title: 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.
authors.affiliation: Institut für Molekularbiologie und Biochemie der Freien Universität, Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Graessmann
authors.forename: G
authors.initials: G
authors.lastname: Sandberg
authors.forename: E
authors.initials: E
authors.lastname: Guhl
authors.forename: M
authors.initials: M
authors.lastname: Graessmann
mesh_terms: D044503:5-Methylcytosine; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D012333:RNA, Messenger; D044503:5-Methylcytosine; D003596:Cytosine; D013937:Thymidine Kinase; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific