Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.     

Function of phospholipids in Escherichia coli. Characterization of a mutant deficient in cardiolipin synthesis.

Screening of a collection of temperature-sensitive mutants of Escherichia coli for defects in phospholipid metabolism led to the isolation of a mutant deficient in cardiolipin synthesis. The defective gene, named cls, is closely linked to the trp marker and maps at about Minute 27 on the E. coli chromosome. After transfer of cls to a defined genetic background by transduction, the mutant has the following properties as compared to an isogenic wild type. Exponentially growing cells show a reduction in cardiolipin content by a factor of at least 15 (less than 0.2 mol % of the total phospholipids). A crude membrane fraction derived from the mutant is unable to synthesize cardiolipin from phosphatidylglycerol in vitro. The mutant has no distinctive phenotype regarding its growth properties, membrane-associated respiratory functions, or the ability to insert bacteriophage M13 coat protein into the cell envelope. The cls mutation confers a 5-times reduction in the turnover of the phosphate moiety of phosphatidylglycerol.