Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycinresistant strains contained the same two plasmids: pABl18A of molecular weight 4.9 x lo6 (7.0 kilobases) and pAB118B of molecular weight 3-0 x lo6 (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 x los (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2-5 x lo6 (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis 168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.
Using gene-manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine-Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5' end of the message and the Shine-Dalgarno sequence drops below 15 bases. Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine-Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter-distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shows that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.
A restriction endonuclease cleavage map of the tetracycline resistance plasmid pAB 124, originally isolated from Bacillus stearothermophilus, was constructed using ten enzymes. Tetracycline resistance was associated with a 1-95 megadalton (Md) region of pAB124 lying between two EcoRI sites, and this region was circularized to produce a viable tetracycline resistance plasmid (pAB224), with two EcoRI fragments of pAB124 deleted amounting to 0.95 Md. A second plasmid (pAB524) with one EcoRI fragment (0-6 Md) of pAB124 deleted was also constructed. Restriction endonuclease cleavage maps of pAB224 and pAB524 were constructed.
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