Czank, A. ; Häuselmann, R. ; Page, A. W. ; Leonhardt, H. ; Bestor, T. H. ; Schaffner, W. ; Hergersberg, M. (1991)
Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase.
In: Gene, 109 (2)
Article, Bibliographie
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.
| Item Type: | Article |
|---|---|
| Erschienen: | 1991 |
| Creators: | Czank, A. ; Häuselmann, R. ; Page, A. W. ; Leonhardt, H. ; Bestor, T. H. ; Schaffner, W. ; Hergersberg, M. |
| Type of entry: | Bibliographie |
| Title: | Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase. |
| Language: | English |
| Date: | 1991 |
| Journal or Publication Title: | Gene |
| Volume of the journal: | 109 |
| Issue Number: | 2 |
| URL / URN: | http://www.cardoso-lab.org/publications/Czank_1991.pdf |
| 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. |
| Divisions: | 10 Department of Biology ?? fb10_zoologie ?? 10 Department of Biology > Cell Biology and Epigenetics |
| Date Deposited: | 05 Mar 2010 14:54 |
| Last Modified: | 02 Nov 2018 10:46 |
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