TRANSFORMATION OF CA⁺⁺-TREATED <i>recA</i> DERIVATIVE OF <i>ESCHERICHIA COLI</i> K12 BY λ<i>dv</i> DNA

Abstract

Plasmid Adv is an autonomously replicating circular DNA, which was derived from coliphage A, and which is perpetuated in E. coli cytoplasm (Matsubara and Kaiser 1968) . This plasmid, consisting only of those genes and structures that act in the process of regulation and replication, can neither be wrapped in phage coat protein, nor conjugally transferred by itself between cells. The non-transmissibility, or "inertness" of this and several other similarly non-transferable plasmids (for a review, see Clowes 1972), has limited the studies on genetic organization of these genomes and on regulatory mechanisms acting in replication of the plasmids. Recently it was discovered that bacterial cellular surface is altered by treatment with Ca++ in such a way that the cells can uptake phage DNA (transf ection) (Mandel and Higa 1970;Sjorstrom et al. 1972). Cohen et al. (1972) applied this method and transferred purified R-factor DNA into E. coli cells. Cosloy and Oishi (1973) found that the Ca-treated E. coli cells can be transformed by chromosomal markers if the recipient cells lack recB, C pathway, but carry recF, J, K pathway.In this communication, we will report that Ca-treated recA-recB+, C+ derivatives of E. coli K12 cells are good recipients for transformation by "inert" plasmid Adv DNA. Advl (Matsubara and Kaiser 1968) and Adv021 (Berg 1974) DNA were prepared as cytoplasmic circular DNA as reported elsewhere (Matsubara and Kaiser 1968). Transformation was done by a modification of the procedure described by Cohen et al. (1972) : E. coli K12 strain Km723 (F-, strR, his, recAl-, su-, galde'), or its thymine-requiring derivative, Km960, was grown at 37° in P medium (Cohen et al. 1972) to a concentration of 6 X 108 cells per ml. Cells were chilled, collected by sedimentation and washed with 0.5 volume of 0.01 M NaCI. The cells were resuspended in half the original volume of chilled 0.05 M CaC12, kept at 0° for 20 min, sedimented and then resuspended in 0.1 times the original volume of chilled 0.075 M CaC12. An aliquot (0.2 ml) of the CaCl2-treated cells was added to 0.1 ml of chilled DNA sample in TEN buffer (0.02 M Tris-HCI, pH 8.0; 0.001 M EDTA; 0.02 M NaCI) by using chilled pipettes in cold room, and incubated for 60 min at 0°. This reaction mixture was diluted 10 fold with PBB medium (Matsubara 1974) at 0°, and aliquots (0.2 to 0.8 ml) were taken, admixed with 7 x 106 particles of each of 2c190 and Ah8OimmAcl90 phages (Ac190 phage whose "left arm", viz, genes A, B through J is substituted by that of X80), and spread over PBB agar

Transfection of Enterobacteriaceae and its applications.

“…In fact, shifting the infected cells to 300C produces a decline of infective centers (149,395). At 00C, 40% of the OA DNA infective centers be-203 VOiL 42, 1978 on July 6, 2020 by guest http://mmbr.asm.org/ Downloaded from come resistant to DNase only seconds after addition of DNA to calcium-shocked cells (393,395).…”

In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities

“…Transformation. Xdv DNA in the circular form can transform Ca2+-treated RecA-cells to new carriers of Xdv (12). The efficiency of transformation is about 10-6/molecule.…”

“…We constructed Xdvl molecules in different oligomeric forms in an in vitro system, by incising this plasmid DNA at a single site per monomer unit using EcoRI endonuclease (13) and joining the "cohesive ends" with DNA ligase (20). The resulting molecules were then used to transform cells (12). In this way, strains that perpetuate monomeric Xdvl were obtained and their properties were examined.…”

Transfection of Enterobacteriaceae and its applications

“…In fact, shifting the infected cells to 300C produces a decline of infective centers (149,395). At 00C, 40% of the OA DNA infective centers be-come resistant to DNase only seconds after addition of DNA to calcium-shocked cells (393,395).…”