Studies on bacteriophage M13 DNA. 2. The gene order of the M13 genome.

The double-stranded replicative form DNA of bacteriophage M13 was cleaved into 13 specific fragments by the restriction endonuclease from Haemophilus aphirophilus. The individual DNA fragments from wild-type replicative form molecules were then annealed to circular, single-stranded DNAs of phage M13, bearing amber mutations as genetic markers. When such DNA hybrids infected competent Escherichia coli cells, only those duplexes which were genetically heterozygous gave rise to wild-type phages in the progeny. In this way, the genetic markers carried on the individual DNA fragments could be determined. In addition, marker rescue in each gene was obtained with the 10 specific fragments of M13 replicative form DNA, produced by cleavage with the restriction endonuclease from Haemophilus aegyptius. From these results and the enzyme cleavage maps of both types of restriction fragments a distribution of genetic markers along the physical map could be obtained, which allowed an arrangement of M13 genes into a genetic map. Evidence is presented that the gene order of M13 is: IV-(I,VI)-III-VIII-VII-V-II with II and IV being contiguous on the circular map.