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Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

Abstract：

:In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

journal_name

Mol Cell Biol

journal_title

Molecular and cellular biology

authors

Graessmann A,Sandberg G,Guhl E,Graessmann M

doi

10.1128/mcb.14.3.2004

subject

Has Abstract

pub_date

1994-03-01 00:00:00

pages

2004-10

issue

eissn

0270-7306

issn

1098-5549

journal_volume

pub_type

杂志文章

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Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.