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).     

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.     

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).     

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     

Recurrent t(11;22) breakpoint mapping by chromosome flow sorting and spot-blot hybridization

Summary The breakpoint of the recurrent t(11;22) translocation, one of the most frequent chromosome anomalies encountered in human population, always involves bands 11q23.2 and 22q11.2. The involvement of the C locus of the immunoglobulin gene cluster on chromosome 22 has been suggested: however, in situ hybridization experiments have yielded conflicting results. In order to solve these discrepancies by another approach, we have used bivariate flow sorting to separate the chromosomes of interest and to map the specific breakpoints by direct spot-blot hybridization with the gene-specific radiolabelled DNA probes, Alu, V, ets. The results showed unambiguously that in the t(11;22) patient analysed, a set of C and V genes was translocated to the der(11) chromosome. Since V genes are situated proximally to C genes, we demonstrate that, in the case studied here, the chromosome 22 breakpoint is not located within or even immediately close to the C region.Presented at the 7th International Congress of Human Genetics, Berlin, September 22–26, 1986     

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.     

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.     

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.     

Bacteriophage lambda DNA-dependent synthesis of endolysin in a membranous cell-free system of E. coli

Summary We have previously shown that a membranous cell-free system derived from uninfected penicillin spheroplasts of E. coli transcribes early and late messenger RNA's from DNA.This in vitro system will also transcribe and translate the endolysin gene R of DNA. The enzyme activity that results from in vitro synthesis corresponds to endolysin (a typical late protein) by several criteria.DNA from C_I857 sus R5 ts 9B and C_I857 sus S7 pgal, mutants carrying nonsense mutations in genes involved in the host lysis, are inactive in the synthesis of endolysin with an extract of non permissive cells, although they are fully active with an extract of permissive cells. Furthermore, suppression of these mutations is entirely dependent on addition of supernatant from suppressor strains.The endolysin synthesis from a thermosensitive C_I mutant is observed at 40°C and not at 30°C. This suggests that the product of C_I gene is formed and acts in the in vitro system at 30°C.Enzymatic activity is detected after a 15 min lag period.Membranes and double stranded DNA are absolutely required for the enzyme synthesis. Ribosomes and supernatant highly stimulate the in vitro system.Inhibitors of RNA, DNA and protein synthesis (actinomycine D; cytosine arabinoside; DNA-ase; and chloramphenicol respectively) will prevent endolysin production when added at zero time. If DNA-ase or actinomycine D are added after 20 min of incubation, only partial inhibition of endolysin synthesis occurs. It is therefore concluded, according to our previous observations, that messengers are stable enough to allow enzyme synthesis after delayed addition of the inhibitors in the in vitro system.It appears that there is a complete regulation in the membranous system like in vivo and which starts with the early and late messenger formation and leads to active late protein synthesis.     

Chromosome Localization of the λ20p1.4 clone of the Drosophila melanogaster Nuclear Lamina DNA in the melanogaster Species Subgroup of the Genus Drosophila (Sophophora)

Chromosome localization of sequences homologous to 20p1.4 of the Drosophila melanogaster nuclear lamina DNA (nlDNA) was established by in situ hybridization in species of the melanogastersubgroup. DNA of the 20p1.4 clone was shown to be located in the chromocenter in all the species examined. Laboratory strains of D. simulans, D. mauritiana, andD. sechellia exhibited interspecific differences in localization of 20p1.4 nlDNA on chromosome arms. In eight natural populations, intraspecific polymorphism of 20p1.4 nlDNA chromosome localization was shown to be present in D. simulans but absent in D. melanogaster. The possible participation of transposable elements in 20p1.4 nlDNA relocation is discussed.     

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.     

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.     

Spectral regions in which aquatic insects see reflected polarized light

For diverse water insects (species of Hydrophilidae, Hadraenidae, Dytiscidae, Haliplidae and aquatic Heteroptera), the attractiveness of an artificial water surface was found to vary when the polarization of the reflected light, the property by which these insects identify water, was abolished in different regions of the spectrum. The sensitivity maxima of their reflection-polarization visual systems (max(POL)) thus determined were in various spectral regions, between < 360 nm (UV) and ca. 550 nm (yellow-green). Species with max(POL) at the short-wavelength end of the spectrum would be able to identify bodies of water by polarization regardless of whether the subsurface reflection was bright or dark; nevertheless, this group includes forms that avoid water with a bright subsurface because of the intensity of the reflected light. Species with max(POL) in the long-wavelength region fail to use certain bodies of water with a bright subsurface as habitats because the light they reflect at the longer wavelengths is insufficiently polarized. That the POL system of a species has a large max could affect habitat choice; on the other hand, it could also be that systems operating in the long-wavelength region were produced in the course of adaptation to the light conditions in or above the habitat.Abbreviations max(POL) spectral sensitivity maximum for polarization vision     

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.     

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.     

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).     

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.     

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.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).