Chromosomes Drosophila melanogaster

The DNA polymerase in crude extracts of Drosophila melanogaster embryos sedimented at 9.0, 7.3, and 5.5 S on glycerol velocity gradients. The relative proportions of these enzymes depended on the method used to prepare the extract. Extracts of whole embryos contained the 7.3S and the 5.5S DNA polymerases and extracts of dechorionated embryos contained the 9.0S and 7.3S DNA polymerases. The porportion of the 5.5S DNA polymerase increased relative to the 7.3S DNA polymerase during storage of the extract of whole embryos. The protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the formation of the 5.5S DNA polymerase, suggesting that it was proteolytically produced from the 7.3S DNA polymerase. This was demonstrated directly by converting the 7.3S DNA polymerase to the 5.5S DNA polymerase by treatment in vitro with trypsin. The degradation of the enzyme occurred without significant loss of DNA polymerase activity. It is further demonstrated that endogenous proteolysis reduced the chromatographic heterogeneity of the Drosophila DNA polymerase on diethylaminoethyl-Sephadex. When endogenous proteolysis was reduced, three forms of DNA polymerase were isolated by diethylaminoethylcellulose chromatography; two of these enzymes sedimented at 7.3S and the third sedimented at 9.0S. These results demonstrate the physical heterogeneity of the Drosophila DNA polymerase and suggest its similarity to vertebrate DNA polymerase-alpha.

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...

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