Regional replication of the bacterial chromosome induced by derepression of prophage lambda

Summary Derepression of prophage in E. coli strain K12 results in constitutive synthesis of the enzymes directed by the nearby bacterial operon, gal (escape synthesis). Phage 82 fails to cause escape synthesis despite that it lysogenizes the strain K12 at the site identical to that of on the host chromosome. The reason for the observed difference between 82 and is studied in the light of the recent finding that escape synthesis in -lysogen is closely associated to phage-promoted replication of bacterial chromosome contiguous to the prophage including gal operon (escape replication). Excision-defective mutants from 82, 82int or 82xis, do initiate escape synthesis, suggesting that the prophage 82 is normally excised too quickly after induction to allow sufficient escape replication. In support of this, much more DNA hybridizable to bacterial DNA contained in gal accumulates after induction of 82int than after induction of 82. Studies with various hybrid phages between 82 and have suggested: 1. The occurrence of gal escape synthesis depends on the nature of the region between b2 and N in the map. 2. Regions of the 82 genome on both sides of the attachment site contribute independently to prevent gal escape synthesis. Implications of these results are discussed with regard to the factors involved in the prophage excision.The IIIrd article of this series is in Molec. Gen. Genet. 159, 185–190 (1978)     

Regional replication of the bacterial chromosome induced by derepression of prophage lambda

Summary We have demonstrated previously by DNA-DNA hybridization that induction of phage with wild type O and P genes results in an increase of bacterial DNA in the chromosomal region adjacent to the left of the prophage, that is a segment between gal and att (gal DNA) (Imae and Fukasawa, 1970). Evidence is presented in this report that such an increase of bacterial DNA is also seen in the region to the right of the prophage; a segment between bio and att (bio DNA). We postulate therefore that the bidirectional replication of DNA extends beyond the prophage and copies the neighboring host DNA until the prophage is excised. The model is verified by making use of excision-defective phages. The synthesis of gal DNA (or bio DNA) slows down to a halt within 40 min after the induction in the normal lysogens. The results are attributed to the prophage excision: (1) In lysogens for int, synthesis of the bacterial DNA continues for longer times. (2) The synthesis of the bacterial DNA slows down to a halt in lysogens for xis or b2 as in the control. However DNA synthesis also slows down in parallel so that the amount of the bacterial DNA relative to that of DNA synthesized by a given time stays constant from 20 min to 80 min. During that time the relative amount of the bacterial DNA rapidly decreases in the normal lysogen.The first article of this series is in J. molec. Biol. 54, 585 (1970).     

Physical characterisation of the "Rac prophage" in E. coli K12.

Summary We confirm the hypothesis of Low (1973) that many E. coli K 12 strains contain a prophage (the Rac prophage) located a few minutes clockwise of the trp operon on the genetic map. We have used restriction endonucleases and ³²P-labelled probes to construct a physical map of this prophage. Some E. coli K 12 strains, including AB1157, have lost the entire prophage, apparently by a specific deletion. This is consistent with prophage excision by site-specific recombination. reverse (rev) phages (Zissler et al., 1971) are recombination proficient derivatives of phage in which the phage recombination functions have been replaced by analogous functions (RecE) derived from the host chromosome (Gottesman et al., 1974; Gillen et al., 1977). Our data support the origin of rev phages by recombination between and the Rac prophage following excision of the Rac prophage from the E. coli chromosome.Important experimental data are included in the Figure legends.     

W reactivation is inefficient in repair of the bacterial chromosome

Summary UV-inducible SOS processes associated with W reactivation of phage lambda were studied for their effect on repair of lambda prophage integrated in the bacterial chromosome. For this purpose, lambda cI857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30° to 42°. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30° after UV exposure W reactivation should be fully expressed and should also exert an effect on the bacterial chromosome and the prophage inside it. When heat-induction is delayed until the time at which W reactivation reaches its maximum, a considerable increase in phage survival might then be expected. The results presented in this report show, however, that the delayed induction had only a small effect on the survival of prophage in the wild-type strain (possibly attributable to excision repair) and no detectable effect on prophage in a uvrA strain. From these results we conclude that W reactivation is largely irrelevant to the repair of UV-damaged bacterial DNA.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Electron microscopic band-interband pattern of the X chromosome in Drosophila hydei

The band-interband pattern of the salivary gland X chromosome in Drosophila hydei was studied by electron microscopy (EM) using the technique of surface-spread polytene (SSP) chromosome preparation. We observed 526 chromosome bands, i.e. 135 additional bands as compared with the original light microscopic chromosome map (Berendes 1963). Individual interband lengths and band thicknesses were measured for the entire X chromosome in electron micrographs of ten SSP chromosome preparations. Average values were used to plot an EM chromosome map. The average interband had an axial length of 0.38 m. Depending upon the extension of the DNA packing ratio in interbands, this indicates 1.1 kb of totally extended DNA or 3.8 kb, if a DNA packing ratio of 0.10 m/kb is assumed for SSP chromosomes (Kress et al. 1985).     

Direction of SPP1 DNA replication in transfected B. subtilis cells

Summary The origin and the direction of replication of the SPP1 chromosome, which has a unique, nonpermuted sequence of markers, was established by determination of the frequency distribution of various markers along the SPP1 map. For this purpose replicating DNA was isolated from transfected competent B. subtilis cells. Marker frequencies were measured by means of helper mediated transfection. In the range defined by the genetic map, replication is unidirectional, originating from a point in the left part of the map. Shearing the DNA into halves prior to transfection permits only one round of replication of that half molecule which carries the origin.     

Prophage lambda induction caused by mini-F plasmid genes

Summary When bacterial cells harboring a temperaturesensitive replication plasmid, which carries the particular ccd segment of a mini-F plasmid, are transferred to 42°C, cell division is inhibited after incubation for an appropriate time. The inhibition occurs, when the copy number of the plasmid decreases to become critically low, about one per cell (Ogura and Hiraga 1983 b). In phage lysogens carrying this type of plasmid, the prophage is induced in a small portion of the cell population under the same conditions, in addition to the inhibition of cell division in most of cells. The prophage induction, but not the inhibition of normal cell division, depends on normal recA function. Both induction of prophage and inhibition of cell division are suppressed by the simultaneous presence of a replication proficient plasmid carrying the ccdA gene. We discuss molecular mechanisms of the ccd function that couples host cell division to plasmid proliferation and induces the prophage. Additionally, we propose a hypothesis that the ccd mechanism of F plasmid contributes to indirect induction of prophage by an F plasmid damaged by UV-irradiation and then introduced into a lysogen via conjugation.Abbreviations kb kilobase pairs. m.o.i., multiplicity of infection     

Prophage induction in Escherichia coli K12 cells deficient in DNA polymerase I.

Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA ⁺ polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.     

Effects of prophage Mu induction on expression of adjacent host genes

The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of -lactamase. The expression of thelacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.