Isolation of a Polyoma-Nucleoprotein Complex from Infected Mouse-Cell Cultures

Abstract

A complex containing polyoma (py) DNA and protein (py complex) was isolated from polyomainfected mouse-cell cultures. The The small size of the polyoma virus genome (about 3 X 106 daltons) makes it an ideal prototype for studies on the mechanism of viral-induced tumorigenesis. Elucidation of the function of each of the proteins coded by the virus would be a great stride toward an understanding of the molecular basis of cancer (1-3). However, the problem becomes more complex when we consider the fact that polyoma infection stimulates cells to synthesize various enzymes and basic proteins that were repressed prior to infection (4-7). These proteins, as well as those coded by the virus, may play a role in the type of response of the cell to infection; i.e., in determining whether the cell is killed or becomes transformed.In an effort to gain information regarding the mechanism of polyoma replication and development, we have taken the approach of isolating those proteins that remain associated with the viral DNA upon extraction from infected mouse-cell cultures. A modified Hirt procedure (8), employing a neutral detergent (Triton X-100) and low salt concentration, is used to separate a polyoma nucleoprotein complex from cellular DNA. The method is quite similar to our method for the isolation of XDNA-RNA polymerase complex from Escherichia coli (9). MATERIALS AND METHODS Infection of cell culturesLarge-plaque polyoma (py) virus stocks were prepared by infection of secondary mouse embryo (ME) or mouse kidney cells growing in tissue culture (10 ,1 1). Non-purified virus was used for most of the experiments. ME, 3T3 Balb/C, or Ts-a-3T3 cells were used as hosts for polyoma virus. They were grown as monolayers in 100-mm plastic tissue-culture plates in Dulbecco's modification of Eagle's medium containing 10% calf serum, except for the 3T3 Balb/C cells, which were grown with 20% calf serum. Plates were seeded such that confluence was reached within 30 hr after seeding.Cells were infected soon after confluence by adding 0.4 ml of virus [10 plaque-forming units (PFU) per cell] to the cell cultures drained free of medium. After incubation (1 hr) at 37°C for adsorption, the cultures were covered with 5 ml of fresh growth medium that contained 1% horse serum. DNA was labeled with [3H]thymidine (5 .sCi/ml, 12-15 Ci/mmol) at the times designated. from Rohm & Haas], 1 ml/plate, was added at room temperature. The plates were gently swirled for 10 min and NaCl was added to a final concentration of 0.2 M. The salt produced noticeable lysis of the cells. The lysate was poured or scraped with a Teflon "policeman" into a polyallomer centrifuge tube and cleared bycentrifugation in the SW39 rotor (Spinco) at 4°C for 30 min at 20,000 rpm in order to pellet the cellular DNA. The supernatant, containing the py DNA, was decanted and stored in a glass vial at 4°C. It is important not to subject the lysate to shearing forces at any stage in order to prevent the release of cellular DNA into the supernatant.In recent experiments, centr...

Retrieval of Latent Herpes Simplex Virus Type 1 Genetic Information from Murine Trigeminal Ganglia by Superinfection with Heterotypic Virus in vivo

“…1 x 108 cells) were infected with 0.1 p.f.u, of HSV per cell in 10 ml of medium at 37 °C for 1 h. Eagle's MEM (20 ml) was then added to each bottle, which was incubated at 37 °C for 48 h. The infected cells were scraped off with a rubber policeman, washed twice in phosphate-buffered saline (PBS), and then collected by low-speed centrifugation (800 g for 10 min at 4 °C). HSV DNA was extracted by a modification of methods described previously (Green et al, 1971 ;Buchman et aL, 1978 ;Lonsdale et al, 1979 ;Pignani et al, 1979). Five ml of lysing solution (0.25 ~ Triton X-100, 10 mM-EDTA, 10 mM-Tris HC1 pH 7.9) was added to the infected cells.…”

Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

“…We originally expected SV40 minichromosomes isolated from monkey cells by the 0.2 M NaCl procedure of Green et al (9) to synthesize virus-specific RNA within minutes after injection into oocytes. However, even though they possessed a full complement of nucleosomes (Fig.…”

“…SV40 DNA containing chromosomal proteins was isolated by using several procedures. To obtain them from Xenopus oocytes, nuclei were manually dissected as described previously (27) minichromosomes were extracted from the nuclei of the infected cells, using either the Triton-EDTA-0.2 M NaCl procedure of Green et al (9) or the low-salt (buffer A) method of Su and DePamphilis (35) as modified by Fanning and Baumgartner (7). Where indicated, a-amanitin (100 ,ug/ml) was added to the cells immediately before incubation of the nuclei at 0°C for 1 h with the extraction buffer.…”

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

“…In cells which have been lytically infected by polyoma or SV40 viruses, essentially all of the non-encapsidated viral DNA exists as a 55S nucleoprotein complex (1)(2)(3), which can be easily separated from cellular chromatin by our Triton extraction procedure (1). The viral complex has been shown by electron microscopic (4)(5)(6) and biochemical (5,(7)(8)(9)(10) analyses to possess a structure which closely resembles that of cellular chromatin.…”