Document Detail

Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.
MedLine Citation:
PMID:  7509450     Owner:  NLM     Status:  MEDLINE    
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.
A Graessmann; G Sandberg; E Guhl; M Graessmann
Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Molecular and cellular biology     Volume:  14     ISSN:  0270-7306     ISO Abbreviation:  Mol. Cell. Biol.     Publication Date:  1994 Mar 
Date Detail:
Created Date:  1994-03-25     Completed Date:  1994-03-25     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  8109087     Medline TA:  Mol Cell Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2004-10     Citation Subset:  IM    
Institut für Molekularbiologie und Biochemie der Freien Universität, Berlin, Germany.
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MeSH Terms
Antigens, Polyomavirus Transforming / genetics*
Base Sequence
Cell Line
Cell Transformation, Viral
Cytosine / analogs & derivatives*,  metabolism
DNA, Viral / metabolism
Deoxyribonuclease HpaII
Deoxyribonucleases, Type II Site-Specific / metabolism
Gene Expression Regulation, Viral*
Molecular Sequence Data
RNA, Messenger / genetics
Simian virus 40 / genetics*
Simplexvirus / genetics*
Thymidine Kinase / genetics*
Reg. No./Substance:
0/Antigens, Polyomavirus Transforming; 0/DNA, Viral; 0/RNA, Messenger; 554-01-8/5-Methylcytosine; 71-30-7/Cytosine; EC Kinase; EC 3.1.21.-/Deoxyribonuclease HpaII; EC, Type II Site-Specific

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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