Editor’s Note
Fig. 1. Autoradiography of unsynchronized CHO cells exposed to3H-TdR for 0.5 min. A and B, grains distributed over entire nucleus. C and D, grains distributed around nuclear membrane. E, clustered grain distribution. F, mixed grain distribution. CHO cells were grown on Petri plates in Joklik-modified MEM (Grand Island Biological Company) supplemented with 7% foetal calf serum and non-essential amino-acids. Pulse-labelling was performed by first adding 5-fluorouridine deoxyriboside (FUDR; Hoffmann-LaRoche) to a final concentration of 1.6 μg ml.–1to inhibit further biosynthesis of dTTP. After 1 min,3H-TdR (51 Ci mmol–1; New England Nuclear) was added to 17 μCi ml.–1. After 30 s the plates were removed from 37℃, and the medium rapidly sucked off. The plates were then washed with ice-cold isotonic saline containing FUDR at 0.1 μg ml.–1(two changes). Cells were removed from the plates by trypsinization at room temperature in isotonic saline still containing FUDR. The cells were pelleted, then fixed with glutaraldehyde and OsO4, dehydrated, embedded in epoxy resin29and cut into gold-purple sections. The sections were mounted on grids and autoradiographed by the technique of Caro and Van Tubergen20except that the acetic acid stop bath was replaced with distilled water. Exposure time was 3.5 months. The bar in each figure represents 1 μm.
Fig. 2. Autoradiography of a HeLa cell exposed to3H-TdR for 1.5 min. The cells were grown in suspension culture in Joklik-modified MEM (Grand Island Biological Company) supplemented with 5% horse serum. Pulse-labelling was done by adding3H-TdR (20 Ci mmol–1, New England Nuclear) to a concentration of 20 μCi ml.–1. Exactly 1.5 min after addition of3H-TdR, the pulse was terminated by addition of an equal volume of ice-cold isotonic saline containing 1% glutaraldehyde. Subsequent processing was as in Fig. 1. Exposure time was 9.5 months. The bar represents 1 μm.
Fig. 3. Distribution of grains over unsynchronized CHO cells exposed to3H-TdR for 0.5 min. Prints at about×15,000 magnification were made of autoradiograms of individual randomly chosen cells similar to and including those in Fig. 1. A line was drawn within each nucleus which was at all points 1.25 μm away from the nearest nuclear membrane. This line divided the nucleus into a central area and a peripheral area. Grains were counted over each area, and the size of each area was measured. In these determinations, grains lying over the nucleolus, and nucleolar areas, were ignored because the nucleolus has so much less DNA than the rest of the nucleus. Grains lying outside the nucleus were also ignored. “Central activity” was calculated as the ratio of the fraction of grains which were central to the fraction of area which was central. Thirty nuclei were measured for the histogram.
Table 1. Extent of DNA Synthesis in Synchronized CHO Cells
* Cells were synchronized as in Fig. 4. At 2 h intervals after release from mitosis,3H-TdR (20 Ci mmol–1; New England Nuclear) was added to each plate (17 μCi ml.–1final concentration). After 10 min the cells were washed twice with cold isotonic saline, collected by trypsinization at room temperature, allowed to swell in hypotonic medium (2 mM MgCl2, 1 mM EDTA, 10 mM KPO4,pH 7.7) for 10 min, pelleted, fixed with methanol-acetic acid (3∶1), spread onto subbed glass slides and allowed to dry. The slides were coated with autoradiographic stripping film (“Kodak AR-10”) and exposed for 7 days. After development, the slides were stained with Giemsa stain, then mounted under “Permount”. At least 312 cells were scanned to determine the percent of cells labelled at each time point.
† The total number of grains over each nuclear section was determined for the autoradiographs used in Fig. 4. These figures were divided by the exposure time in months. Extra data for 2 h and 8 h after release from mitosis are included here.
Fig. 4. Distributions of grains over synchronized CHO cells exposed to3H-TdR for 0.5 min. A, Early S phase (4-6 h after release from mitosis). B, Late S phase (10-12 h after release from mitosis). CHO cells were synchronized by the “Colcemid” reversal method of Stubblefield. Pulse-labelling (performed at 2 h intervals after release from mitosis), preparation for electron microscopy, and autoradiography were performed as in Fig. 1. Exposure time was 2-4 months. Histograms were prepared as in Fig. 3. For A, eight nuclei pulse-labelled at 4 h and eleven nuclei labelled at 6 h after mitotic release were used. For B, thirteen nuclei labelled at 10 h and sixteen nuclei labelled at 12 h were used.
Fig. 5. Distribution of grains over CHO cells after pulse-chase or long pulse-labelling with3H-TdR. A, Synchronized cells (see Fig. 4) were pulse-labelled 6 h after release from mitosis as in Fig. 1. Then 30 s after addition of label, the medium was sucked off and the plates were washed, then replaced with medium containing 2.5 μg ml.–1of TdR. After 6 h the cells were collected by trypsinization and prepared for electron microscope autoradiography as in Fig. 1. Exposure time was 1.5-4 months. B, Unsynchronized cells were pulse-labelled as in Fig. 1 and chased for 6.75 h as in A. Exposure time was 3.25 months. C, Unsynchronized cells were pulse-labelled as in Fig. 1 except that the3H-TdR was left in contact with the cells for 10 min instead of 30 s. Exposure time was 1.5-6 months. In all cases, histograms were prepared as in Fig. 3. For A, twenty-four cells were used, whereas for B, twenty-nine cells were used, and for C, thirty-two cells were used.
(241, 32-36; 1973)
Joel A. Huberman, Alice Tsai and Robert A. Deich
Departments of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received May 4; revised December 15, 1972.
References:b5e2iTpo4F135WgQ9AIa2pUnYgxyRvowo9s/WS4MdzdPjTSzPIlMSH+CJa6UfZoi