A temperature‐sensitive cell cycle mutant of the BHK cell line

Abstract

A temperature-sensitive growth mutant derived from the BHK 21 cell line, ts AF8, was found to have greatly reduced DNA synthesis at the nonpermissive temperature. This reduction is mainly due to a decrease in the frequency of cells synthesizing DNA. Upon shift up, ts AF8 becomes blocked in the G1 phase of the cell cycle. The cells acquire elevated CAMP levels and a unimodal distribution of DNA content, equivalent to that of G1 cells at the permissive temperature. Ts AF8 cells blocked at the Gl/S boundary with hydroxyurea will enter S when shifted to the nonpermissive temperature. On the other hand, ts AF8 cells arrested in G1 by serum deprivation and shifted to the nonpermissive temperature at the moment of serum addition do not enter S, while those synchronized by isoleucine deprivation and shifted at the time of isoleucine addition will enter S . These data suggest that the cycle arrest point of the ts AF8 mutation is located in G1 between the blocks induced by serum starvation and isoleucine deprivation. The reduction in DNA synthesis caused by the ts AF8 mutation is not reversed by infection or transformation with Polyoma virus. Mitochondria1 DNA continues to be synthesized at wild-type levels at the nonpermissive temperature.

Transformation by polyoma virus alters expression of a cell mutation affecting cycle traverse.

“…The few tsAF8 cells that enter S under these conditions are capable of reaching mitosis. The duration of S appears to be somewhat elongated, as already reported (1). The data for PyAF8 show that, although the number of cells synthesizing DNA is much higher than in tsAF8, a large proportion of these do not reach mitosis ( Table 2).…”

“…tsAF8 cells are incapable of growing at 390, but remain viable for long periods of time. Experiments designed to investigate the effect of this mutation on the cell cycle, including cell synchronization-shift up experiments, show that tsAF8 cells at 390 are blocked in cell cycle traverse and accumulate in G1 (1).…”

“…Metaphase cells were prepared after incubation with Velban and processing as previously described (1). Labeled mitoses were evaluated using mitotic preparations dipped in Kodak nuclear track emulsion and processed for radioautography.…”

“…To test whether PyAF8 cells entering DNA synthesis would have a higher probability of dying and coming off the plate than the rest of the population, we shifted tsAF8 and PyAF8 cells to 39.5°. Thirty-five hours later they were labeled for 1 *The data are from the experiment described in Figure 5. A is the frequency of DNA-synthesizing cells measured by autoradiography at the end of the pulse.…”

Synthesis of labile, serum-dependent protein in early G 1 controls animal cell growth

“…Even more severe inhibition could lead to cell death. These considerations also apply to the consequences of amino acid deprivation, which can put some cells into Go (14), like serum deprivation (1), or other cells into a state from which they can more rapidly emerge (25)(26)(27).…”