Second EcoRI fragment of F capable of self-replication

Abstract

The cloning of fragments of F' plasmid deoxyribonucleic acid produced by restriction endonuclease EcoRI has revealed that fragment f7, not previously suspected to have replicative properties, is able to replicate autonomously. The ability of f7 to replicate was observed when it was cloned with fragments coding for resistance to either kanamycin or streptomycin and sulfonamide. Such f7 miniplasmids have been obtained from an F'lac+ and two F'gal+ temperature-sensitive mutant plasmids and from the unmutated F plasmid. Plasmids containing both f5 and f7 fragments were also obtained. Expression of resistance to "female-specific" bacteriophages requires that f5 and f7 be present in the same plasmid since cells containing separate f5 and f7 plasmids are not resistant to bacteriophage phi II. f7 plasmids were less stable than miniplasmids containing f5, particularly at fast growth rates. The bearing of these results on the isolation and behavior of temperature-sensitive F mutants is discussed.

Molecular Homology and Incompatibility in the IncFI Plasmid Group

“…Media, agarose gel electrophoresis, nick translation of fragments. and DNA preparation have been described previously (Jamieson & Bergquist, 1977;Jamieson et al, 1979;Lane & Gardner, 1979;Bergquist et al, 1980).…”

“…the assignment of the functions and the restriction enzyme cleavage sites in the f5 and f 7 fragments are described in detail in Lane ( 198 1). ori-l and ori-2 are the primary and secondary origins of replication; 2" rep is from Gardner (1979); pif is from Skurray et al (1976); the incFI homology region (here termed incD) from Palchaudhuri & Maas (1977); sta is the stability function associated with incD (Lane & Gardner, 1979). Regions of sequence homology with the F replication regions, as determined by heteroduplex analysis (Palchaudhuri & Maas, 1977), are shown as hatched blocks in the lower part of the figure.…”

“…The genes responsible for inhibition of the growth of female-specific bacteriophage (Pi0 also are located in the f7 fragment in the vicinity of F kilobase coordinate 38 (Skurray et al, 1976). The expression of the Pif+ phenotype by mini-F plasmids depends on the f5-f7 junction of F remaining intact (Lane & Gardner, 1979). Hence the genetic loci are located at the right and left ends of the two fragments (Fig.…”

“…This result suggests that ColV3-K30 replicates from a region analogous to the secondary replicon of F, and expresses the incompatibility associated with this region. We examined the incompatibility relationships between ColV3-K30 : : Tn5 and PAC 184-f7, since plasmids composed only of f 7 and an antibiotic resistance determinant are unstable (Lane & Gardner, 1979). By itself, pAC184 is compatible with ColV3-K30, pHH507 and p307.…”

Identification of the pifC gene and its role in negative control of F factor pif gene expression

“…The pif region of the F factor and substituted F' factors contains at least two genes, pifA and pifB, that interfere with the normal development cycle of bacteriophage T7 (1,16,20,22). Studies with mini-F plasmids containing portions of the F factor cloned into pSC101 (5,22,24) or existing as independent replicons (15) have established that the pif region is located within EcoRI fragments f5 and f7 of F. We have recently described the subcloning of the pifregion into pSC101 (22). One of the constructed Pif+ plasmids, pGS103, contains a 5.2-kilobase (kb) PstI fragment with coordinates 38.9 through 44.1 kb on the F factor physical map, derived from DNA on both sides of the EcoRI site between fragments f5 and f7 as shown in Fig.…”