The high-molecular-weight DNA polymerase fraction of calf thymus, rat liver or rat spleen can be resolved into further activities differing from one another in molecular weight and charge properties. The use of Sepharose 6B columns brings about partial resolution of these activities although DEAE-cellulose achieves a more effective separation. The high-molecular-weight fraction from calf thymus has so far yielded four species of activity, A, B, C and D sedimenting at 7.9 S, 5.2 S, 7.3 S and 6.0 S, respectively. In rat liver and spleen, 7.6-S (I), 7 . 2 3 (11) and 5-S enzymes have been detected although smaller and more variable amounts of 5-S enzyme occur in the rat tissues compared with calf thymus where the 5.2-5 species can represent 20-

1. A rapid, mild method for isolating nuclear membranes from isolated rat liver nuclei is described. The method employs a double digestion of nuclei with a low level of DNAase I at approximately 0.1 mM Mg2+ and slightly alkaline pH, to release the membranes.2. Electron microscopy shows excellent preservation of the nuclear membrane morphology. Large membrane fragments consisting of a double membrane with ribosomes adhering to the outer layer are produced. The layers are connected at nuclear pores, which have distinct annular subunits and occasional central granules. Contamination by other subcellular structures is minimal.3. The recoveries of nuclear protein, DNA, RNA and phospholipid in the membrane are approximately 8O/,, I -3O/,, So/, and 550/,, respectively. The DNA associated with the membrane does not appear to result from the reassociation of released DNA. The enzymic content of the membrane is similar to that of the microsomes. It is concluded that the nuclear membrane is the sole nuclear location of glucose-6-phosphatase.4. About lo/, of the total nuclear DNA polymerase was recovered associated with the nuclear membrane. By three criteria this enzyme was identical with pure authentic nuclear DNA polymerase. This finding is discussed against the background of the suggestions that the nuclear membrane is involved in DNA replication.A role for the nuclear membrane has been postulated in the widely differing fields of DNA replication [I, 21, the organisation of chromatin [ 3 ] , the formation of the herpes virus nucleocapsid envelope[4], nucleo-cytoplasmic exchange of macromolecules and nuclear respiration [ 5 ] .InveAtigation of some of these postulated functions would be considerably facilitated by suitable membrane preparations. Existing preparative methods are slow, taking a t least 12 h from the isolation of the nuclei and involve either sonication or high salt extraction or both r6-91. Sonication causes membrane fragmentation and in some cases fusion between different membranes [lo], while high salt is likely to extract many proteins associated with the membrane.We describe here a rapid, mild method of isolating nuclear membranes from the isolated nuclei which takes approximately 80 mjn and avoids both sonication and high salt extraction. The isolated membranes have been characterised chemically, biochemically and by microscopy and have the features expected for the nuclear membrane.There exists considerable controversy as to the site of nuclear DNA replication [1,2,11,12]. It, has been suggested that the association of a DNA polymerase activity with the nuclear membrane indicated this to be the site of DNA replica.tion [13]. I n a previous publication we found that newly replicated DNA was not concentrated in the nuclear membrane fraction of regenerating rat liver nuclei and so concluded that this was probably not the site of all DNA replication [Ill. We report here that the small proportion of the total nuclear DNA polymerase found associated with the nuclear membrane is identical by three criteria ...

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Small samples of the 8‐S species of enzymes (A1 and A2) occurring in the DNA polymerase‐a fraction of calf thymus, have been extensively purified using non‐denaturing (normal) polyacrylamide gel electrophoresis. When peak fractions of activity on normal gels were subjected to dodecylsulphate‐polyacrylamide gel electrophoresis, a polypeptide at 155000 correlated with polymerase activity. Samples of the 7.3‐S (C) enzyme prepared from A2 by treatment with 2.4 M urea or isolated directly without exposure to urea, also showed the presence of a 155000‐Mr polypeptide. It is concluded that the 7.3‐S (C) enzyme, of previously estimated molecular weight 155000–170000, is a single polypeptide and that the 8‐S enzymes A1 and A2 contain an additional subunit of 50000–70000 molecular weight.

1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1.35 at 5 degrees .