Autoradiography of chromosomal DNA fibers from Chinese hamster cells.

Ignorance of the true length of the DNA molecules in the chromosomes of higher organisms has always been a major obstacle to understanding chromosome structure. Consequently, attempts have been made, usually with the aid of electron microscopy, to estimate the size of DNA in higher organisms. Solari' has reported the longest such DNA measured before now-a DNA fiber from a sea urchin sperm at least 93 , long. The autoradiographic technique developed by Cairns2 for visualizing DNA has allowed measurement of fibers much longer than 93 A. It has been used successfully by Cairns2 with bacterial DNA, and by Riggs and Mitchell3 with DNA from PPLO. This paper presents the results we obtained by applying the Cairns technique to Chinese hamster cells grown in tissue culture. Methods.-Incorporation of H3-thymidine: Cells of Chinese hamster fibroblast strain B14FAF28 (a gift from Dr. T. C. Hsu) were grown as monolayer cultures on plastic Petri dishes in Eagle's medium supplemented with 10% calf serum. At a cell density of 105 cells/ml of medium, 5fluorodeoxyuridine (FUDR, courtesy of Hoffmann-LaRoche Laboratories, Inc.), an inhibitor of thymidine biosynthesis, was added to make 0.05 pg/ml. Uridine was added to 2.5 pg/ml at the same time. About 10 hr later H3-thymidine (14 c/mmole, New England Nuclear Corp.) was added to 4 ,g/ml. Incubation was continued for 35-40 hr. Then the cells were harvested by a 10-min treatment at 370C with 0.05% trypsin in TD (0.137 M NaCl, 0.005 M KCl, 0.007 M NaH2PO4, 0.025 M tris, pH 7.4, containing 100 mg/liter of streptomycin sulfate and 5 X 105 units/liter of penicillin G) and diluted to about 400 cells/ml in TD. Lysis and spreading procedure: The method usually used was a modification by Riggs and Mitchell3 of the procedure developed by Cairns.2 The cells suspended in TD were diluted tenfold into "lysis medium" (1.0 M sucrose, 0.05 M NaCl, 0.01 M EDTA, pH 8.0). Usually calf thymus DNA was added at this point to 5 ug/ml. One ml of cell suspension (about 40 cells) was then introduced through a polyethylene tube into a dialysis chamber. Construction of the dialysis chambers is outlined in Figure 1. The cells were lysed by dialysis for 3 hr at 34°C against 250 ml of 1% sodium dodecyl sulfate (SDS) in "lysis medium." Further dialysis (6 changes of 2 hr each) against "dialysis medium" (0.05 M NaCl, 0.005 M EDTA, pH 8.0) served to remove SDS and unincorporated thymidine. Finally, the dialysis chambers were removed from the "dialysis medium" and emptied, either by draining through a small hole pierced in one of the VM filters, or by siphoning through the glass inlet tube. In the process of emptying, some DNA was trapped on the VM filters and was spread out as the liquid meniscus moved past. Single cell method: In some cases, cells were suspended in "lysis medium" at an average concentration of 0.5 cell/sl. Drops of 2 ul were placed (one drop per filter) on VM filters which had been coated with a thin film of silicone grease around the outer edge and soaked in "lysis medium." The drops were examined microscopically. Those drops containing single cells were diluted to 0.1 ml with "lysis medium." The drops, still on the filters, were dialyzed first against 1% SDS in "lysis medium" (3 hr) and then against "dialysis medium" (6 changes of 2 hr each) by floating the filters on the surface of the appropriate solutions. The liquiC remaining on top of each filter was drained off through the filter by transferring the filter to a dry surface and then placing the point of a wedge-shaped piece of bibulous paper underneath its center. In this way all DNA was trapped on the filter. Pronase digestion: In some cases, after the fifth change of dialysis medium, dialysis was continued for 14.5 hr against SSC-tris (0.15 M NaCl, 0.015 M trisodium citrate, 0.01 M tris, pH 8.0) containing pronase (Calbiochem, B grade) at a concentration of 50 pg/ml. Under these condi-