Abstract
The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites around -57 base pairs from the major start site for transcription and HhaI sites around +9, +24, or both. All other HpaII or HhaI sites in the DHFR coding region or in the plasmid sequences remained consistently methylated. The DHFR minigene, after methylation with HhaI methylase, was also introduced without selection by cotransfection with pSV2neo and selection for neor clones in G418. Preferential demethylation of the same sites was observed even without selection for the DHFR+ phenotype. Analysis of the chromatin structure of the integrated minigene revealed characteristic proximal and distal hypersensitive regions of the promoter, as previously observed in human cells. Correctly initiated DHFR mRNA was detected in all of the transformants studied. These results suggest that formation of the characteristic chromatin structure is an intrinsic property of the DHFR promoter sequence and that demethylation of specific sites accompanies gene expression.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.