Electron Micrographic Mapping of Deletions, Insertions, Inversions, and Homologies in the DNAs of Coliphages Lambda and Phi 80

Abstract

The study of the functional organization of the DNA molecules of several bacteriophages has been the primary concern of this laboratory during the past few years (see the reviews of Szybalski, 1969see the reviews of Szybalski, 1970, and Szybalski et al., 1969, and Szybalski et al., 1970). A major effort has been devoted to the elucidation of the patterns and controls of transcription in the lambdoid coliphages (e.g., Szybalski et al., 1966; Taylor et al., 1967; Kumar et al., 1969; Lozeron and Szybalski, 1969; Bøvre and Szybalski, 1969; Nijkamp et al., 1970). Physical mapping of the genes of these phages is an indispensable tool in the quantitative study of the transcription patterns, and has led us to develop a novel technique, designated electron micrographic mapping of heteroduplex DNA (Westmoreland et al., 1969). In this procedure the strands of two distinct phage DNAs are preparatively separated, one strand of the first DNA is annealed to the complementary strand of the second DNA in a formamide-containing solution, and the resulting heteroduplex DNA molecules are examined by electron microscopy. The regions of homology in the heteroduplexes appear as double-stranded, more rigid DNA segments, whereas unpaired single strands, thinner and kinkier, are visualized in the nonhomologous regions. Simple deletions or insertions result in single-stranded “loops” emanating from one point of the otherwise uninterrupted double-stranded DNA (see point 1 in Fig. 2), whereas substitutions or inversions appear as “bubbles,” i.e., interruptions in the double-stranded DNA composed of two single unpaired strands (see region 2 to 3 in Fig. 2). Applying this technique, which...