SUMMARY The 11.8 kilobase pair (7-8 x 10 6 mol. wt.) genome of mycoplasma virus L2 in lysogenic Acholeplasma laidlawii cells was examined. For this study, DNAs were analysed by agarose gel electrophoresis and viral DNA sequences identified by DNADNA hybridization. L2 DNA was found to be integrated into the lysogenic host cell chromosome at a unique site in both viral and cellular DNA. The viral DNA site was roughly mapped and the approximate time of integration during the L2 non-cytocidal infectious cycle was determined. Mycoplasma virus L2 is an enveloped virus containing circular superhelieal double-stranded DNA of 11-8 kilobase pairs (7.8 x 106 mol. wt.) (Lombardi & Cole, 1979; Nowak & Maniloff, 1979). L2 infection of Acholeplasma laidlawii is non-lytic and leads to the establishment of a lysogenic state (Putzrath & Maniloff, 1978). Clones of lysogenic cells were found to be resistant to superinfection by homologous virus, but able to be infected by heterologous virus. These cells had the potential to produce virus and transmitted this potential as a stable heritable trait. Mitomycin C and u.v. light induced an increase in infectious centres in cultures of lysogens. Therefore, in the studies reported here, such cells were examined by DNA-DNA hybridization to determine the state of the L2 viral genome they carry. This study used A. laidlawii strain K2 host cells, mycoplasma virus L2 and tryptose growth medium, as described previously (Haberer et al., 1979; Putzrath & Maniloff, 1977). Cells were assayed as colony-forming units (c.f.u.) and viruses as plaque-forming units (p.f.u.). To obtain virus, 200 ml overnight cultures of A. laidlawii strain K2 were infected with L2 (m.o.i. 1 to 10) and diluted to 1 litre with fresh tryptose broth. After overnight incubation at 37 °C, cells were removed by centrifugation (7 min at 9000 rev/min at 4 °C in a Beckman JA-14 rotor), and the virus pelleted (30 min at 25 000 rev/min at 10 °C in a Beckman SW27 rotor). The virus was resuspended in 5 ml TES buffer (0.01 M-Tris-HC1 pH 7.8, 0-1 M-NaCI, 0.001 MEDTA) and purified by sedimentation (3 la at 25 000 rev/min at 10 °C in a Beckman SW27 rotor) in a linear 15 to 30~o (w/v) sucrose gradient. Fractions containing L2 were pooled and concentrated by centrifugation. After resuspension in 0-5 ml TES, the virus titre was 1013 to 1014 p.f.u./ml. Virus DNA was obtained by extracting twice with equal volumes of phenol saturated with TES. The aqueous layer was made 0.2 M in sodium acetate and DNA precipitated by addition of 2 vol. 95~ ethanol. After overnight storage in the cold, DNA was harvested by centrifugation (1 h at 30000 rev/min at 0 °C in a Beckman SW50.1 rotor) and resuspended in 0.5 ml TES. DNA concentrations were typically about 1 mg/ml. Restriction endonucleases were obtained from Bethesda Research Laboratories and the digestion conditions were as recommended. The protocol for mapping cleavage sites in L2 DNA by use of agarose gel electrophoresis was described previously (Nowak & Maniloff, 1979). Several restriction endonucleases were used to construct a physical map of the L2 genome (Fig. 1). The HindlII and HpaI sites confirm previously published results (Nowak & Maniloff, 1979). Although only one XbaI site was originally identified (Nowak & Maniloff, 1979), two close XbaI sites were resolved in these studies (Fig. 1). Using L2 virus grown on A. laidlawii strain JA1, the single BgllI site was chosen as the map zero-point (Nowak & Maniloff, 1979).