To the Editor: Application of DNA or RNA probes to microbiology and virology requires probes free of vector sequences, particularly vectors of bacterial origin, such as pBR322. Incomplete purification of inserts from vector sequences can produce a falsely positive result when nucleic acid probes are applied to tissue or body fluids that may be containmated with bacteria. A plasrnid vector such ...

The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures. Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322. No segregation of the primase-encoding RP4 derivative was observed.

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduc...

Transcripts originating from the SV40 late promoter of pSV2-neo or pSV2-cat contain pBR322 sequences and are polyadenylated at the SV40 late poly(A) site, resulting in an RNA of 3500 nt. If the SV40(L) poly(A) signal is destroyed, late orientation transcripts are polyadenylated at a site within pBR322 sequences, yielding in an RNA of 2500 nt. This cryptic poly(A) site is located 42-46 nucleotid...

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replic...

Proteins cross-linked to pBR322 mRNAs and DNA by formaldehyde treatment of intact Escherichia coli cells have been detected with the use of a novel detection method. Among the proteins cross-linked to pBR322 mRNAs were S1, S21, and at least six other proteins of the small ribosomal subunit, initiation factor 1, elongation factor (EF) Tu, and very small amounts of EF-G and EF-Ts. The single stra...

We have demonstrated in vitro the existence on the plasmid pBR322 of a promoter signal that is strictly dependent on cAMP and its receptor protein CRP. Transcription initiates with pppG at nucleotide 2270 and proceeds counterclockwise on the standard pBR322 map. DNase protection studies show that CRP selectively binds to the -35 region of the promoter. This region exhibits strong structural hom...

Entrapment of pBR322 DNA within liposomes was demonstrated by (i) its comigration with liposomes on Sepharose 4B columns, (ii) resistance of its biological activity to DNase digestion, and (iii) identification of plasmid DNA on agarose gels after lipid extraction. The biological activity of the liposome-entrapped plasmid was determined by transformation assays. The incubation of intact liposome...

Plasmid pBR322 DNA isolated from topoisomerase I mutants of Escherichia coli and Salmonella typhimurium exhibits a distinctive supercoiling distribution characterized by an extremely heterogeneous distribution of linking numbers that contains highly negatively supercoiled topoisomers. Analysis of the supercoiling distributions of deletion and insertion derivatives of pBR322 shows that the prese...