Molecular characterization and identification of antigen 32-5B6 as enz

Diplomarbeit aus dem Jahr 1996 im Fachbereich Biologie - Genetik / Gentechnologie, Note: keine Angabe, Eberhard-Karls-Universität Tübingen (Max-Planck-Institut für Entwicklungsbiologie, Tübingen), Veranstaltung: -, Sprache: Deutsch, Abstract: Molecular characterization and identification of antigen 32-5B6 as enzyme S-Adenosyl-L-Homocystein-Hydrolase from Xenopus laevis oocyte nucleiClaudia MohlDiploma thesis in Biochemistry, Eberhard-Karls-University, Tübingen, 1996and Max-Planck-Institut for Development Biology, Tübingen(Title and Abstract in english; Original work in german)Abstract:The aim of my diploma thesis work (1996) was molecular characterization and identification of late migrating antigen32-5B6 on a molecular level. Antigen 32-5B6 is a protein that is distributed in cytoplasm during Blastula stage and transported into oocyte cell nuclei at Gastrula stage 12 during embryonic development of Xenopus laevis (Dreyer et al. 1982; 1983). To isolate cDNA sequences that encode the late migrating antigen32-5B6, I screened, isolated and sequenced five cDNA clones from placques with positive antibody reaction from a Xenopus laevis ovar lambda zap II cDNA expression library. Sequence analysis showed that two cDNA clones encode the enzyme S-Adenosyl-Homocystein-L-Hydrolase (clone 10) from Xenopus laevis (Seery et al. 1994) and an isoform of this enzyme (clone 8). Molecular weight and IEP of S-Adenosyl-Homocystein-L-Hydrolase are nearly identical with those of antigen 32-5B6. To get further evidence in regard to sequence of antigen 32-5B6, proteins were isolated from original Xenopus laevis oocyte nuclei for protein microsequencing. For this reason I established a new protein purification strategy purifying proteins from original oocyte nuclei of Xenopus laevis proteom by means of anion-exchange chromatography and 2-dimensional gel electrophoresis. By means of western blot analysis I could detect two enzyme isoforms those IEPs lie in range between pH 6.02 - 6.2 and with molecular weights between 46 and 47.7 kDa. Protein microsequencing were performed by Prof. Dr. Klaus Weber and Uwe Plessmann (MPI for Biophysical Chemistry, Göttingen, Germany) and confirmed result of screening of Xenopus laevis ovar lambda zap II cDNA expression library. Eight peptides of different lenght were to 100% identical with protein sequences of enzyme S-Adenosyl-L-Homocystein-Hydrolase from Xenopus laevis (Seery et al. 1994) and peptides derived from cDNA sequences of clone 8 and 10. Following the antigen 32-5B6 is identical with the enzyme S-Adenosyl-Homocystein-L-Hydrolase from Xenopus laevis (Seery et al. 1994) and a new isoform of this enzyme could be identified during this diploma thesis.

Diplomarbeit aus dem Jahr 1996 im Fachbereich Biologie - Genetik / Gentechnologie, Note: keine Angabe, Eberhard-Karls-Universität Tübingen (Max-Planck-Institut für Entwicklungsbiologie, Tübingen), Veranstaltung: -, Sprache: Deutsch, Abstract: Molecular characterization and identification of antigen 32-5B6 as enzyme ...