The origin of Q-independent derivatives of phage lambda

Summary qsr (Q-independent) phages are characterised by the replacement of the region of the genome that contains Q, S, R, and the late gene promoter, P_R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that p4 and qin A 3, two such Q-independent phages, are the product of recombination between and a defective lambdoid prophage (the qsr prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K 12 strain AB1157 from which qsr phages cannot be generated, the qsr prophage has suffered an internal deletion. That the qsr prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.     

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.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

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.     

Evidence that pretreatment of Escherichia coli cells with N-methyl-N''-nitro-N-nitrosoguanidine enhances mutability of subsequently infecting phage lambda

Summary The frequency of mutations in is significantly higher for host cells of a rec ⁺ or recA ^- strain pretreated with N-methyl-N-nitro-N-nitrosoguanidine (NG) than for untreated control cells. Direct NG treatment of DNA-injected host cells results in about tenfold higher mutation frequency in than NG treatment of host cells alone. Since mutability of is enhanced by UV preirradiation of host cells in the rec ⁺ strain but not in the recA ^- strain, indirect NG mutagenesis is different in mechanism from indirect UV mutagenesis. It is concluded that NG mutagenesis in consists of at least two processes: induction of rec ⁺-independent mutagenic activity in the host bacterium and production of rec ⁺-independent mutational damage to intracellular DNA.     

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.     

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).     

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.     

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.     

Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor

Summary Transfer of a UV-damaged F sex factor to a recipient lysogen induces prophage development. Under these conditions RecA protein synthesis was induced and repressor cleaved, as observed upon direct induction, that is, when the recipient lysogen was directly exposed to UV-light. The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction. We observed the simultaneous disappearance of repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of repressor was cleaved. Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis. A phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of repressor. Indirect induction by UV-damaged F sex factor or phage oriF resulted in biochemical cellular reactions similar to those observed upon direct induction. LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did repressor.     

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.     

Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

A difference in UV-sensitivity between genes in the repressed and genes in the de-repressed state

Summary The sensitivity of the capacity for forming galactokinase inE. coli was measured under various conditions. Differences were found between strains W 8 and Hfr 7 () and between these strains and mutants, derived from them, which are partially constitutive for galactokinase. The same behavior as that of the constitutive mutants was also obtained for Hfr 7 () induction of the genes for galactokinase or -galactosidase prior to UV-irradiation. It was concluded that these genes exhibit different sensitivities toward UV, depending on whether they are in the repressed or de-repressed state. Pre-induction effects on UV-sensitivity are observed only in strain Hfr 7 () and only if the latter is lysogenic for phage .With 6 Figures in the Text     

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.