Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes.
pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for </=130 cell generations after transfection. Furthermore, after </=172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.
Nielsen
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Department of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Cossons
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Zannis-Hadjopoulos
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Price
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GB
D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics*; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic
D004270:DNA, Circular; D005839:Gentamicins; C010680:antibiotic G 418; D001973:Bromodeoxyuridine