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Summary Activity of some fifty potential alkylating agents on respiration and anaerobic glycolysis and upon RNA, DNA and protein, synthesis of Walker 256 ascites tumour cells has been investigated. The sulphonylhydrazone moiety proved to be selectively toxic to hypoxic cells in some structures; use of carbohydrate residues as “carriers” led to loss of activity. One halo-deoxy sugar showed an effect upon glycolysis and RNA synthesis.

After infection of primary cultures of green monkey kidney cells or of an established green monkey kidney cell line (CV-1) with SV40, thymidine kinase activity was induced. Increased enzyme activity was first detectable 12–16 hours after inoculation and was 4–15 times greater at 28–48 hours in infected than in noninfected cultures. Thymidine kinase has been partially purified from noninfected and SV40-infected cells. With deoxyuridine (UdR) as substrate, the Michaelis constant of the enzyme from infected cells was 2–3 times greater than that from noninfected cells. However, the kinetics of inactivation of the enzymes were about the same at 38 °. 1-β- d -Arabinofuranosylcytosine (ara-C) inhibited the incorporation of tritiated thymidine (H3-TdR) into the deoxyribonucleic acid (DNA) of noninfected or SV40-infected monkey kidney cultures by 95% or more. Ara-C treatment increased greatly the thymidine kinase activity of noninfected monkey kidney cultures. The Michaelis constant of thymidine kinase purified from ara-C treated CV-1 cultures was identical to that from untreated cultures. Ara-C did not prevent the induction of thymidine kinase by SV40. Indeed, the effects of ara-C and SV40 on enzyme induction were approximately additive in drug treated and infected cultures. Bromodeoxyuridine (BUdR) treatment did not prevent thymidine kinase induction by SV40, although virus growth was inhibited by 99.9%. Unlike ara-C, BUdR did not have a stimulating effect on thymidine kinase activity in noninfected cells. Cycloheximide inhibited the incorporation of radioactive amino acids into proteins of monkey kidney cells. Cycloheximide prevented thymidine kinase induction when added to cultures early after SV40 infection and arrested further enzyme induction when added after thymidine kinase induction had begun. A gradual twofold increase in thymidylate synthetase activity occurred in SV40-infected cultures but thymidine 5′-monophosphate (TMP) kinase and uridine kinase activities increased by less than 50%. Radioautographic and biochemical experiments on the incorporation of radioactive precursors into DNA demonstrated that the percentage of cells with labeled nuclei and the uptake of either tritiated deoxyadenosine or H3-TdR into DNA was sharply increased beginning at about 16–20 hours after infection. The total DNA content of SV40-infected cultures was also increased.

Summary Polyoma virus particles contain three small basic polypeptides. These appear to be host cell histones which can be synthesized in mouse cells before infection. Comparison of peptides of polypeptides derived from [35S]-methionine labelled virus with those from mouse cell histones showed correspondence of two major and several minor peptides. It is concluded that these polypeptides are therefore not virus coded.

Mutant sublines of HeLa S3 cells resistant to growth inhibition by bromodeoxyuridine (BUdR) have been isolated. The resistant cell lines (HeLa BU-10, HeLa BU-15, HeLa BU-25, HeLa BU-50, and HeLa BU-100) proliferated in the presence of 10, 15, 25, 50, and 100 μg/ml BUdR, respectively. Extracts from HeLa BU-25, HeLa BU-50, and HeLa BU-100 cells exhibited 2–5 per cent of the thymidine-3H, deoxyuridine-3H, and bromodeoxyuridine-3H phosphorylating activities of parental HeLa S3 cells. HeLa BU-10 and HeLa BU-15 cell extracts were also deficient in thymidine kinase activity, yielding approximately 43 per cent and 8 per cent, respectively, the thymidine kinase activity of parental HeLa S3 cells. The deficiency in thymidine kinase activity of HeLa BU-100 cells was not due to negative feedback inhibition by high levels of BUdR or to interference with the thymidine kinase assay by inhibitors or competing enzymes in the HeLa BU-100 cell extracts. Following 5 weekly passages in media lacking BUdR, the HeLa BU-100 cells did not exhibit increased thymidine kinase activity. Moreover, mixtures of extracts from HeLa S3 and HeLa BU-100 cells displayed a thymidine kinase activity equivalent to the sum of the activities of extracts prepared, respectively, from the HeLa S3 and HeLa BU-100 cells. Radioautographic studies have shown that after HeLa S3 cells were incubated for 6 hours with thymidine-3H, 35–45 per cent of the nuclei were heavily labeled with radioactivity. However, fewer HeLa BU-100 cells displayed labeled nuclei and the nuclei were only lightly labeled. HeLa BU-100 cell extracts contained normal amounts of thymidylate synthetase, thymidylate kinase, and uridine kinase activities. Following infection by vaccinia virus, high levels of thymidine kinase activity were induced in HeLa BU-100 cells. La deficience de l'activite thymidine-kinasique des cellules HeLa BU-100 n'etait pas due a une inhibition du feedback negatif par de fortes concentrations de BUdR ni a une perturbation du dosage de la thymidine kinase par des enzymes inhibiteurs ou concurrents dans les extraits de cellules HeLa BU-100. En effet, apres cinq passages hebdomadaires dans des milieux prives de BUdR, les cellules HeLa BU-100 ne montraient aucun accroissement de l'activite thymidine kinasique. En outre, les melanges d'extraits de cellules/HeLa S3 et HeLa BU-100 presentaient une activite thymidine kinasique equivalant a la somme des activites des extraits prepares a partir des cellules HeLa S3 et HeLa BU-100 respectivement. L'observation autoradiographique a montre que, lorsque les cellules HeLa S3 etaient mises en incubation pendant 6 heures avec de la thymidine-3H, 35 a 45 pour cent des noyaux etaient fortement marques par la radioactivite. Par contre, les noyaux marques etaient plus rares dans les cellules HeLa BU-100 et ceux qui etaient marques ne l'etaient que legerement. Les extraits de cellules HeLa BU-100 presentaient des activites normales de la thymidylate synthetase, de la thymidylate kinase et de l'uridine kinase. L'infection des cellules HeLa BU-100 par le virus de la vaccine a suscite une forte activite de la thymidine kinase.

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation. Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis. Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.