The strands of DNA from lambda and related bacteriophages: Isolation and characterization

Abstract

Each of the intact complementary strands of DNA from bacteriophage λ has been isolated by virtue of the different buoyant densities they exhibit in alkaline CsCl. Preparations of a given strand are free (< 1%) of the other strand and do not contain detectable amounts of fragments. Individual strands exhibit no significant activity for the genes of λ in the helper-phage assay of Kaiser & Hogness (1960), but renaturation of equimolar mixtures of the two isolated strands yields duplex molecules active for genes which span the λ genome. The difference in density observed in alkaline CsCl ( Δ ϱalk ) is not seen in neutral CsCl and is presumed due to a difference between strands in the sum of the frequencies of the titratable bases, G and T (ΔGT). This ΔGT has been estimated to involve about 1500 out of 50,000 base pairs in λ DNA, and essentially all of these have been located in its left half. Comparison of these results with the binding of poly rG to the strands in whole and half molecules of λ DNA indicate that in a given segment of this DNA the strand with the lesser GT content binds the greater amount of poly rG. The DNA's from a λdg variant and from the λ-related phage, 424, exhibit values of Δ ϱalk sufficient for the isolation of their strands. Four different types of 424 phage particles were observed on the basis of their buoyant densities in neutral CsCl. The most and least dense of these contain DNA that is 97 and 83% the size of λ DNA, respectively, and the estimated number of base pairs responsible for the ΔGT in each is about 1900. Heteroduplex molecules can be formed between strands of DNA from λ and the most dense 424 particle, providing the strand of highest GT content from one phage is combined with that of lowest GT content from the other. Such heteroduplexes exhibit densities in neutral CsCl midway between the densities of native and denatured DNA, indicating an extent of homology of about 50% between the two phages.