Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment.

A procedure is described that converts the pre-existing transposon insertion libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions. To enhance the efficiency of crossing-in the FRT-oriV cassette, we transiently increased the copy number of our retrofitting plasmids using a temperature-sensitive TrfA-supplying helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10KmR and three Tn10CmR insertions. Subsequently, the FRT-oriV retrofitted insertions were crossed with each other in pairs (KmRxCmR), using P1 phage transductions. The resulting CmRFRT-[28-65-kb]-KmRFRT strains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled Ptet promoter. Induction of this tightly repressed promoter by autoclaved chlortetracycline (cTc) resulted in the efficient excision and amplification of genomic fragments located between FRT sites, but only in productive strains, i.e. having two parallel FRTs. We have shown that genomic fragments of 28-, 40-, 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FRTs in non-parallel orientation), to productive combination of parallel FRTs, because one of the FRT elements was flanked by two convergent loxP sites, and thus could be inverted by the Cre function delivered either by the P1 phage or by a specially constructed temperature-sensitive Plac-cre plasmid. Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (prepared without the conventional cloning and its artifacts) for refined sequence comparison among strains and species, and for further analysis of uncharacterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated like any other large plasmids but only in hosts that could supply the appropriate Rep function. Our original 'pop-out' method [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398] was already employed for sequencing of the E. coli genome [Blattner et al. (1997) Science 277, 1453-1462]. Moreover, the Flp-mediated recombination between two FRT elements resulted in bacterial strains with large deletions (for parallel FRT orientations) or with large inversions (for inverted FRT orientations).

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing.

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

In vivo excision and amplification of large segments of the Escherichia coli genome.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

Comparison of the action of ionizing radiation and UV-light on lambda phage. Influence on phage adsorption, DNA injection, replication, and DNA repair.

The influence of gamma radiation, X-rays, UV-light at 254 nm and 365 nm, the latter combined with furocoumarin sensitizers has been studied on plaque forming ability, phage adsorption, DNA injection, and replication processes. UV-light (365 nm) plus furocoumarin treatment of phage particles gave rise to two types of DNA crosslinks. Type I crosslink corresponded to furocoumarin mediated covalent linkage between adjacent sites in opposite strands of the double helix. Crosslink type II (hairpin crosslink) required a highly condensed DNA and corresponded to the covalent linking of adjacent sites in double helical segments of a folded DNA molecule. The relationships of the type I crosslinks to inhibition of DNA replication and of the type II crosslinks to suppression of the DNA injection process are discussed. Pronounced deviations in phage inactivation have been obtained by X-ray radiation alone compared with UV-light (254 nm) pretreated and subsequently X-ray irradiated probes. The observed protective effect of the latter was described in terms of an inducible repair mechanism. The same protection has been observed by combination of gamma radiation with a sublethal UV-light (254 nm) dose.

The role of the direct repeat in qut-controlled antitermination in phage lambda.

For antitermination of transcription from the late p'R promoter of phage lambda, a cis-acting qut sequence, which overlaps with p'R, is required, together with the product of lambda gene Q. Using our BspMI-mediated multicycle technique for generation of precise deletions, we have confirmed that deletions removing DNA downstream of +18 bp (counted from the p'R-controlled transcriptional start point s'R = +1) do not affect the efficiency of qut antitermination; at the same time we found that deleting one more bp (shifting the right-hand boundary to bp +17) reduces antitermination by only 20%. Deleting another 5 or 6 bp (+11 or +12 bp right-hand qut boundary), decreases antitermination by about 80%. These deletions reduce the 9/10-bp-direct repeat (5'-TGGGT(A or T)AATT)2 in qut to only the five italicized bp. Similar strong reduction in antitermination (by about 68%) was obtained with +16 bp qut boundaries, in constructs which also contained 2- or 3-bp insertions between bp +11 and +12 or between +12 and +13. Since the latter deletions retain only 5/10 bp of the direct repeat, it appears that antitermination is dependent on the length and intactness of the direct repeat.

Photobiology of furocoumarins. Various types of crosslinking with DNA and their interference with the development of lambda phage.

It was shown that the multiplication of phage lambda was strongly suppressed by furocoumarins after irradiation with near ultraviolet light of 365 nm wavelength. Using xanthotoxin or angelicin there was a marked inhibition of the phage DNA injection and replication but adsorption was unaffected. This inhibition was attributed to various types of DNA crosslinking produced in the phage heads. Type I crosslink corresponded to covalent binding between adjacent sites in opposite strands of the double helix. Crosslink type II (hairpin crosslink) required a highly condensed DNA and corresponded to covalent binding between adjacent sites on double-helical segments of a folded DNA molecule. The relationships of the type I crosslinks to the DNA replication and of the type II crosslinks to DNA injection are being discussed. Like type II crosslinks, the nucleic acid--protein crosslinks hinder injection.

Cross-link formation of phage lambda DNA in situ photochemically induced by the furocoumarin derivative angelicin.

The combined action of 365 nm ultraviolet light and xanthotoxin or angelicin inhibits the injection of phage lambda into the host. For both furocoumarin derivatives the inhibition of injection is discussed in terms of photochemically induced cross-linking of the DNA inside the phage heads; Cross-linking of DNA has previously been described for xanthotoxin (Musajo, L. and Rodighiero, G. (1972) in Photophysiology (Giese, A.C., ed.), Vol. VII, pp. 115-147, Academic Press, New York and Scott, B.R., Pathak, M.A. and Mohn, G.R. (1976) Mutation Res. 39, 30-74) but not for angelicin. The electronic structures in the first excited states calculated by means of quantum chemistry according to the Pariser-Parr-Pople method are very similar for xanthotoxin, psoralen and angelicin. Hence angelicin should be capable of acting bifunctionally like xanthotoxin, but for sterical reasons such reaction should be possible only for the folded DNA, as in phage heads, but not for diluted aqueous solution of DNA.

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.

Fractionation of the complementary strands of coliphage lambda DNA based on the asymmetric distribution of the poly I,G-binding sites.

Poly I,G interacts preferentially with one of the two complementary DNA strands of lambda and lambda-related phages 21, 434, and phi80, thus permitting preparative separation of the "dense" fraction, consisting of a complex between the intact strands C and poly I,G, from the less dense ("light") fraction containing the intact strands W, which bind 3-4 times less poly I,G. The isolated and self-annealed fractions are over 99% pure as far as their hybridization properties with complementary RNA are concerned. The interaction of poly I,G with intact and fragmented DNA of lambda and its deletion mutants could be interpreted as indicating the asymmetric distribution of poly I,G-binding deoxycytidine-(dC)-rich clusters between the strands of lambda DNA; on the left arm (55% G + C) the dC-clusters seem to be restricted to the C strand, whereas both complementary strands of the right arm (46% G + C) of lambda DNA contain these dC clusters. Thus, the two arms of lambda DNA differ not only in their average base composition (Hershey, 1966), but also in the mode of distribution of the dC-rich clusters, the latter possibly related to the initiation, termination and orientation of the DNA-to-RNA transcription from the complementary DNA strands.