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To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.

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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 10(8) 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.

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DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.

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The activity of a 7.3S-8.3S Drosophila DNA polymerase was characterized in detail using poly dA.p(dT)[unk] and poly rA.p(dT)[unk]. With poly dA.p(dT)[unk], Mg(2+) ion was the preferred divalent cation, and enzyme activity was inhibited by K(+) ion and by spermidine. With poly rA.p(dT)[unk], Mn(2+) ion was the preferred divalent cation and enzyme activity was stimulated by K(+) ion and by spermidine. The dependence of enzyme activity on the concentration of primer-template and on the ratio of primer to template was the same in both reactions. The two enzyme activities were identically inhibited by N-ethylmaleimide. Poly dA was replicated extensively and poly rA was replicated partially. The activation energy for poly dA replication was twice that for poly rA replication. Enzyme activity with poly dA.p(dT)[unk] was more stable to thermal inactivation than was enzyme activity with poly rA.p(dT)[unk]. These studies suggest that the same enzyme responds to both the deoxy- and the ribohomopolymer template but that the mechanisms of replication may be different.

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We synchronized Drosophila cell lines (Schneider's line 2 and Kc) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S = 14 to 16 hr; G2 = 6 to 8 hr; M = 0.4 hr) were similar to those of Kc cells.

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The size of DNA replication intermediates from Drosophila cells pulse-labeled with 3H-thymidine for 30-120 sec was determined by electrophoresis in formamide-polyacrylamide gels. Replication intermediates were formed in three discrete size classes, with median lengths of 61, 125 and 240 nucleotides. Replication intermediates in the 125 nucleotide size class occurred most frequently. Two of the three size classes may contain discrete species of replication intermediates about 90-400 nucleotides long. The data also suggested that some larger replication intermediates accumulate in pulse-labeled cells. We concluded that 61 nucleotide molecules give rise to 125 and 240 nucleotide molecules, which then form high molecular weight DNA. Mechanisms for forming these replication intermediates are discussed.

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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.

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