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Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.

Czank, A ; Häuselmann, R ; Page, A W. ; Leonhardt, H ; Bestor, T H. ; Schaffner, W ; Hergersberg, M :
Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
[Online-Edition: http://www.cardoso-lab.org/publications/Czank_1991.pdf]
In: Gene, 109 (2) pp. 259-63. ISSN 0378-1119
[Artikel], (1991)

Offizielle URL: http://www.cardoso-lab.org/publications/Czank_1991.pdf

Kurzbeschreibung (Abstract)

Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNA for the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping cDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in controls, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.

Typ des Eintrags: Artikel
Erschienen: 1991
Autor(en): Czank, A ; Häuselmann, R ; Page, A W. ; Leonhardt, H ; Bestor, T H. ; Schaffner, W ; Hergersberg, M
Titel: Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
Sprache: Deutsch
Kurzbeschreibung (Abstract):

Mammalian DNA cytosine-5-methyltransferase (MTase, EC 2.1.1.37) is an essential component for establishing and maintaining cell-type specific methylation patterns in the genome. The cDNA for the murine enzyme was previously cloned in segments. We have reconstructed the entire gene, encoding a protein of 1517 amino acids, from a set of overlapping cDNA clones. We report the assembly of two expression constructs in bacterial/mammalian shuttle vectors. Transcription in the first construct (pEMT) is driven by the cytomegalovirus enhancer/promoter and encodes a fusion protein with 15 additional aa at the N terminus, while the second construct (pJMT) is driven by the simian virus 40 early promoter/enhancer upstream from the natural ATG codon. Immunofluorescence microscopy and immunoblot analysis have shown that both constructs direct the synthesis of MTase in COS-1 cells. Enzyme activity in whole-cell lysates of transfected COS-1 cells transfected with pEMT and pJMT are on average tenfold and fivefold higher than in controls, respectively. The specific activities of the recombinant and endogenous mouse-cell enzyme are similar. These expression constructs will be of use in studies of DNA methylation in mammals.

Titel der Zeitschrift, Zeitung oder Schriftenreihe: Gene
Band: 109
(Heft-)Nummer: 2
Fachbereich(e)/-gebiet(e): Fachbereich Biologie, Biology > Zellbiologie und Epigenetik, Cell Biology and Epigenetics
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Fachbereich Biologie, Biology
Hinterlegungsdatum: 05 Mär 2010 14:54
Offizielle URL: http://www.cardoso-lab.org/publications/Czank_1991.pdf
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