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.

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

[This corrects the article on p. 100 in vol. 41.].

Energy conservation in chemotrophic anaerobic bacteria.

With a stepwise degradation and terminal labeling procedure the 3′-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-AOH. It is suggested that this region of the RNA is able to interact with mRNA and that the 3′-terminal U-U-AOH is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA·30S subunit complex.

https://www.pnas.org/content/pnas/71/4/1342.full.pdf

The 3′-Terminal Sequence of Escherichia coli 16S Ribosomal RNA: Complementarity to Nonsense Triplets and Ribosome Binding Sites

A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

Culture Medium for Enterobacteria

The penicillin method (Davis, 1948; Lederberg and Zinder, 1948) has permitted convenient isolation of auxotrophicl mutants of Escherichia coli with specific growth requirements for most of the known water-soluble vitamins, as well as amino acids, purines, and pyrimidines. Accordingly, when crystalline vitamin B12 became available, a search was made for mutants requiring this nutrilite. Several strains of the desired type were promptly recovered. In all cases methionine, but not homocysteine, could be substituted for the vitamin. This paper describes certain biochemical properties of these mutants, as well as of others responding to methionine but not to B,2.

Mutants of escherichia coli requiring methionine or vitamin b12

https://science.sciencemag.org/content/sci/170/3964/1279.full.pdf

Prospects for genetic intervention in man

Aromatic biosynthesis. VII. Accumulation of two derivatives of shikimic acid by bacterial mutants

Binding of sulfonamides by plasma proteins.

Effect of spectinomycin on polypeptide synthesis in extracts of Escherichia coli

To determine the length of secreted nascent polypeptide chain that is surrounded by membrane, we digested labeled nascent chains protruding from protoplasts of Bacillus subtilis with Pronase and isolated the residual ribosome-attached chains from the membrane-polysome fraction. Gel chromatography revealed a sharp major peak that had been protected by membrane plus bound ribosomes. The ribosomes themselves protected half as great a length. Because no free chain between the ribosome and the membrane was detected by Pronase treatment, the difference between the two protected lengths should measure the length protected by the membrane. More accurate measurements of these lengths, obtained by dansylation of the exposed NH2 terminus of the isolated fragments, yielded a difference of 21 amino acids. This value corresponds to an extended chain of 75 A, which is approximately the thickness of the bacterial cell membrane. We earlier presented evidence that bacterial ribosomes are attached to membrane solely by their secreted chain. The present results further show that after loss of the extracellular segment of the chain its attachment persists, at 37 degrees as well as 0 degrees C. These findings suggest that the chain does not slip through a passive membrane but is actively held within a channel.

Bernard D. Davis

Papers

Prospects for genetic intervention in man

Aromatic biosynthesis. VII. Accumulation of two derivatives of shikimic acid by bacterial mutants

Binding of sulfonamides by plasma proteins.

Effect of spectinomycin on polypeptide synthesis in extracts of Escherichia coli

Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis.