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A nonlethal concentration of H202 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H202 and NUV reduces the amount of DNA injected into the bacterial host, but not-the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage '11 with NUV plus H202, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Labeling Techniques. Phage used for adsorption experiments were labeled as reported (9). The method of Hawkins (11) was used in double labeling. Both procedures yielded from 10-6 to 10-7 cpm per phage.Adsorption and Injection Studies. Phage were separated by the polyethylene glycol/dextran sulfate method (12) and further purified by differential centrifugation (13) prior to gradient centrifugal studies. Titers of ca. 5 X 1012 plaqueforming units per ml were routinely obtained. For phage adsorption and injection assays (14) E. coli B was infected with treated phage; 8 min after infection of cells, chloramphenicol was added, a sample was removed to assay radioactivity, and infected bacteria were removed by centrifugation. The pellet was resuspended and an aliquot was withdrawn to determine percentage of phage adsorbed. To determine the fraction of DNA injected into the host, cells were lysed, treated with DNase I, and centrifuged to remove bacterial debris as well as whole phage particles, and the supernatant was then assayed for radioactivity. This procedure is based on the assumption that the solubilized radioactivity represents the amount of DNA that is injected into the bacterial host, because only it is available for digestion by deoxyribonuclease.Genetic Recombination. For genetic crosses between amber mutants (15, 16) E. colh 011' (Su+) was coinfected with either T7 amli (gene 8) or am28 (gene 5), and one of a series of different amber mutants (helper phage) located throughout the T7 geqome. Lysates were assayed on 011' (Su+) and B (Su-) for total progeny and wild-type recombinants, respectively. Results are expressed as percentage of normal recombination (16). Average frequencies of wild-type recombinants for the control crosses were compatible with those in previous reports (15,16).Gel Electrophoresis Studies. E. coli B was grown at 37°C to 4 X 108 cells per ml in M9 medium plus 2 mg of casamino acids per ml, irradiated with 72 J-M2 of far-UV radiation to suppress host cell protein synthesis, and incubated for 30 min at 30°C in the dark. Aliquots (2 ml) were then infected with appropriately treated T7 at a multiplicity of infection of 10 and incubated with 10 ,tCi (1 Ci = 3.7 X 1010 becquerels) of [-5S]-methionine (New England Nuclear) per ml for 24 min at 30°C. Samples were washed and concentrated 10-fold by centrifugation and applied to polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gradient slab gels were prepared 1.5 mm thick with a linear gradient of 8-13% acrylamide and a 5% acrylamide stackin...

We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na,K-ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na,K-ATPase inhibitor ouabain, ranging from 2.9 X 10(6)/cell to 11.8 X 10(6)/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 X 10(6) to 10.5 X 10(6) ouabain-binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids. The Na,K-ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.

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Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.

An enrichment selection method using repeated pulses of low drug concentration (1 ,ug/ml) was

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