SYNOPSISMagnetic latex particles were prepared by the emulsion polymerization of styrene at 70°C in the presence of a commercial ferrofluid containing surfactant-stabilized magnetite particles in the aqueous phase or its modification by ultrafiltration, using potassium persulfate as an initiator. The effects of diversified variables such as the amounts of initiator, monomer, and additive (calcium chloride and fluorescent dyes) on the polymerization reaction and particle characteristics were investigated. The general polymerization features were analogous to those of ordinary emulsion polymerizations. Transmission electron microscopy revealed that when the commercial ferrofluid was used the magnetite particles localized in the latex particles and the magnetite content varied from particle to particle and that when the ferrofluid was used after ultrafiltration the magnetite particles were dispersed well in the latex particles.

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The effect of bleomycin on the molecular integrity of SV40 DNAwas determined under various conditions by taking advantage of the fact that a single scission produced in such a superhelical form of DNAcould be clearly manifested by analysis of samples with centrifugation in alkaline sucrose gradients. Conditions were found where the reaction velocity, the rate of disappearance of the characteristic form of DNA,depended on the concentration of bleomycin as well as on the reaction time. In contrast to general enzymatic reactions, bleomycin activity was greater at 0°C than at 37°C. The optimum pH was 9.1 while no activity was observed below pH 6 or above pH 13. Additional DNA,irrespective of its source, inhibited the reaction, while RNAhad no effect. Cu++ion at 1 mMalmost completely inhibited the reaction while 2-mercaptoethanol at 1 mMstimulated the reaction by about 20-fold. No sulfhydryl compound was included in our standard assay system. EDTAat 10 mM showed no effect.Bleomycin treatment produces a scission of DNAstrands both in vivo and in vitrol~~6\ In this action, bleomycin neither requires the native conformation^nor a particular base composition of DNA18). The fragmentation ofDNAby bleomycin, which can be enhanced by addition of reducing or oxidizing agents8'9*, is accompanied by release of free bases10) and aldehyde functions3). However, the detailed reaction sequence remains to be elucidated. To characterize bleomycin action, it seemed of primary importance to find reaction conditions where the rate of DNAfragmentation could conveniently be followed as a function of time and drug concentration. Use of SV40DNAas substrate is of major advantage since its native structure is double-stranded, covalently closed-circular and superhelical. It sediments with 53S at an alkaline pH, and if a single scission is produced on either strand of the native form, the products sediment with 18S (single-stranded, closed-circular DNA)and 16S (single-stranded, linear DNA) under the same conditions11*. Under restricted conditions, 16S and 18S DNAwere found to be the major products of bleomycin action because amounts of slower sedimenting DNAfragments or acid soluble material were negligible. Since bleomycin can act efficiently on DNAeven at 0°C, as will be described below, a critical aspect of our assay system was the use of an extremely alkaline pH to terminate the reaction. With this assay, reaction velocities as a function of bleomycin concentration were determined as was the effect of temperature, pHand various additives on the bleomycin activity. In the present paper, these results are presented and the probable mechanismof bleomycin action is discussed.

Cleavage of SV40 DNA by bleomycin was assayed quantitatively in vitro in the presence of various polynucleotides. SV40 DNA was protected from bleomycin-induced cleavage by native or denatured DNA of other origins, poly dG-C•poly dG-C, poly dA-T•poly dA-T and poly dA-T (denatured) but not by tRNA of E. coli, apurinic acid, poly dA, poly dT and various deoxyribooligonucleotides.Various bleomycins and their derivatives and various fragments of bleomycin were tested for possible activity in cleaving SV40 DNA and from the results some structure-activity relationships for the action of bleomycin to act on DNA were outlined. Actinomycin D stimulated bleomycin action while ethidium bromide inhibited it.

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