10 A possible explanation of the role of ATP in the transformation of II to III is that it gives rise to additional phosphorylated products, which, however, are too rapidly converted to III to be detected.

The recent application of electrophoretic techniques to the separation of ribosomal proteins has provided a means by which differences in the protein composition of ribosomes may be readily detected. '-4 In a survey of the ribosomal proteins from various bacterial strains by one such technique, electrophoresis on polyacrylamide gel columns, it was observed that of all the strains of E. coli examined, K12 strains were distinctly different from the rest. To simplify the discussion we refer to this difference in K strains as "K-ribosomal character" or "K-character." It was obvious that such electrophoretic differences might prove to be useful for genetic studies involving the chromosomal localization of a ribosomal character.The present paper describes first the nature of the "K-character" and its prevalence among various strains of E. coli. Secondly, the electrophoretic difference has been used for the assignment of a genetic locus for a ribosomal protein on the bacterial chromosome.Materials and Methods.-Bacterial strains and growth media: K12 strains: HfrC, W1485, W1485(X), KB (Benzer), C600 (Appleyard), JC12A (Hfr), AB313 (Hfr), AB331, CR63, P678, K12 (Tatum), 58, Y-9, 679-680; B strains: B, B/r, B Berkeley; C strains: C20, C129 (Hfr), C409; other strains: 15, W, ML35. In addition, a K12 strain, AB312PL, was prepared for this study since a strain with the Hfr characteristics of AB312 but strs was desired. It was derived from a 2-hr uninterrupted mating between AB312 (Hfr) and AB470 (F-)j and has the following characters: Hfr: thi-, strs, xyl-, mt-, gal-, lac-, pro-, Xa-. The origin and direction of insertion of its chromosome are indicated by the inner circle in Figure 3.Bacteria, either for the preparation of ribosomes or for genetic crosses, were grown in L broth. Minimal media employed contained M9 salts solution,6 0.2% glucose, 5 jug/ml thiamine, 1.5% Bacto-agar, and when appropriate, amino acids at 20 ,g/ml. EMB agar: 2.74% Levine eosinmethylene blue agar without lactose (Baltimore Biological Lab.) supplemented with 1.0% of the appropriate sugar.Bacterial crosses: Hfr and F-strains were grown at 370 in L broth to a density of about 2 X 108 cells/ml. Hfr, 0.8 ml, and 4.2 ml of the F-culture were mixed in a blank Petri dish and incubated at 370 without agitation. After 1 hr, samples of the mixture were centrifuged, resuspended in saline, and suitable dilutions were plated on minimal agar supplemented with the appropriate amino acids. Colonies appearing after 48 hr were picked and restreaked on the same media to purify them. Single colonies were then checked for nonselected nutritional markers, sensitivity to dihydrostreptomycin (100 gg/ml), and fermentation characteristics. The latter were checked by streaking on EMB-agar containing either maltose, xylose, or mannitol. Transduction with phage Plkc: Lysates of bacteriophage Plkc, prepared by growth on a strRdonor bacterium using the plate lysis method of Arber,7 were added to a strs-recipient at a virus/ bacteria input of 2 in the presence of 2.5 X 10-3 M CaC...

The binding of dihydrostreptomycin to ribosomes and ribosomal subunits of a number of different Escherichia coli strains was studied, and the Mg2+ and pH dependence, as well as the effect of salts and polynucleotides, was determined. The only requirement for binding with ribosomes and subunits from susceptible strains was 10 mm Mge. Monovalent salts weakened the binding in a manner similar to the effects on ribonucleic acid secondary structure, and this was antagonized to some extent by increased amounts of Mg2+. Bound dihydrostreptomycin could be readily exchanged by streptomycin and any antibiotically active derivative, but not by fragments of the antibiotic or any other aminoglycoside. With native (run-off) 70S ribosomes from streptomycin-susceptible strains, the binding was rapid and relatively temperature independent over the range from 0 to 37 C. Polynucleotides did not stimulate the binding. With concentrations of dihydrostreptomycin up to 10-5 M, greater than 95% of native 70S ribosomes bound exactly 1 molecule of the antibiotic tightly, with a KdiS, for the bound complex at 25 C of 9.4 X 10-8 M. The following thermodynamic parameters were found for the binding with 70S ribosomes at 25 C: AG0 = -9.6 kcal/mole, AH°= -6.2 kcal/mole, and AS0 = + 11.4 entropy units/mole. Differences in affinity for the antibiotic were found between ribosomes of K-12 strains and those of other E. coli strains. There was insignificant binding to 70S ribosomes or subunits from streptomycin-resistant or -dependent strains, and to 50S subunits from susceptible strains. The binding to 30S subunits from susceptible strains was weaker by an order of magnitude than that to the 70S particle, with a Kdi,, at 25 C of 10-6 M. Polyuridylic acid stimulated this binding slightly but did not influence the affinity of the bound molecule. At antibiotic concentrations above 10-5 M, streptomycin-susceptible 70S and 30S particles bound additional molecules of the antibiotic, and binding also occurred to ribosomes from streptomycin-resistant and -dependent strains, as well as to 50S subunits from all strains. Kdi8, for all of these binding equilibria were > 10-4 M. This weaker nonspecific binding coincided with the beginning of aggregation phenomena involving the particles, and occurred at sites distinct from the single site which binds the antibiotic tightly. This latter site was completely lost after the one-step mutation to highlevel resistance or dependence.

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