To determine the time of entry by conjugation the Hfr strain is mixed with a recipient strain carrying a defective copy of a particular gene, "a". After conjugation has proceeded for a specific time, a sample of the mixture is removed. This is plated on agar that prevents growth of the Hfr and only allows growth of strains carrying the wild type version of gene "a". Survivors are derivatives of the recipient that have gained the wild type version of gene "a" from the Hfr. This is repeated for several time points. The whole procedure is then repeated for the other genes. In strain Hfr 1 (left panel), the integrated F-plasmid is closest to gene "d" and only begins transferring gene "a" after about 20 minutes. In strain Hfr 2 (right panel), the F-plasmid is integrated closer to gene "a" which therefore begins to appear in the recipient as early as five minutes after transfer begins.
Integrated F-plasmids may excise themselves from the chromosome by reversing the integration process. Sometimes they excise with pieces of chromosomal DNA, much like the way lambda forms specialized transducing phages. Recombination may occur between homologous sequences in the chromosome outside the F-DNA. The example shown in (Fig. 18.18) uses the IS1 sequences that are found in multiple copies in the E. coli chromosome but are absent from the F-plasmid. This creates F'- or F-prime plasmids which retain all of the F-plasmid and gain extra chromosomal DNA. F-prime plasmids may be transferred to recipient cells, carrying with them the chromosomal genes from their original host cell. F-primes are often used to carry part of the lacZ gene in the alpha-complementation method used for screening recombinant plasmids (see Ch. 22).
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