The p lambda CM system: phage immunity-specific incompatibility with IncP-1 plasmids

Summary A pCM2 replicon derived by an N^– deletion from ::Tn9 which carries the imm434 immunity region is incompatible with some (but not all) IncP-1 plasmids. The imm pCM1 replicon does not show the same incompatibility behavior.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

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).     

Studies on lambda virulent mutants

Summary The clearish plaque mutants virC which were isolated from true-virulent, virLvirCvirR (virLCR), do not complement CI mutants but CII, CIII and mutant (c ₄₂) for lysogenization. No complementation for lysogenization was observed between virCR and any CI, CII, CIII or y mutants. No lysogen was obtained when virC or virC carrying susN, susO or susP was infected to -sensitive sup ^- host. This was also true for virCR. Infection of ind ^- lysogen with virCRsusNO(P) or virCsusNO(P) results in marked prophage induction. Effect of virCRsusNO(P) on prophage induction is stronger than that of virCsusNO(P). These results suggest the existence of gene(s) for anti-repressor. When virCsusNO(P) or virCRsusNO(P) was infected to W3350 sup ^- at high m.o.i., lysogen in anti-immune state and that in weak-immune state was obtained, respetively. Wild type phage forms clear plaque on virCsusNO(P) lysogen with e.o.p. of one and no plaque on virCRsusNO(P) lysogen. T4rII can plate on both lysogens. This weak-immunity caused by virCRsusNO(P) prophage is different from CI immunity and not abolished by irradiation of ultraviolet light (hereafter this is referred to as the vir-immunity). Action of anti-immunity and vir-immunity are almost specific. Possible functional sites for anti-and vir-immunity substances are suggested to be virL and virR regions. A hypothesis was presented that the vir-immunity may caused by the overproduced anti-immunity substance coded from x region.This material has been published as an abstract in Jap. J. Genetics 45, 479 (1970).     

Normal expression of the viral gene N interferes with growth of bacteriophage lambda in Escherichia coli 15T-.

Summary Growth of and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T^-. Mutants of which overcome this inhibition map in gene N. Some of these hty mutants are temperature sensitive for growth on E. coli K12. Thus plating of on strain 3057 allows one to isolate temperature sensitive N mutants. The hty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus ^- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate can be at least partially reversed by addition of thymidine to the medium and the growth difference between hty and in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by in 3057 at low thymidine concentration is lower than that produced by hty under the same conditions. Only a small percentage of the DNA produced by in 3057 is packaged into viable phage particles. This suggests that not only produces less DNA in 3057 than hty but that an important part of the DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable to grow on E. coli 15T^- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over N ⁺ which confirms the idea that N activity or the activity of a gene under N control interferes with growth in 3057 at low thymidine concentration.     

Directed integration of an F'' plasmid by integrative suppression: isolation of plaque forming lambda transducing phage for the dnaC gene.

Summary A new approach for isolation of a plaque forming specialized transducing phage is described. It consists of directed transposition of an F plasmid into the gal region of a dnaAts galE ^- Escherichia coli strain by integrative suppression and deletion of the chlD region in order to shorten the distance between the marker of interest on the F and the prophage serving to prepare an LFT¹ lysate.An F danC ⁺ thr ⁺ plasmid was used here and dthr and ddnaC phages were isolated. In addition, pdnaC was obtained from a double lysogen for ddnaC and b2.     

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA

Summary The purified bacteriophage replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of O and one molecule of P. The O-P oligomer is active in the in vitro replication of ori-containing DNA.Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the replication proteins and ori DNA. It was found that the P protein binds specifically to ori-containing plasmid DNA only in the presence of O protein. About 100 molecules of O and 10 molecules of P form a complex with the ori DNA. The DNA-O-P complex was shown to be active in an in vitro replication system.Since the physical interactions between ori and O and between P and the Escherichia coli dnaB replication protein are well documented, the evidence for a O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables to direct the host replication machinery to the replication of its own DNA.     

Transduction of nitrate reductase loci of Escherichia coli by phages P-1 and lambda

Summary A lactate-nitrate medium suitable for genetic studies with nitrate reductase mutants (nar ^-) of Escherichia coli was devised. This permitted the selection of nar ^- strains by their failure to use nitrate as terminal electron acceptor during anaerobic growth, in addition to the selection procedure based on the chlorate resistance of nar ^- mutants. Transduction studies with phage P1 and nar ^- mutants from both sources demonstrated the existence of at least three nar genes in the gal region of the E. coli linkage map, their relative positions being: gal .... narF .... bio .... narD .... narE. Using phage cotransduction of narD with bio was observed and several independently-isolated defective -transducing phages were examined. Phage also transduced the narF gene with gal linkage but the narE gene was not -transducible.     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Summary Genetically marked and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the protein coat were selected on WR4027 (), a -immune, P22-resistant derivative of WR4028. In these immP22 hybrids, at least the c through P genes of were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (, immP22). One such hybrid, immP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that immP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for immP22dis. In addition, immP22dis contains the P22 a1 locus responsible for somatic 0–1 antigen conversion in Salmonella. Although the immP22dis phage particle has the head and tail, the phage genome also carries P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the immP22dis prophage near the proA locus on the bacterial chromosome.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

Induction of lambdoid prophages by amino acid deprivation: Differential inducibility; Role of recA

Summary Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and i²¹ are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of repressor with a cI carrying-plasmid prevented amino acidless induction of as did the ind ^– mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. i⁸⁰ was inducible both by amino acid and thymine deprivation.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

Tandem duplication induced by an unusual ampA1-, ampC-transducing lambda phage: A probe to initiate gene amplification

Summary Secondary attachment site -lysogens were isolated in an Escherichia coli strain carrying multiple tandem 9.8 kb repeats. The repeat carried the structural gene for chromosomal -lactamase, ampC. One lysogen produced lysates with amp-transducing activity. Three types of phages with different densities were obtained from this lysogen. The one with the lowest density was found to be a helper cI857S7 phage. The other two phages showed identical restriction endonuclease fragmentation patterns. The difference in density was due to the presence or absence of phage tail. In damp the right cohesive end segment was deleted in a random fashion with the majority ending between 81.0% and 82.4% of . The chromosomal segment of damp was most likely located at the attachment site. The damp DNA was compared to that of a ColE1 hybrid carrying the chromosomal amp segment and a ColE1 hybrid carrying the same 9.8 kb amp repeat as the lysogen from which damp was isolated. It was found that the chromosomal part of damp constituted 9.8 kb, i.e. the size of one repeat. Moreover, the novel joint between adjacent repeats was present. In a attB-deleted E. coli K-12 strain, lysogenic for damp, highly ampicillin-resistant mutants occurred at an exceedingly high frequency. They were found to contain in the chromosome an amplified 9.8 kb repeat. This suggested that integration of the novel joint from damp into the amp region gives rise to an amplifiable duplication. In E. coli lysogenized for damp at attB highly ampicillin-resistant clones were also found at a high frequency. These clones carried multiple tandem repeats of damp DNA, each with an intact right end segment.     

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

Summary Temperature sensitive dnaAts46 mutants, in which initiation of chromosome replication is blocked at 42° C, are unable to maintain a dv plasmid at the permissive temperature unless the plasmid carries a mutation in gene P of the type permitting phage to grow in groP (dnaB) bacteria. The growth rate of dnaAts46 mutants seems to be impaired by the presence of the dvP mutant plasmid.Cold sensitive dnaAcos mutants which overinitiate replication at low temperature and grow normally only at 40° and above, can maintain efficiently dvP ⁺ plasmids as well as dvP mutants. Cold sensitivity of dnaAcos mutants is suppressed by the presence of the plasmid dvP ⁺ and by certain dvP mutants, but not by others.The gene P product seems to act by reducing the initiation potential of both types of dnaA mutants, aggravating the initiation defect in dnaAts46 and correcting the overinitiation of dnaAcos.     

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification

Summary Bacteria with A-specific restriction plate unmodified phage with an efficiency of 10^-2. One mutational event can produce restriction insensitive (sA^o) mutants of . These differ from the original sA form of by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sA^o, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on DNA with affinity for A-specific restriction. DNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of DNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, ³H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified DNA. No association of radioactivity was observed in control experiments with DNA from either modified ·A or from asA^o mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sA^o mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.     

Physiological relationship between the temperate phages lambda and phi80

Summary This work deals with the ability of phage 80 to provide defective mutants of with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of 80 cannot excise prophage . However, 80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between 80 and . Maturation of DNA by 80. The Ter recombinants excised by 80 from tandem dilysogens are packaged into a 80 protein coat. This contrasts with the fact, already mentionned by Dove, that 80 is extremely inefficient for packaging phage superinfecting a -lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of (80hy4 or 80hy41 — see Fig. 1). However, the maturation of the superinfecting is much more efficient if the 80hy used as a helper has the att-N region of (like 80hy1). Conversely a with the att-N region of 80 (hy6 — see Fig. 1) is packaged more efficiently by 80 or 80hy4 than by 80hy1. It is suggested that the maturation of chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by 80. That N mutants are efficiently helped by 80 does not tell that 80 provides the defective with an active N product; the chromosomes are simply packaged into a 80 coat. This shows that 80 is unable to switch on the late genes of . That neither 80 nor any of the 80hy tested can provide an active N product is shown in a more direct way by their complete failure to help N ^-r₁₄; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a N ^- by 80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by 80. This suggests that the products coded by these genes are not interchangeable between 80 and , and that packaging of DNA into 80 coats is possible but inhibited when late proteins are present in the cell. Activation of the Late Genes. Among the im ₈₀ h ⁺ hybrids tested, only 80hy41 is able to switch on the late genes of a N defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of 80 and of the 80hy1 and 80hy4 to activate the late genes of would imply that these phages are unable to provide an Q product active on the chromosome Reciprocally, switches on the late genes of prophage 80hy41, but not of prophages 80hy1 and 80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

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Ce travail a été réalisé dans le cadre du contrat d'association Euratom-U. L. B. 007-61-10 ABIB et avec l'aide du Fonds de la Recherche Fondamentale Collective.     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

Human λ light chain locus: Organization and DNA sequences of three genomicJ regions

Evidence for the genomic organization of human lambda light chain joining (J) region gene segments is presented. A mouse J probe was used in Southern hybridizations to localize joining region sequences in a cosmid clone containing the genomic cluster of six human lambda constant (C) region gene segments. The results of these hybridizations suggest the presence of at least one J gene segment upstream from each constant region gene segment. The DNA sequences indicate that the human JI, J2, and J3 gene segments have consensus nonamer and heptamer sequences, proposed to be involved in V-J joining, are capable of encoding the known amino acid sequences for the respective J peptides, and have a sequence which could give functional RNA splice site at the end of their coding regions. Our data show that a single functional J is located 1.3 or 1.6 kb upstream of each of the C gene segments known to encode the Mcg, Kern^– Oz^–, and Kern^–Oz⁺ isotypes. Therefore, the gene organization of this region of the human lambda locus is J1 CI -J2C2-J3C3. The DNA sequences ofJ 1,J 2, andJ 3 presented in this paper establish that a singleJ gene segment precedes each expressed C gene segment, and support a model for the evolution of the human JC clusters where JICI andJ2C2-J3C3. arose from different ancestral JC units.