The fate of newly made DNA in Escherichia coli mutants thermosensitive in DNA synthesis

Summary E. coli mutants exist in which DNA synthesis is thermosensitive. In one class of these mutants DNA synthesis stops immediately if a critical temperature (42°C) is reached. When DNA replication in such mutants is followed by ³H thymidine incorporation at 33°C, it is found that 1. only the newly made DNA is degraded at 42°C, 2. the discontinuously replicated DNA is lost predominantly at 42°C, 3. 1–3% of the chromosomal DNA is rendered acid soluble at 42°C without concomitant loss of viability of the cells at 33°C.Replication of phage DNA is inhibited in the same mutant at 42°C. However, when DNA synthesis is followed in infected cells at 33°C it is found that 1. no degradation of specific DNA seems to occur at 42°C in the early phase of infection, 2. replicating DNA molecules in the late phase of infection are completed at 42°C before DNA synthesis comes to a halt.     

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).     

Analysis of a temperature sensitive mutation in gene cII of bacteriophage lambda

Summary The mutation cIIts612 was found to map outside the immunity region of phage imm21 hybrid. As expected of a cII mutation, cIIts612 is unable to stimulate either cI repressor or Int synthesis during the establishment of lysogeny. These results indicate that part of the cII gene of is homologous to that of imm21 phage.     

A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations

Summary A mutant of was isolated that grows in the Escherichia coli himA/gyrB-him320(Ts) double mutant at 42°C; conditions which are non-permissive for wild-type growth. The responsible mutation, ohm1, alters the 40th codon of the Nul reading frame. The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging. The Nul-ohm1 gene product acts in trans to support growth in the double himA/gyrB mutant, and cos154 growth in the single himA mutant. The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA: protein interactions that occur at the initiation of packaging.     

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.     

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.     

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).     

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.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

In vitro insertion of the lambda attachment site into the plasmid RP4.

Summary The region of the phage lambda chromosome containing the attachment site (P · P) and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site · P. The construction and properties of the hybrid plasmid RP4att are described.     

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

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

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.     

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.     

Regional replication of the bacterial chromosome induced by derepression of prophage lambda

Summary Derepression of prophage in E. coli strain K12 results in constitutive synthesis of the enzymes directed by the nearby bacterial operon, gal (escape synthesis). Phage 82 fails to cause escape synthesis despite that it lysogenizes the strain K12 at the site identical to that of on the host chromosome. The reason for the observed difference between 82 and is studied in the light of the recent finding that escape synthesis in -lysogen is closely associated to phage-promoted replication of bacterial chromosome contiguous to the prophage including gal operon (escape replication). Excision-defective mutants from 82, 82int or 82xis, do initiate escape synthesis, suggesting that the prophage 82 is normally excised too quickly after induction to allow sufficient escape replication. In support of this, much more DNA hybridizable to bacterial DNA contained in gal accumulates after induction of 82int than after induction of 82. Studies with various hybrid phages between 82 and have suggested: 1. The occurrence of gal escape synthesis depends on the nature of the region between b2 and N in the map. 2. Regions of the 82 genome on both sides of the attachment site contribute independently to prevent gal escape synthesis. Implications of these results are discussed with regard to the factors involved in the prophage excision.The IIIrd article of this series is in Molec. Gen. Genet. 159, 185–190 (1978)     

The control of multi-muscle systems: human jaw and hyoid movements

A model is presented of sagittal plane jaw and hyoid motion based on the model of motor control. The model, which is implemented as a computer simulation, includes central neural control signals, position- and velocity-dependent reflexes, reflex delays, and muscle properties such as the dependence of force on muscle length and velocity. The model has seven muscles (or muscle groups) attached to the jaw and hyoid as well as separate jaw and hyoid bone dynamics. According to the model, movements result from changes in neurophysiological control variables which shift the equilibrium state of the motor system. One such control variable is an independent change in the membrane potential of -motoneurons (MNs); this variable establishes a threshold muscle length () at which MN recruitment begins. Motor functions may be specified by various combinations of s. One combination of s is associated with the level of coactivation of muscles. Others are associated with motions in specific degrees of freedom. Using the model, we study the mapping between control variables specified at the level of degrees of freedom and control variables corresponding to individual muscles. We demonstrate that commands can be defined involving linear combinations of change which produce essentially independent movements in each of the four kinematic degrees of freedom represented in the model (jaw orientation, jaw position, vertical and horizontal hyoid position). These linear combinations are represented by vectors in space which may be scaled in magnitude. The vector directions are constant over the jaw/hyoid workspace and result in essentially the same motion from any workspace position. The demonstration that it is not necessary to adjust control signals to produce the same movements in different parts of the workspace supports the idea that the nervous system need not take explicit account of musculo-skeletal geometry in planning movements.This article was processed by the author using the LAT_EX style file pljour2 from Springer-Verlag.     

Repressor and int synthesis of bacteriophage lambda in the E. coli host mutant ER437.

Summary Analysis of phage infection of the host mutant ER437 by SDS polyacrylamide gel electrophoresis and autoradiography has revealed altered expression of repressor and integration function (Int). We show that in this host Int as well as repressor synthesis is not dependent upon the cIII gene product in the usual manner, nor is their synthesis turned off in the normal way.