Location of the endA gene coding for endonuclease I on the physical map of the Escherichia coli K-12 chromosome.

The endonuclease I of Escherichia coli is an RNA-inhibitable DNase which is located in the periplasmic space of the E. coli cell (3); it cleaves native DNA into oligonucleotides (7). It is assumed that in the noninhibited, RNA-free state the enzyme introduces nucleotide sequence-independent doublestrand breaks into DNA (2, 9), whereas the inhibited, RNAcomplexed enzyme probably causes only nicking ofDNA in the presence of high concentrations of salt (5). The enzyme is not essential for the cell, and its physiological role is unknown. Endonuclease I-deficient mutants resemble the wild type in growth rate, ability to propagate phage, and conjugational properties (4). The endA mutations were mapped near 64 min on the genetic map of the E. coli chromosome (1). We report the location of this gene on the physical map of E. coli and the direction of its transcription. We devised an improved method to locate genes which determine a nonselectable phenotype on X clones of the Kohara library of E. coli (6). Cells of an endA mutant (AB1886 endA; strain H520, obtained from H. Hoffmann-Berling) were coinfected with one of the Kohara X clones and a X cI857 helper phage carrying a partially deleted TnlO insertion with a functional tetracycline resistance gene (11). Phage suspensions (107 PFU/ml) of the Kohara clones and the helper were mixed in a 2:1 ratio, and 10 ,ul of the mixture was spotted on the top agar of a Luria-Bertani agar plate seeded with 109 stationaryphase H520 cells. After incubation of the plate for 24 h at 30°C, cells from the lysis zone were suspended and grown at 30°C overnight on LB plates containing 15 jig of tetracycline per ml. The colonies obtained were replicated to filter paper and stained with methyl green by the method of Wright (13). Green colonies are endA; colorless colonies are endA . Among the tetracycline-resistant colonies, about 3% were EndA+ when the coinfecting phage was X474 and about 2% were EndA+ when the coinfecting phage was X475, indicating that they carry the endA4 gene. No EndA+ clones were obtained with the flanking Kohara X clones 471, 472, 473, 476, 477, 478, and 479. Selecting lysogens in this way avoids the necessity of screening X-resistant clones, which normally constitute more than 90% of the survivors in the lysis zones. The inserts in Kohara X clones 474 and 475 overlap by about 10 kb (Fig. 1). The approximately 1.3-kb HindIIl fragment of the X475 insert (6) was subcloned into a pBluescript vector. The resultant plasmid complements the endA mutation in the methyl green staining assay (13). Sequencing of the HindIII fragment and identification of the endA gene indicate that transcription occurs in a clockwise direction on the standard E. coli map (5a). Thus, the endA gene is located at position 3104 of the physical map (6), between genes metK and mutY near 63.6 min of the genetic map (1), as shown in Fig. 1.