Two-Hit Kinetics of Focus Formation in Cells Transfected with Rous Sarcoma Provirus

Abstract

DNA was purified from an established line of SR-E-transformed quail cells and was assayed for transfection in turkey cells. Transfection events were determined from foci of transformed cells grown under agar overlay which was used to prevent the spread of the progeny virus. It was found that transfection with Rous sarcoma provirus follows two-hit kinetics, indicating that at least two (unlinked) proviruses are required to give rise to a focus of transformed cells.

Tandem Integration of Avian Retroviruses in Double-Infected Cells

“…For instance, in the ex periments of Akiyama and Vogt [37] about 10% cotransfections occurred when chicken cells were treated with mixed DNAs purified from two single RSV-infected cell cultures. We have seen that chicken cells, unlike turkey cells, require a high amount of the DNA to produce foci under agar overlay, and no ki netics of focus formation could be established in these cells when the DNA was serially di luted [14]. According to the specific infectivity data, the chicken cells used as DNA receivers by Akiyama and Vogt were also poor focus producers.…”

“…An estab lished line. Q54, of SR-E-transformed virus-producing quail cells has been described [14], These cells were grown for more than 1 year in vitro in Eagle's MEM containing 4 times amino acids and vitamins and supplemented with 10% TPB, 5% heated calf serum, 5% fetal calf serum and 1% DMSO.…”

“…Hirt fractionation [21] was performed as described elsewhere [22]. The DNA was purified according to the procedure of Marmur [23], with modifications [ 14]. Care was taken to avoid break age of DNA molecules by mechanical shearing.…”

“…In this transfection procedure [14], turkey fibro blasts w'ere plated in 5 ml of growth medium at 1.2 x 101 ' cells per Petri dish (60 mm diameter), exposed to DNA using the calcium phosphate technique as above, and incubated at 38 in Eagle's medium supplemented with 10% heated calf serum for 14 h. The medium was then removed and replaced by agar overlay containing 0.74% agar( Bacto, Difco), 10% TPB.0.06% NaHCOs. and 10% heated calf serum, and the cultures were further incubated at 38 in a humidified atmosphere and 5% CO> for about 6-8 days.…”

“…Genetic experiments have revealed, however, many unusual character istics of retrovirus recombination and repli cation processes [9], and the mechanism of We reasoned that transfection assay [13] could be used to carry out genetic analysis of integrated proviral forms. In particular, the elaboration of a technique for transfection un der agar overlay to prevent virus spread with out inhibiting the transfection process [14] has enabled us to clone viruses by transfection.We have been able to analyze the host range and transforming ability of some 250 clones derived by transfection of DNA purified from superinfected and double-infected cells. We show that in transfections using the DNA of double-infected, but not superinfected, cells, a small fraction of virus clones contained both…”