[171] On the infection of bacteria with λDNA—the helper-infected system

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This chapter Active DNA can be isolated from phage particles by several different methods if care is taken to avoid degradation by mechanical shear or by nuclease action. The following methods yield products of comparable activities—extraction with aqueous phenol at 5°, lysis by sodium dodecyl sulfate 7, and lysis with 2 M Mg⁺⁺. The third method of lysis consists of warming phage to 45° in 2M MgC1₂ until the light scattering, measured around 300 mμ, falls to a minimum. All the strains of bacteria, sensitive to λphage, which have been tried, are sensitive to XDNA; these include K12 strain W3104, K12 strain C600, and Escherichia coli strain C. Cultures may be grown in broth, in salts plus glucose, or in salts plus glycerol. The cultures may be in the exponential or the resting phase. As exponential cultures give more reproducible results, they are preferred. The efficiency of infection of whole bacteria with λDNA is less than 10^–4 as high in the absence as in the presence of complete phage particles, the helper phage. Genetic markers can be used to identify the phage offspring of the DNA in the presence of the offspring of the helper phage. The mechanism by which helper infection generates competence is obscure. The helper phage DNA, as well as its protein, seems to play a role because the sensitivity to ultraviolet light of helping activity is only two and one-half times greater than the sensitivity of its plaque-forming ability.