Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli

Abstract

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coliC600r-m- with the ligated mixture after enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin E1. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin E1 factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. Coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.

New cryptic plasmid of Bacillus subtilis and restriction analysis of other plasmids found by general screening

“…In an attempt to attach a genetic marker to one of the cryptic plasmids, we constructed a composite plasmid pTA2010 (12) between E. coli plasmid RSF2124 (ampicillin resistance and colicin El productivity) and pTA1060 of B. subtilis. (The plasmid prefix of our laboratory was changed from pAT to pTA according to the assignment by the Plasmid Reference Center.)…”

“…(The plasmid prefix of our laboratory was changed from pAT to pTA according to the assignment by the Plasmid Reference Center.) The composite pTA2010 cloned in E. coli conferred ampicillin resistance to the host (12), but it could not be introduced into a restrictionless and highly transformable mutant strain RM125 of B. subtilis 168 (29), in spite of many trials to get an ampicillin-resistant transformant (S. Horinouchi, T. Uozumi, and T. Beppu, unpublished data). This failure is not unexpected, because E. coli genes such as the ampicillin resistance gene of pBR322 (5) so far tested have been reported not to be expressed in B. subtilis, although some genes of B. subtilis can be expressed in E. coli (6, 22).…”

Cloning and Expression of a Gene for Resistance to Butirosin both inEscherichia coliandBacillus subtilis

“…10 ) Plasmid DNAs were isolated from B. subtilis as previously described. 11) Plasmid DNAs in E. coli were prepared according to Horinouchi et al 8 ) Large scale preparation of plasmid DNAs was performed by CsCI-ethidium bromide density gradient centrifugation. In this figure, we describe five recombinant plasmids.…”

Electron Microscopy and Restriction Endonuclease Analysis of RSF2124-pLS11 Composite Plasmid ConstructedIn Vitro

“…3) In forming a heteroduplex DNA molecule by annealing the mixture of related different DNAs of single-stranded form, the region that is identical in base sequence or very near so forms a double helix, while the region of nonhomology does not anneal and remains singlestranded. The nonhomologous regions are distinguishable in electron micrograms.…”