Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12920-5.

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene.

Author information

  • 1Whitehead Institute for Biomedical Research, Cambridge, MA, USA.

Abstract

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.

PMID:
8917520
[PubMed - indexed for MEDLINE]
PMCID:
PMC24021
Free PMC Article

Images from this publication.See all images (4)Free text

Figure 1
Figure 3
Figure 4
Figure 2
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk