Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Changes in transepithelial potential and spike responses of ampullae of Lorenzini of the skate to temperature stimulation

Response of the sensory epithelium of single ampullae of Lorenzini and spike responses of nerve fibers connected to them to temperature stimulation of the region of the sensory epithelium were studied in experiments on Black Sea skatesRaja clavata. Electrically isolated ampullae with input resistance R=500–800 k, to which an external load (a controllable resistance R_ext) could be connected through a feedback circuit, were investigated. Heating the ampulla was accompanied by the appearance of a negative potential in the canal, and other conditions being the same, its magnitude was an almost linear function of the resultant inward resistance of the preparation [R_in=(R_a·R_ext)/(R_a+R_ext)]. The character and intensity of the spike response of the nerve fiber also was determined by the magnitude of R_in. With a resistance of more than 400–500 k, quickening of spike activity occurred in response to heating, and the degree of quickening increased with an increase in R_in. With a smaller value of R_in, the discharge was inhibited, and the inhibition strengthened as the resistance decreased. The presence of two sources of potential, evoked by a change of temperature and giving rise to opposite spike responses, is discussed.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR. Leningrad I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 1, pp. 11–18, January–February, 1982.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

Genetic study of prophage excision with a temperature inducible mutant of Mu-1

Summary Escherichia coli strains, carrying F'episomes with a thermoinducible prophage of Mu were used to study the effect of heat induction on the ability of the cells to transfer these episomes to recipient cells. With this method information was obtained on the excision of the prophage after induction. Control studies were done with an episome containing a thermoinducible lambda prophage.The heat induction has opposite effects on the transfer of episomes containing Mu or : in the case of the number of sexductants in a -immune recipient is not significantly affected while sexduction into a non-immune recipient is increased by a factor 10–20. In contrast, the transfer of Mu-containing episomes into Mu-immune and also non-immune recipients is decreased by a factor of 100–200.The presented data exclude a precise -like excision of prophage Mu upon induction. A possible model for Mu excision is discussed.Colony forming units.     

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.     

Changes in the EEG parameters related to parkinsonism and aging

The EEG parameters were analyzed in 73 patients with parkinsonism characterized by different levels of clinical disorders and duration of the disease (44- to 77-year-old persons, mean 58.1 years; group P) and in 60 practically healthy persons (control group, group C). Group C was divided into two subgroups: Cy and Co (persons younger and older than 40 years, respectively). In the P group, the powers of fluctuations in 1- and 2-ranges and of -waves in anterior recording sites, (F,C), was noticeably higher than those in the C group. These parameters were in positive correlation with the disease intensity; there was a trend toward a lower level of the above EEG components in older patients. Patients with a lower level of clinical symptoms showed lower power of -waves in posterior recording sites, (P,O), than that in the Co and, moreover, Cy subgroups. Yet, a higher level of clinical symptoms correlated with a higher level of this parameter. A decrease in the frequency of (F,C) and (P,O) components in the P group was found. These findings show that there are obvious shifts in spatial distribution, and relative levels and frequency parameters of brain electrical activity in parkinsonian patients, which reflects the multilevel pattern of brain disorders related to this disease.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 396–402, September–December, 1995.     

Nonadrenergic inhibition of human intestinal smooth muscle

The effects of ATP, ,-methylene-ATP, ,-methylene-ATP and adenosine on longitudinal and circular smooth muscle of the human large and small intestine were investigated using the sucrose gap technique. Applications of ATP to the smooth intestinal muscle produced an effect resembling that of stimulating the nonadrenergic nerve fibers in most cases. Desensitization of the purinoreceptors by ,-methylene-ATP selectively reduced the amplitude of nonadrenergic inhibitory synaptic potentials. The findings presented confirm the purinergic hypothesis of nonadrenergic inhibition in the smooth muscle of human intestine.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 3, pp. 373–381, May–June, 1986.     

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

Summary The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca⁺⁺, Mg⁺⁺ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp ¹, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, ), , , (, ) which in the notation of Downie et al. (1981) reads atpB (E F H) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc^-) mutations. We made use of the fact that specialized transducing phages asn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn⁺, and that the resulting Asn⁺ cells grow slowly if the asn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the asn which either eliminated atp genes or affected their expression (promoter mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, 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.     

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.     

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.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

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.     

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.     

Molecular cloning and sequencing of a cDNA for an auxin-repressed mRNA: correlation between fruit growth and repression of the auxin-regulated gene

A complementary DNA (cDNA) library has been constructed in gt10 from poly(A)⁺ mRNA isolated from auxin-deprived strawberry receptacles. By differential plaque filter hybridization, a cDNA (SAR5) to an auxin-repressed mRNA has been isolated. The expression of the auxin-repressed gene is studied at various stages of normal fruit development and in fruits of variant strawberry genotype using SAR5 as a probe. Northern analyses of RNA isolated from pollinated and unpollinated fruits of various developmental stages revealed that mRNA corresponding to the SAR5 clone is repressed during normal fruit development, and the level of SAR5 mRNA is regulated by endogenous auxin. Furthermore, results with both normal and variant genotype strawberry fruit indicate that there is a positive correlation between growth of strawberry fruit and repression of mRNA corresponding to the SAR5 clone. The SAR5 cDNA has been sequenced and is 723 nucleotides in length. The deduced protein has 111 amino acid residues with a molecular mass of 12.5 kDa. The putative polypeptide starts at nucleotide position 20 and ends at 352. The molecular weight of the predicted polypeptide is in agreement with the molecular weight of the in vitro translated polypeptide of hybrid selected mRNA. A comparison of the nucleotide and deduced amino acid sequence of SAR5 with nucleotide and protein sequences in data banks has not revealed any homology to known proteins.     

On the mode of antirepressor action in anti-immune cells

Summary The mode of antirepressor action in anti-immune cells was analysed in respect to its two main features, low lysogenization and high cell killing. By means of complementation experiments between and i434 in anti-immune cells, it was shown that the antirepressor no longer channels phages towards lysis in such cells if the genes which are needed for lysogenization are provided in trans by the heteroimmune phage i434. Since complementation could be demonstrated, it was possible to exclude that direct action of the antirepressor over repressor production is responsible for the feature under analysis. It was also shown that both int- and cII-product are required to improve lysogenization and to prevent high levels of killing.Recipient of an EMBO predoctoral fellowship.     

The origin of Q-independent derivatives of phage lambda

Summary qsr (Q-independent) phages are characterised by the replacement of the region of the genome that contains Q, S, R, and the late gene promoter, P_R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that p4 and qin A 3, two such Q-independent phages, are the product of recombination between and a defective lambdoid prophage (the qsr prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K 12 strain AB1157 from which qsr phages cannot be generated, the qsr prophage has suffered an internal deletion. That the qsr prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.