Construction of plasmids carrying the cI gene of bacteriophage lambda.

Abstract

XKH54h8o; in some cases, both se~tions were performed simultaneously. The immunity of plasmid containing strains was further tested by streaking single colonies across a streak of phage on an agar plate. Colonies of transformants which constitiltively synthesized f3-galactosidase appeared blue on agar plates containing a chromogenic, noninducing substrate, 5-chloro-4-bromo-3-indolyl-,B-D-galactoside (40 Ag/ml). Assays of X repressor (12), fl-galactosidase (f3-D-galactoside galactohydrolase, EC 3.2.1.23) (13), and the isolation of operator containing DNA fragments on nitrocellulose filters (14) have been described previously. Purified X repressor was a gift of R. Sauer and purified lac repressor was a gift of A. Maxam. Experiments were carried out in a P1 (EK1) facility. RESULTS Preliminary considerationsWe wished to proceed in two steps; first, to clone cI flanked by as little extraneous phage DNA as possible on a plasmid and then to insert a DNA fragment bearing the lac promoter near the beginning of cI. Two problems arose. No single restriction endonuclease cleaves X DNA just outside the ends of cI without also cleaving within it, and no restriction endonuclease site suitable for inserting the lac promoter exists in X DNA near the beginning of cI. We adopted a strategy based on the following considerations: gene cI can be neatly isolated on two DNA fragments, one of which bears two HindIII ends and the other of which bears one HindIII end and one HaeIII end (see Fig. 1). Proper joining of these fragments reconstitutes cI on a larger fragment bearing one HindIII end and one HaeIII end. HindIII ends are staggered and readily anneal to each other, whereas HaeIII ends are flush. Staggered ends which anneal to each other can be joined by T4 polynucleotide ligase (15), and flush ends can be joined to other flush ends by that enzyme (9). To clone our cI fragment, we sought a plasmid which could be opened so as to produce one HindIII end and one flush end. The plasmids we used, pCR11 and pMB9, each have a single HindIII site and a single EcoRI site. Although EcoRI produces staggered ends, we anticipated that these ends could be converted to flush ends by treatment with DNA polymerase I and the four deoxyribonucleotide triphosphates, because the recessed 3' end can be extended by copying the protruding 5' end of the complementary strand (a process we hereafter refer to as filling-in). Precise joining of a filled-in EcoRI end to a HaeIII end should produce a molecule recognized by EcoRI at the junction (see Fig. 2). This regenerated EcoRI site near the beginning of cI could then be used as a site to insert the lac promoter.

Studies on the ω Subunit of Escherichia Coli RNA Polymerase

“…Plasmids used for the preparation of truncated DNA templates were as follows: pKB252 (Backman et al, 1976) for the lacUV5 promoter, pSP261 (Pedersen et al, 1984) for the rpsAPl promoter and pSU"6 (Yoshimura et al, 1984) for the leuX promoter. All the above plasmids were obtained from Prof. Ishihama (National Institute of Genetics, Japan).…”

Probing the active site of flavocytochrome b2 by site‐directed mutagenesis

“…Escherichia coli strain MM294 [9] was used for plasmid propagation and NM522 for propagation of M13 recombinants and preparation of single-stranded plasmid DNA. The bacteria were grown in L-broth [lo] with the addition of ampicillin (100 pg ml-') as appropriate.…”

Inactivation and proteolytic cleavage of phage lambda repressor in vitro in an ATP-dependent reaction.

“…1 illustrates an experiment in which increasing amounts of extract were incubated with a constant amount of repressor. As a convenient source of repressor we used a small amount of crude extract of E. coli strain 294(pKB252), which contains up to 50 times as much repressor as a normal lysogen because it carries a plasmid containing the X repressor gene (9). As a control we used an extract of strain DM1180 (5), which is the parent of DM1187 and does not display constitutive repressor inactivation in vivo.…”