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.     

Studies on lambda virulent mutants

Summary Lambda virC mutant, presumably an operator mutant for the operon including x, y, CII and O genes (Fig. 1), produce clearish plaque on a sensitive bacteria.Four revertants producing turbid plaques were isolated from virC and the mutational sites of which were studied. One (tw ₁) is located very close to and on the left side of virC₃₄, and another (tw ₃₂) is at the almost same site of virC₃₄. The others (tx ₆ and tx ₅₃) are located on the right side of virC₃₄. tx recombinants have been isolated and characterized. These recombinants produce very turbid plaques and the rate of the repressor formation in the presence of CIts repressor is somewhat higher than that of wild type. tx develops very poorly after infection to sensitive cells but CItx develops normally. tx lysogens synthesize two to three times more exonuclease than the wild type lysogen. On a function of x region for the repressor formation and on a presence of a possible anti-repressor were discussed. The mutant tw ₁ might be a promotor mutation of the CI-rex operon.This material has been published as an abstract in Jap. J. Genetics 45, 474 (1970).     

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.     

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.     

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

Summary The amidase genes of Pseudomonas aeruginosa were inserted into a replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant ami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with ami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of ami were made by introducing Q ^-, S ^- mutations. Cultures of E. coli infected with amiQ ^- S ^- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificity, thermal stability and immunological crossreaction.     

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.     

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.     

Molecular genetic analysis of the Ta 25H deletion: Evidence for additional deleted loci

Seventeen linking clones sublocalized to the central region of the mouse X Chromosome (Chr) were screened against genomic DNA from male mice carrying the tabby-25H (Ta ^25H ) deletion. Two of these linking clones, EM131 and EM169, were found to be deleted in Ta ^25H /Y animals. Genetic mapping through Mus musculus domesticus/Mus spretus interspecific backcross progeny, segregating for the original tabby (Ta) gene mutation, was utilized to order these markers and to define nearest flanking markers to the Ta ^25H deletion (EM140 and EM171). The size of the Ta ^25H deletion was thus estimated as up to 4.5 centiMorgans (cM). The order of markers, proximal to distal, was found to be EM140/EM131, mouse androgen receptor gene (Ar)/EM169, Ta/EM171. A putative CpG-rich island and a highly evolutionarily conserved DNA probe were isolated from the DXCrc169 locus which co-segregates with the Ta locus in this study.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

A new bacterial gene (groPC) which affects lambda DNA replication.

Summary A bacterial mutation affecting DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci. Most of the parental DNA does not undergo even one round of replication in this host. Lambda mutants, called , which map in the P gene are able to overcome the inhibitory effect of the groPC756 mutation. It is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C. A -transducing phage, carrying the groPC⁺ allele, was isolated as a plaqueformer on groPC756 bacteria. Upon lysogenization, it restores both the gro ⁺ and temperature resistant phenotypes.     

Intraspecific variation of spectral sensitivity in threespine stickleback (Gasterosteus aculeatus) from different photic regimes

To examine the influence of the spectral characteristics of underwater light on spectral sensitivity of the ON and OFF visual pathways, compound action potential recordings were made from retinal ganglion cells of threespine stickleback from different photic regimes. In fish from a red-shifted photic regime (P₅₀ 680 nm for downwelling light at 1m), peak sensitivity of both the ON and OFF pathways was limited to long wavelength light (_max 600–620). In contrast, the ON pathway of fish from a comparatively blue-shifted (P₅₀ 566 nm) photic regime exhibited sensitivity to medium (_max 540–560) and long (_max 600 nm) wavelengths, while the OFF pathway exhibited peak sensitivity to only medium (_max 540 nm) wavelength light. In a third population, where the the ambient light is moderately red-shifted (P₅₀ 629 nm), the ON pathway once again exhibited only a long wavelength sensitivity peak at 620 nm, while the OFF pathway exhibited sensitivity to both medium (_max 560 nm) and long (_max 600–620 nm) wavelength light. These findings suggest that the photic environment plays an integral role in shaping spectral sensitivity of the ON and OFF pathways.     

Changes in the spectral absorption of cone visual pigments during the settlement of the goatfish Upeneus tragula: the loss of red sensitivity as a benthic existence begins

The goatfish Upeneus tragula undergoes an abrupt metamorphosis at settlement when the pelagic larvae begin a reef-associated benthic mode of life. A microspectrophotometric investigation of the retinal visual pigments was carried out on fish prior to, during, and following settlement. It was found that the visual pigment in the long wavelength-absorbing member of the double cones in the dorsal retina changed rapidly from a rhodopsin with a wavelength of maximum absorption (max) of 580 nm to that of 530 nm. The second member of the double cones always had a rhodopsin with the max absorbing at shorter wavelengths. Prior to settlement the average for this class of cones was 487 nm whereas during and immediately following the settlement period the max recorded from individual outer segments was found to vary between 480 nm and 520 nm, with two possible classes of cone absorbance emerging within this range. These two classes of absorbance had average max values of 487 and 515 nm. The average max of the paired cone classes in one larger wild-settled fish were found to be at 506 nm and 530 nm. No change was detected in the max of the single cones or the rods which were always found to have a max of about 400 nm and 498 nm respectively. The loss of the redabsorbing pigment occurred over the same time scale as the metamorphosis of morphological features associated with the settlement process. It is thought that the loss of this visual pigment is associated with the change in light environment of the fishes as they leave the surface waters to begin a benthic mode of life in deeper water.Abbreviations AIMS Australian Institute of Marine Science - ANOVA Analysis of variance - IR infra-red - max wavelength of maximum absorption - MSP microspectrophotometer - NA numerical aperture - SL standard length     

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.     

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).     

Relationship between the grain pattern in the wood,domain pattern and pattern of growth activity in the storeyed cambium of trees

Summary The relationship between the arrangement of cell events occurring in cambium in a definite configuration and the grain pattern of wood was investigated. Taking into consideration the growth activity of fusiform cell ends, a model of a migrating morphogenetic wave determining an event configuration was made. Waves of length =1 m for the periods T=2 years and T=3 years and waves of lengths =l m and =0.04 m for the period T=10 years were considered. On the model, events from successive annual rings, conventionally comprising 10 cell layers each, were summed. In this way, event maps were obtained. For wave =4 mm, the domain pattern on the modelled map was compatible with the grain pattern. The domain pattern for the wave =1 m was impossible to recreate because the wave migrated too fast. In this case, the pattern of event configuration, incompatible with the grain pattern, formed microareas, which were not domains.     

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.     

Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3() strain. Although the constitutive reversions could be transduced by , the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by .In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3() strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3() strain, and not in gal ⁺, gal ⁺(), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.A gal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (31). Electron micrographs of gal ⁺ and gal ^c 200 31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage ; and (iii) the gal3 insertion appears to inhibit the production of gal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.     

Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance.

Summary In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap^r plasmid: pSC138 (Timmis et al., 1975). F recombinants were shown to be chimerae between the and the F replicons. By genetical tests, we have demonstrated that both and F replication mechanisms are functional: the F recombinant behaves as a non defective plaque forming phage on sensitive bacteria and establishes itself as a stable plasmid on recA F^- homoimmune bacteria. In the extra-chromosomal state, the F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.