The transfecting activity of Bacillus phage 429 DNA, extracted either by sodium lauroyl sarcosine-phenol or by 2 M perchlorate, was destroyed by treatment with proteolytic enzymes, although these enzymes did not effect transfecting DNAs of SPP1, SPOI, and SP50. These facts suggest that a protein is associated with transfective 4)29 DNA. Stabilization of protease-resistance during transfection appeared earlier than that of DNaseresistance, indicating that the protein associated with 429 DNA is necessary for initiation of the incorporation of DNA molecules into competent cells. The physical nature of 4)29 DNA before and after the trypsin treatment was investigated by sucrose and CsCl density gradient centrifugations. The trypsin treatment did not alter the sedimentation rate of the unit 029 DNA; however, it did convert the sedimentation rate of the aggregated material in the untreated DNA to that of the unit 4)29 DNA. The density of the trypsinized DNA was 0.009 g/cm3 greater than that of the untreated DNA. The possible location of the protein on the DNA is discussed.After Avery et al. (1) reported that the transforming principle is probably DNA, efforts were made to prepare DNA free from protein (2, 3). It was then established that small amounts of protein, remaining in DNA preparations, were not important in transformation. Similarly, for transfection by bacteriophage DNA, the protein remaining in the DNA preparation had no influence on its biological activity (4-6).Transfecting 429 DNA, composed of nonpermuted linear duplex molecules of 11 X 106 daltons (7), purified by sodium lauroyl sarcosinate (SLS)-phenol, shows the characteristic DNA absorption spectrum. However, several observations, such as shapes of DNA molecules and thermosensitive infectivity of 429 DNA, obtained in our laboratory (8) (14) were used. The phage-diluting solution was 0.1 M Tris buffer containing 0.1 M NaCl and 0.01 M MgCl2 (pH 7.5). Standard saline citrate (SSC) contains 0.15 M NaCl and 0.015 M Na3 citrate.Enzymes and Reagents. Trypsin (1 X crystallized, 160 EU/ mg), a-chymotrypsin (3 X crystallized), Pronase-P, trypsininhibitor (soy bean), bovine serum albumin, calf-thymus DNA, and phenyl methylsulfonyl-fluoride (PMSF) Complete lysis occurred within 1.5 hr at 37°. Crude phage sediments were collected by differential centrifugation and were purified by centrifugation through a discontinuous CsCl gradient (15). The phage suspension thus obtained had a titer of 5 X 1012 infective centers/ml. SPP1, SP50, and SPO1

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The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the phi FSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.