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Abstract
A repetitive mammalian genetic element, HSAG-1, has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolate reductase (DHFR). LR-73 cells, a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, then subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which specifically inhibits DHFR. Cultures transfected with the HSAG-1-containing construct acquired drug resistance faster than those transfected with the parent vector. This acceleration of acquisition of drug resistance was due to an increased probability of the generation and subsequent selection of cellular variants with increased copy numbers of the vector. The effect has also been observed in CHO(DHFR-) and HeLa cell lines. Possible mechanisms for the effect of the HSAG-1 element on gene amplification are discussed.
Highlights
A repetitive mammalian genetic element, HSAG-1, the phenotype shown to be dominant upon cell fusion (Rolfe has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolatereductase (DHFR).LR-73 cells,a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which inhibits DHFR
Effect of HSAG-1 on the Rate of Acquisition of Drug Resistance-The cis effects of a given genetic element on gene amplification were studied by inserting the element into the EcoRI site of the vector pSV2-DHFR, which will be referred to as "D"
The recombinant vector construct containing the 3.4-kb element, HSAG-1 (Stanners et al, 1981), in the lefthand orientation, termed"HLD", is depicted in Fig. 1.LR-73 cells were transfected with the dominant selectable vector, pSV2-AS,along with a 10-foldexcess of either Dor HLD, and asparagine synthetase-positive transfectants were selected by 10-14 days' growth in asparagine-free medium containing albizziin
Summary
1984), incultured cells inresponseto selective agents a 3.4-kb EcoRI fragment thiastmember of a familyof middle (Schimke, 1988), and in tumors and tumor-derivedcell lines repetitive elements, cloned from a human CLL-CHO’ hybrid (Alitalo and Schwab, 1986; Slamon et al, 1987). cell genomic library (Stanners et al, 1981) Members of this several different mechanismsfor geneamplification have been family have been shown to elicit a CLL-associated cellular proposed (for review see Stark et al, 1989),amplification of a surface antigen in animal cells by transfection (Stanners et specific gene in a particular cell may occur at a frequency of al., 1981), possibly byan indirect, inductive mechanism Protein Analysis-Subconfluent cultures were labeled for 3 h with [:%]methionine a t 10 pCi/min, the cells removed, andtotal cell protein samples containing equal counts/min subjected to sodium dodecyl sulfate-polyacrylamideelectrophoresis (Laemmli, 1970).Gels were treated with En3Hance (Du Pont-New England Nuclear), dried, and exposed to x-ray film for 3 days
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