Genetic exchanges caused by ultraviolet photoproducts in phage λ DNA molecules: The role of DNA replication

Summary Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 () lysogens infected with genetically marked phage . Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed.First, samples of a heteroimmune phage imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P ⁺ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be atributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme.Second, in homoimmune crosses, samples of similarly treated homoimmune P3 phages were allowed to infect lysogens carrying cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by immunity, and was futher blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P ⁺ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light.It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.     

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.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

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).     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.     

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.     

Lambda transducing phages for the nalA gene of Escherichia coli and conditional lethal nalA mutations.

Summary Defective transducing phages for the nalA region of the Escherichia coli chromosome were isolated from a lysogen in which is inserted in the nearby glpT gene. The three classes of transducing phages designated nrdA, dubiG, and dnalA contained bacterial DNA extending from glpT through nrdA, ubiG, and nalA, respectively. The bacterial genes are in the left arm of the chromosome. Of the eleven polypeptides coded by dnalA that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate only one was not also specified by dubiG This 105,000 dalton polypeptide is the nalA gene product. The electrophoretic mobility and isoelectric point of this protein were unaffected by a nalA mutation (nalA48) that confers nalidixic acid resistance. Temperature-sensitive and amber mutations in the nalA gene were isolated using a dnalA48 lysogen which is heterodiploid for nalA. The conditional lethality of these mutations proves that nalA is an essential locus.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

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.     

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.     

Visual pigments in the individual rods of deep-sea fishes

Summary The visual pigments in the rods of 15 species of deep-sea fish were examined by microspectrophotometry. In 13 species a single visual pigment was found. The max of these pigments, which ranged from 475 nm to 488 nm, suggest they give the fish maximum sensitivity to the ambient light in the deep, blue ocean waters where they live. In two species two visual pigments were found in separate rods.Bathylagus bericoides had rhodopsins of max 466 nm and 500 nm andMalacocephalus laevis had two rhodopsins of max 478 nm and 485 nm. It is noted that the species with two visual pigments tend to be dark in colour and live in deeper, darker, water.     

Visual and lenticular pigments in the eyes of demersal deep-sea fishes

We report on the lens pigmentation and visual pigments of 52 species of demersal deep-sea fishes caught at depths ranging from 480 m to 4110 m in the Porcupine Seabight and Goban Spur area of the North-eastern Atlantic. Only one species, caught between 480 and 840 m, had a lens with large amounts of pigment, consistent with the hypothesis that heavily pigmented lenses in deep-sea fish serve to enhance the contrast of bioluminescent signals by removing much of the background radiance, which is only visible to fish living shallower than 1000 m. Low concentrations of lens pigmentation were also observed in a further two species (Rouleina attrita and Micromesisteus poutassou). The retinae of all species except five, contained only a single visual pigment, as determined by microspectrophotometry of individual rods, and/or spectrophotometry of retinal wholemounts and retinal extracts. Those fishes caught between 500 m and 1100 m had wavelengths of peak sensitivity (_max) ranging from 476 nm to 494 nm, while most fish living below 1100 m tended to be more conservative with (_max) values ranging from 475 nm to 485 nm. The only exceptions to this were three deep-living species caught between 1600 m and 2000 m whose retinae contain abnormally short-wave sensitive visual pigments (Cataetyx laticeps — _max 468 nm; Alepocephalus bairdii — _max 467 nm; Narcetes stomias _max 472 nm), suggesting adaptation for the detection of short-wave bioluminescence.     

Construction and physical mapping of plasmids containing the MetA gene of Escherichia coli K-12

Summary Plasmids containing the metA gene of E. coli K-12 were constructed in vitro using pBR322 as the cloning vehicle and metA transducing phage as the source of metA DNA. EcoRI digests of pBR322 and metA20 were joined by ligase and plasmids carrying the metA gene were selected after transformation in a metA deletion strain. Recombinant DNA molecules contained one pBR322 fragment and one metA20 fragment of 12.2 kb which was present in either of two possible orientations. Plasmids constructed by BamH1 digestion of metA2 contained a single bacterial DNA fragment of 5.8 kb inserted in the tet gene. Insertion of the metA fragment led to loss of resistance to tetracycline in one orientation and partial resistance in the opposite orientation.     

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).     

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 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.     

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Summary Three transducing phages have been isolated from pEDR20, an R100:: cointegrate plasmid in which the insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of . Each phage contains IS1b, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA73, contains the entire r-determinant of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.     

Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana

The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector gtB. Three different recombinants (gtRa1, gtRa23 and gtRa5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The gt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.