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A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.

Several spontaneous revertants able to grow in restrictive temperature (39°C) were isolated from a temperature-sensitive mutant (ts3) of murine leukemic cells L5178Y. The revertants showed reduced plating effi-ciencies and prolonged doubling times at 39°C when compared with wild type cells. The alanyl-tRNA synthetase activity was thermolabile in both ts3 cells and the revertants, indicating suppression as the mechanism of reversion. It was also demonstrated that glycerol protected the enzyme from thermal inac-tivation. The specific activities of alanyl-tRNA synthetase and alanine amino-transferase did not differ much in the wild type, mutant, and revertant cells. This indicates the unlikelihood of thermolabile enzyme overproduction or of increased intracellular concentration of L-alanine causing phenotypic suppres-sion in these revertants. The mechanism of sunnressinn is discussed.

A temperature-sensitive mutant (ts2) of murine leukemic L5178Y cells accumulates aberrant mitotic cells and subsequently, multinucleate cells upon exposure to a restrictive temperature. Temperature-shift experiments using synchronized cell populations demonstrate that the execution point of ts2 cells is located between the early and mid S phase in the cell cycle. Mutant cells can achieve cell division when they are shifted down by the time of karyokinetic stage. However, multinucleate cells, once formed, are unable to undergo cytokinesis even though transferred to a permissive temperature. Cytofluorometric analysis indicates that unequal chromosome distribution among daughter nuclei takes place during multinucleation. The mechanism of defective karyo-and cyto-kinesis in ts2 cells is discussed.

A high-voltage generating machine which could generate semi-rectangular pulses in PBS solution was constructed, and the effects of field strength and duration of the pulse on electric pulse-mediated transformation of mouse mammary carcinoma FM3A cells by a linear form of plasmid pSV2neo DNAs were examined. In parallel, cell survival and growth after pulsing were analyzed. When the field strength and duration of the pulse were increased, the transformation frequency increased, although the cell survival rate decreased. Under the best conditions, the transformation frequency was 2 X 10(-4), which was 80 times higher than that obtained by the calcium phosphate coprecipitation method.