Biochemistry of vaccinia-infected mouse fibroblasts (Strain L-M): IV. 3H-thymidine uptake into DNA of cells exposed to cold shock

Abstract

The effects of cold shock at 4 ° and of vaccinia infection on the DNA metabolism of mouse fibroblasts (Strain L-M) were investigated by biochemical and radioautographic methods. The capacity of exponentially growing one-day-old cells to incorporate 3H-thymidine ( 3H-TDr) for one-hour into DNA is initially very active but declines to about a third of the control value if the cells are stored at 4 ° for 2 to 4 days. After the cold shocked cells are returned to 37 ° for 2 to 6 hr, DNA biosynthetic capacity is completely recovered in the case of cells cold shocked for 48 hr, and partially recovered in the case of cells cold shocked for 4 days. Vaccinia inhibited 3H-TDr uptake into the total DNA of one-day-old cold shocked cells. When 2- day-old cells are stored at 4 ° for 4 days, and washed twice with BSS prior to incubation at 37 °, 3H-TDr uptake for 1 hr into DNA is exceedingly low during the initial 7 hr period at 37 ° and less than 20 per cent of the cells incorporate 3H-TDr into DNA. Thereafter, successive waves of DNA biosynthesis and mitoses are observed as the cells gradually recover from the cold shock treatment. Following infection of the cold shocked 2-day-old cells by vaccinia, the percentage of cells incorporating 3H-TDr into the cell nuclei is reduced from about 15 per cent to less than 1 per cent during the 7 hr period of active viral-DNA synthesis. During the 8 to 7 hr period post inoculation of the virus, incorporation of 3H-TDr into the total DNA of the infected cultures is stimulated as compared with the uninfected cultures. The results demonstrate that either stimulations or inhibitions of 3H-TDr uptake into DNA of vaccinia infected cells may be observed, depending on the basal level of host cell chromosomal DNA synthesis during the period of virus DNA synthesis and the ratio of virus DNA to host cell DNA biosynthesis.