Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promotor-cat gene construct in the genome of hamster cells: Gene expression and stability of methylation patterns

Ulrich Mueller, W. Doerfler

Research output: Contribution to journalArticle

Abstract

The late E2A promotor of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promotor. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W.Doerfler, Proc. Natl. Acad, Sci, USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci, USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promotor-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promotor methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promotor controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promotor controls for gene for neomycin phosphotransferase. The pAD2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuraton. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAD2E2AL-CAt construct, the late E2A promotor remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promotor remained almost completely methylated. In five cell lines, the E2A promotor sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylation were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promotor, after this promotor was fixed by integration in the mammalian genome. Were position or other effects important factors in determining the stability or instability of methylation patterns?

Original languageEnglish (US)
Pages (from-to)3710-3720
Number of pages11
JournalJournal of Virology
Volume61
Issue number12
StatePublished - 1987
Externally publishedYes

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chloramphenicol acetyltransferase
Chloramphenicol O-Acetyltransferase
Adenoviridae
hamsters
Cricetinae
methylation
Methylation
cell lines
Genome
Gene Expression
Cell Line
gene expression
genome
Genes
genes
cells
DNA
plasmids
Plasmids
Simian virus 40

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

@article{ae842990824c4427bce3ffbe30a8ab02,
title = "Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promotor-cat gene construct in the genome of hamster cells: Gene expression and stability of methylation patterns",
abstract = "The late E2A promotor of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promotor. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W.Doerfler, Proc. Natl. Acad, Sci, USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci, USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promotor-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promotor methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promotor controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promotor controls for gene for neomycin phosphotransferase. The pAD2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuraton. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAD2E2AL-CAt construct, the late E2A promotor remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promotor remained almost completely methylated. In five cell lines, the E2A promotor sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylation were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promotor, after this promotor was fixed by integration in the mammalian genome. Were position or other effects important factors in determining the stability or instability of methylation patterns?",
author = "Ulrich Mueller and W. Doerfler",
year = "1987",
language = "English (US)",
volume = "61",
pages = "3710--3720",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promotor-cat gene construct in the genome of hamster cells

T2 - Gene expression and stability of methylation patterns

AU - Mueller, Ulrich

AU - Doerfler, W.

PY - 1987

Y1 - 1987

N2 - The late E2A promotor of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promotor. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W.Doerfler, Proc. Natl. Acad, Sci, USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci, USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promotor-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promotor methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promotor controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promotor controls for gene for neomycin phosphotransferase. The pAD2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuraton. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAD2E2AL-CAt construct, the late E2A promotor remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promotor remained almost completely methylated. In five cell lines, the E2A promotor sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylation were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promotor, after this promotor was fixed by integration in the mammalian genome. Were position or other effects important factors in determining the stability or instability of methylation patterns?

AB - The late E2A promotor of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promotor. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W.Doerfler, Proc. Natl. Acad, Sci, USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci, USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promotor-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promotor methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promotor controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promotor controls for gene for neomycin phosphotransferase. The pAD2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuraton. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAD2E2AL-CAt construct, the late E2A promotor remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promotor remained almost completely methylated. In five cell lines, the E2A promotor sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylation were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promotor, after this promotor was fixed by integration in the mammalian genome. Were position or other effects important factors in determining the stability or instability of methylation patterns?

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