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细菌PaeR7甲基化酶基因在哺乳动物细胞中的导入与表达。

Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells.

作者信息

Kwoh T J, Kwoh D Y, McCue A W, Davis G R, Patrick D, Gingeras T R

出版信息

Proc Natl Acad Sci U S A. 1986 Oct;83(20):7713-7. doi: 10.1073/pnas.83.20.7713.

DOI:10.1073/pnas.83.20.7713
PMID:3463994
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC386791/
Abstract

An approach is devised for studying the role of DNA methylation in eukaryotic gene expression. The approach is based on the expression of site-specific bacterial methylase genes in animal cells. A model system using the cloned PaeR7 (an isoschizomer of Xho I) methylase gene was constructed to test the feasibility of this approach. Expression plasmids for the PaeR7 methylase gene were introduced into mouse Ltk- cells by cotransfection with the cloned chicken thymidine kinase (tk) gene. Several of the cell strains derived from Tk+ colonies were found to express the PaeR7 gene as judged by four criteria: the cellular DNA of these strains showed increased resistance to cleavage by Xho I; these strains contained cellular proteins that comigrated with pure PaeR7 methylase protein, as visualized by immunoblotting; PaeR7 methylase activity was found in vitro in crude extracts of total cellular protein from these strains; and murine adenovirus genomes grown on cells expressing PaeR7 methylase showed resistance to cleavage to PaeR7 endonuclease. The potential applications of this approach for the study of cellular and viral gene regulation, DNA repair, and restriction modification are discussed.

摘要

设计了一种研究DNA甲基化在真核基因表达中作用的方法。该方法基于位点特异性细菌甲基化酶基因在动物细胞中的表达。构建了一个使用克隆的PaeR7（Xho I的同裂酶）甲基化酶基因的模型系统来测试该方法的可行性。通过与克隆的鸡胸苷激酶（tk）基因共转染，将PaeR7甲基化酶基因的表达质粒导入小鼠Ltk-细胞。从Tk+菌落衍生的几个细胞株被发现通过四个标准来表达PaeR7基因：这些细胞株的细胞DNA对Xho I切割的抗性增加；这些细胞株含有与纯PaeR7甲基化酶蛋白共迁移的细胞蛋白，通过免疫印迹可观察到；在这些细胞株的总细胞蛋白粗提物中体外发现了PaeR7甲基化酶活性；在表达PaeR7甲基化酶的细胞上生长的鼠腺病毒基因组显示出对PaeR7内切酶切割的抗性。讨论了该方法在细胞和病毒基因调控、DNA修复以及限制修饰研究中的潜在应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/672a15c23a64/pnas00324-0160-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/7e94414f1086/pnas00324-0159-a.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/a3afb0e21e9d/pnas00324-0159-e.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/cf420d914eca/pnas00324-0159-f.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/061d4c95cf58/pnas00324-0159-g.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/6794ad847175/pnas00324-0160-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/672a15c23a64/pnas00324-0160-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/7e94414f1086/pnas00324-0159-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/350f6db6555c/pnas00324-0159-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/b4b5a047fed6/pnas00324-0159-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/525a1997da06/pnas00324-0159-d.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/a3afb0e21e9d/pnas00324-0159-e.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/cf420d914eca/pnas00324-0159-f.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/061d4c95cf58/pnas00324-0159-g.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/6794ad847175/pnas00324-0160-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ad0/386791/672a15c23a64/pnas00324-0160-b.jpg

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本文引用的文献

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Sequential Estimation of p with Squared Relative Error Loss.具有平方相对误差损失的p的序贯估计
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Analysis of substrate specificity of the PaeR7 endonuclease: effect of base methylation on the kinetics of cleavage.PaeR7核酸内切酶的底物特异性分析：碱基甲基化对切割动力学的影响。
Nucleic Acids Res. 1990 Sep 11;18(17):5063-8. doi: 10.1093/nar/18.17.5063.
Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063-7. doi: 10.1073/pnas.77.2.1063.
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Methylation of foreign DNA sequences in eukaryotic cells.真核细胞中外源DNA序列的甲基化
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6463-7. doi: 10.1073/pnas.77.11.6463.
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Mutational analysis of the cloned chicken thymidine kinase gene.克隆的鸡胸苷激酶基因的突变分析。
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Clonal inheritance of the pattern of DNA methylation in mouse cells.小鼠细胞中DNA甲基化模式的克隆遗传。
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DNA methylation and gene activity: the adenovirus system as a model.DNA甲基化与基因活性：以腺病毒系统作为模型
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Proc Natl Acad Sci U S A. 1983 Jan;80(2):402-6. doi: 10.1073/pnas.80.2.402.
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The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.大肠杆菌DNA腺嘌呤甲基化酶（dam）基因的分离与鉴定。
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