| Structure and organization of a stable extrachromosomal element in human cells. | |
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MedLine Citation:
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PMID: 7518788 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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We have determined the structure and organization of a 630-kb extrachromosomal element (amplisome) containing the dihydrofolate reductase-encoding gene (DHFR) in a methotrexate (MTX)-resistant human cell line, HeLa-Bu25-10B3. The size and copy number of amplisomes have previously been found to remain remarkably stable with or without selection. Both linear and open circular 630-kb amplisomes are present in these cells. We have been able to isolate the linear amplisomes after pulsed-field gel electrophoresis (PFGE), and transfect the amplisomes into MTX-sensitive recipient cells by electroporation, thus demonstrating that DNA as large as 630 kb can be transfected into mammalian cells. The NotI restriction site immediately upstream from DHFR on the circular amplisome is devoid of methylation, suggesting that it is transcriptionally active. Restriction mapping by PFGE reveals that there is only one copy of DHFR per amplisome and no repetitive structure is observed. The small size of the amplisomes, their stability and our ability to transfect large DNA molecules provide the necessary ingredients for the development of mammalian cloning vectors for large DNA fragments. |
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Authors:
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C Esnault; H Lee; E Lai |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Gene Volume: 144 ISSN: 0378-1119 ISO Abbreviation: Gene Publication Date: 1994 Jul |
Date Detail:
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Created Date: 1994-08-24 Completed Date: 1994-08-24 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 7706761 Medline TA: Gene Country: NETHERLANDS |
Other Details:
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Languages: eng Pagination: 205-11 Citation Subset: IM |
Affiliation:
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Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Azacitidine DNA / chemistry Electrophoresis, Gel, Pulsed-Field Extrachromosomal Inheritance Gene Amplification Hela Cells Humans Methylation Nucleic Acid Conformation Restriction Mapping Tetrahydrofolate Dehydrogenase / genetics* Transfection |
| Grant Support | |
ID/Acronym/Agency:
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R29-GM45943/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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320-67-2/Azacitidine; 9007-49-2/DNA; EC 1.5.1.3/Tetrahydrofolate Dehydrogenase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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