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The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-L-lysine, poly-L-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamineor protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10-3 and 3 x 10-4 for lambda DNA; between 10-6 and 3 x 10-8 for T7 DNA; and between 3 x 10-6 and 10-7 for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.

The addition of 25 ,ug of protamine sulfate per ml to lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for Ti phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10-3 infective centers per DNA molecule; 4X174 replicative form, 300-fold to an efficiency of 5 x 10-2; fd replicative form, 300-fold to 10-6; T7, 300-fold to 3 x 10-1. Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10-5; T5, 3 X 10-1; and P22, 3 X 10-9. The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.

Arber2 has shown that bacteriophage fd, which contains single-stranded circular DNA (Marvin and Schaller12) is restricted by E. coli B and E. coli (P1) +. This report presents detailed data on the restriction of both the infectious, singlestranded fd-phage DNA and the infectious, double-stranded fd-replicative form (RF)18 DNA. It also describes an infectivity assay for in vitro host-controlled restriction and modification. This assay has been used to find restriction enzymes in fractionated extracts of E. coli (Linn and Arber11). Materials and Methods.-Bacterial strains: E. coli F+meth-gal-rK -mK-18 E. coli F+ metK-leuK-rB+mB + threoB+, E. coli F+met-gal-(P1) +, and E. coli B gal-met-rB -mB+ were kindly provided by Prof. Werner Arber (for derivation of strains, see Arber2). Phage strains: Bacteriophage fd was donated by Prof. Werner Arber; a phage mutant which is only partially restricted by E. coli B (Smith, Arber, and Kuehnlein'7) was the gift of Urs Kuehnlein.Purification and characterization of phage and phage DNA's: The 6X174 phage was purified to the first high-speed centrifugation step described by Rueckert, Zillig, and Huber."1 The fd phage was harvested from the medium of Fraser and Jerrel7 and purified by polyethylene glycol precipitation and CsCl density-gradient centrifugation. The spectrum of the purified phage coincided with the spectrum reported by HoffmannBerling, Marvin and Duerwald.8 Phage DNA was extracted as described by Benzinger, Delius, Jaenisch, and Hofschneider.3 Following exhaustive dialysis against 0.01 M Tris buffer, pH 8, containing 1 mM EDTA, the DNA was further purified by CsCl density gradient centrifugation.The fd RF was isolated according to slight modifications of the procedure of Jansz, Pouwels, and Schiphorst.'0 The filtered RF was sedimented on linear 5-20% sucrose gradients and fractions were collected. Electron micrographs of these fractions (kindly prepared by Dr. H. Delius) showed that more than 80% of the DNA was circular. The fast-sedimenting RF fractions were mostly twisted circles; the slower-sedimenting fractions were mostly open circles.'4 Contamination by single-stranded phage DNA was ruled out as described below. Infectious DNA assays: Lysozyme-EDTA spheroplasts were prepared according to the following modifications of the procedure of Ray, Bscheider, and Hofschneider:13 the harvested cells were resuspended in storage medium (plus 10 ,g/ml of required amino acids) at a titer of 1 X 1010 to 2 X 1010 cells/ml. They were then aerated vigorously for 30 min at 370 (Bscheider, personal communication). The bacteria were treated with lysozyme-EDTA in the presence of Povite albumin for 1 min as described by Ray et al."3 Following addition of MgSO4, the spheroplasts were incubated without aeration for another 30 min at 370 (Bscheider, personal communication). After the spheroplasts had been chilled in an ice bath, they were ready for testing with infectious fd DNA's. The titer of infectious DNA with these preparations remained constant for at least 5 days and did not va...