Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.