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.     

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.     

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.     

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.     

Waves in a simple,excitable or oscillatory,reaction-diffusion model

A simple one variable caricature for oscillating and excitable reaction-diffusion systems is introduced. It is shown that as a parameter, , varies the system dynamics change from oscillatory ( > ₀) to excitable ( < ₀) and the frequency of the oscillation vanishes as for ₀. When such dynamics are coupled by continuous diffusion in a ring geometry (1-space dimension), propagating wave trains may be found. On an infinite ring excitable devices lead to unique solitary waves which are analogous to pulse waves. A solvable example is presented, illustrating properties of dispersion, excitability, and waves. Finally it is shown that the caricature arises in a natural way from more general excitable/oscillatory systems.     

Comparative susceptibility and possible detoxification mechanisms for selected miticides in banks grass mite and two-spotted spider mite (Acari: Tetranychidae)

The susceptibility and possible detoxification mechanisms of the Banks grass mite (BGM), Oligonychus pratensis (Banks), and the two-spotted spider mite (TSM), Tetranychus urticae Koch, to selected miticides were evaluated with and without synergists. BGM was 112-fold more susceptible to the organophosphate dimethoate, and 24-fold more susceptible to both the pyrethroids bifenthrin and -cyhalothrin than TSM. The synergist triphenyl phosphate (TPP) enhanced the toxicities of bifenthrin and -cyhalothrin against BGM by 3.0- and 4.2-fold, respectively, and enhanced the toxicities of bifenthrin, -cyhalothrin, and dimethoate against TSM by 6.2-, 1.9-, and 1.7-fold, respectively. The synergist diethyl maleate (DEM) enhanced the toxicities of bifenthrin and -cyhalothrin against BGM by 2.2- and 2.9- fold, respectively, and enhanced the toxicity of bifenthrin against TSM by 4.1-fold. On the other hand, the synergist piperonyl butoxide (PBO) increased the toxicities of bifenthrin and -cyhalothrin by 6.0- and 2.6-fold, respectively, against BGM, and by 4.5- and 1.9-fold, respectively, against TSM. The significant synergism with these pyrethroids of all three tested synergists (except for DEM with -cyhalothrin against TSM) suggests that esterases, glutathione S-transferases, and cytochrome P450 monooxygenases all play important roles in their detoxification. However, the toxicity of dimethoate was not enhanced by these synergists in either mite species (except for TPP against TSM). Apparently, these metabolic enzymes play less of a role in detoxification of this organophosphate in these mites.     

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.     

Requirement of protein and RNA synthesis for lambda repressor inactivation by tif-1: effects of chloramphenicol, neomycin and rifampicin.

Summary The inactivation of repressor was followed by the specific DNA binding assay during the course of lysogenic induction provoked by incubation at 42°C of an E. coli tif-1 lysogenic strain. The presence of up to 400 g/ml chloramphenicol during the inducing treatment did not impair the loss of repressor binding activity, whilst concentrations of 200 g/ml neomycin and 100 g/ml rifampicin effectively inhibited the inactivation of repressor.Residual protein synthesis in the presence of chloramphenicol, neomycin and rifampicin was 5%, 5% and 27% respectively of that observed in the drug-free control. This residual synthesis did not appear to involve amplification of the X-protein. These results suggest that tif-mediated inactivation of the repressor requires the activation of some specific gene(s), the translation of which appears to be resistant to chloramphenicol.     

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.     

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.     

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).     

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.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

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.     

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     

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.     

The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functionalin vivo

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete ^2FS precursor protein or only for the subunit have been placed under the control of the P_R promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the P_R promoter, the complete ^2FS constructions constitutively express a smaller polypeptide of 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (²P_F and ²P_S) located in the duplicated ^2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp² cDNA sequence, when fused upstream to the cDNA coding for ¹-antitrypsin, constitutively promotesin vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against ¹-antitrypsin or haptoglobin.     

Different binding of RNA polymerase to individual promoters

Summary The amount of E. coli RNA polymerase which can be bound to individual promoters on pgal and dgal phage DNA in a stable heparin-resistant form was measured by assessing its capacity to transcribe, upon addition of the nucleoside triphosphates, the RNA sequences starting at these promoters. These RNA species were analysed by competition hybridization to separated single strands of , pgal and dgal phage DNA.Individual promoters bind, at saturation, different numbers of polymerase molecules. From the amount of polymerase necessary to saturate all promoters (Fig. 3), from the proportions of RNA synthesized at the individual promoters (Table 1) and from the amounts of -³²P-ATP or-GTP label incorporated into the different RNA species (Tables 2 and 3) we calculate polymerase storage capacities for the promoters as follows: gal: 6 molecules; l-strand specific: 3–5 molecules starting with ATP and 1 molecule starting with GTP; r-strand specific: 3–5 molecules starting with ATP (and perhaps one molecule starting with GTP); these estimates are lower limits and may be too small by a factor of up to three.The heparin resistant binding of six polymerase molecules to the gal promoter is dependent on CGA protein and cAMP, but ATP and GTP can allow one polymerase to bind to the same site or to a very close one.Several parameters of polymerase binding are different with the individual promoters tested.