytoskelett-verwandtes Protein [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Steppan2001Molek-8594,
  year={2001},
  title={Molekularbiologische Untersuchungen über ein neues Cytoskelett-verwandtes Protein},
  author={Steppan, Sonja},
  address={Konstanz},
  school={Universität Konstanz}
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