Biological characters of palindromic DNA.

Abstract

We have prepared three kinds of plasmids containing two lactose promoters in palindromic positions. In the first, structure I, the promoters are joined tail to tail and transcription proceeds from head to tail. In structure II they are joined head to head. In structure III a foreign DNA is located between the two lactose promoters. The plasmid of structure I can not multiply in wild-type Escherichia coli but can in E. coli recB recC sbcB recF. Since a plasmid with structure III can grow in the wild-type strain, a linear DNA fragment which is prepared from structure III by removing the foreign DNA can be used as a very efficient cloning vector. Plasmid DNA with structures I and II isolated from E. coli recB recC sbcB recF were shown to have a relaxed form by agarose gel electrophoresis. However, in contrast to the structure I plasmid, structure II plasmid can multiply in wild-type cells, though it is unstable and present in a low copy number. Excised head to head dimer, but not tail to tail dimer, moved at an unusual rate in polyacrylamide gel electrophoresis. The difference between the physical structures of the two dimers of the lactose promoter may be reflected in the biological character of the plasmids.Several laboratories have reported that a plasmid carrying palindromic DNA longer than 73bp could not multiply in wild-type Escherichia coli (1-3). LEACH and STAHL (4) reported that phage 2 carrying a long palindrome could not multiply in wild-type E. coli but could in E. coli recB recC sbcB. MIzuuCHI et al. prepared a palindromic DNA and showed it became cruciform when it was treated by DNA gyrase in vitro (S).We have tried to construct a plasmid containing two lactose promoters in palindromic positions. The five possible structures of a plasmid with two lactose promoters are shown in Fig. 1. Structures I and II have the same lactose promoters but their direction of joining is opposite. This minor difference had a significant effect on the viability of the plasmids and their physical structure. Structure IV was

Biological characteristics of palindromic DNA. II.

“…The 0.5 kb band corresponds to the selfannealed product. This is confirmed by heat denaturation as shown in lane 4 (8). The 0.4 kb fragment is derived from pDLAE535, for the purified pDLAE535 produces this fragment after BamHI digestion as shown in lane 4.…”

“…The total length of the former is 2.9kb and the latter is 5.5 kb. pDLl84 and pDLantl84 contain the lactose promoter and their characters are described in a previous paper (8). pDO184 is the same as pDLl84 except the total length is 11.2 kb.…”

“…Structural analysis of the plasmid DNA by agarose gel 394 YOSHIMURA et al VOL. 32 electrophoresis indicates that most of the extracted DNA had a closed but relaxed form (8).…”