Expression in mammalian cells of a cloned gene encoding murine DNA methyltransferase
| Autor: | Remo Häuselmann, Heinrich Leonhardt, Martin Hergersberg, Andreas Czank, Walter Schaffner, Andrea W. Page, Timothy H. Bestor |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
DNA-Cytosine Methylases
Recombinant Fusion Proteins Genetic Vectors Cytomegalovirus Fluorescent Antibody Technique Gene Expression Simian virus 40 Biology DNA methyltransferase law.invention Mice Transcription (biology) law Complementary DNA Gene expression Escherichia coli Genetics Animals Cloning Molecular Promoter Regions Genetic Enhancer Gene Cell Line Transformed General Medicine Molecular biology DNA methylation Recombinant DNA Plasmids |
| Zdroj: | Gene. 109:259-263 |
| ISSN: | 0378-1119 |
| DOI: | 10.1016/0378-1119(91)90618-l |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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