Metaphase chromosomes with high molecular weight DNA were isolated from Chinese hamster ovary (CHO) cells in a neutral buffer containing polyamines and chelators. The individual, unfixed chromosomes retained their centromeric and secondary constrictions, distinct sister chromatids, and complex banding patterns. The DNA from these chromosomes was 100-fold larger (2 x l0 s daltons) than DNA from chromosomes isolated by other procedures. These characteristics indicate preservation during isolation of considerable native structure. In contrast to chromosomes produced by other methods, these chromosomes were stable in storage and did not aggregate, thus providing useful material for studies of the structure and biochemistry of individual chromosomes.KEY WORDS chromosomes DNA Chinese hamster cells 9 metaphase Several procedures have been described for isolation of metaphase chromosomes (4-7, 10, 13). Most use acidic isolation buffers (8) or a neutral buffer in the presence of 2-methyl-2,4-pentanediol plus divalent cations (15) to stabilize chromosome morphology. These isolation conditions lead to the degradation of chromosomal DNA (16) either by depurination at low pH or by nuclease activity at neutral pH in the presence of divalent cations. High molecular weight DNA has been obtained from metaphase chromosomes by raising the pH of the isolation buffer to 9.6-10.5 (14, 16). This high pH, however, may have undesirable effects on the recovery of acidic proteins from metaphase chromosomes. In this report, we present a procedure for rapidly obtaining mammalian metaphase chromosomes, at neutral pH, that contain high molecular weight DNA. We employed the buffer system described by Wallace et al. (12), which uses polyamines rather than divalent cations to stabilize chromosome structure and heavy metal chelators to prevent nuclease activity. This buffer has been used successfully in isolating Drosophila polytene chromosomes and diploid nuclei without changing their native structure (9). MATERIALS AND METHODS Cell LinesChinese hamster ovary (CHO) cells were grown in McCoy's 5A medium supplemented with 15% calf serum, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) buffer, 50/~g/ml of streptomycin, 50 U/ml of penicillin, and 50 /zg/ml of chlorotetracycline, in either Falcon plastic T-flasks (Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, Calif.) or glass roller bottles. Cultures were labeled for 12-15 h with 0.2 p.Ci/ml of [14C]thymidine (sp act, 10 Ci/mmol) or 0.1 /zCi/ml of [3H]thymidine (50 Ci/mmol) before harvesting of mitotic cells.When the cell cultures in T-flasks were -80% confluent (doubling time was ~ 12 h), loosely attached cells were removed by gentle shaking, and fresh medium containing colcemid (2 x 10 -~ M) was added. Cultures were then incubated for 3--4 h at 37~ and mitotic cells were harvested by gentle shaking. Cells in roller bottles were grown to -50% confluence. Loosely attached cells or cells incubated with colcemid, as described above, were removed by rotating the bot...

Chromosomes were isolated from Chinese hamster ovary cells that had been pulse-labeled with 3H-thymidine and bromodeoxyuridine and allowed to proceed to metaphase in the presence of colcemid. The chromosomes were then irradiated with 313 nm light and the length of the pulse-labeled DNA segments was estimated by the distribution of 3H-DNA in alkaline sucrose gradients. The average rate of fork displacement, calculated from this measurement, was 0.9 micrometer/min, which was in excellent agreement with measurements from interphase cells. By the same method, hydroxyurea was shown to retard DNA chain growth by 50--60% in both isolated chromosomes and interphase cells.