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.     

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.     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

Transient suppression of F-plasmid incompatibility in a strain of Escherichia coli

Summary The incompatibility between F plasmids is transiently suppressed in Escherichia coli strain CSH54. As a result this strain is able to maintain two F factors or an F factor and a mini-F plasmid for considerably longer periods than normal strains. When selective pressure for two markers carried by two separate Fs (or an F and mini-F) is imposed on normal strains, the two plasmids tend to form a cointegrate structure which can be detected genetically by the joint transfer of both the markers upon mating. This does not happen in CSH54; instead, the two plasmids are maintained and transferred independently. Physical evidence for the maintenance of an F and a mini-F plasmid is provided by agarose gel electrophoresis.     

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. II. Evidence that large N-terminal amber fragments of the beta subunit interfere with RNA polymerase function

Summary A collection of 95 independent, spontaneously-occurring mutants carrying amber lesions that affect expression of the gene, rpoB, has been isolated (see accompanying paper (Nene and Glass 1982)). Certain rpoB amber mutations act in trans, preventing a functional allele present on an F plasmid from acting at high temperature. Two such temperature-sensitive rpoB(Am) strains are shown to produce large, N-terminal amber fragments. The possibility that these truncated polypeptides are the cause of this trans-dominant conditional-lethal phenotype is supported by analysis of fragment levels in thermoresistant survivors: the nonsense fragments are degraded at a significantly faster rate (half-lives 1.4- to 2.6-fold reduced) in Ts⁺ derivatives likely to carry second-site mutations within rpoB. We suggest that the fragments interfere with RNA polymerase function by interacting with one or more of the polymerase subunits.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Ongoing ultradian activity rhythms in the common vole,Microtus arvalis,during deprivations of food,water and rest

Summary The timing mechanism underlying ultradian (2–3 h) activity patterns in the common vole, Microtus arvalis, was studied using behavioural deprivation experiments. These were aimed at distinguishing between a homeostatic control mechanism, in which the rhythmic behaviour itself is part of the causal loop, and a clock mechanism, independent of the behaviour.In 175 experiments, deprivation of food during 3 ultradian cycles in (subjective) daytime did not result in significant changes in the ultradian periodicity of attempts to obtain the food, compared with ad lib. access to food and water. A minor, but significant increase in ultradian activity time () occurred in the course of the deprivation, but this was compensated by a shorter ultradian rest (). These results were obtained both in intact animals (n = 24), which showed ultradian and circadian rhythmicity in behaviour, and in animals (n = 21) with electrolytic lesions aimed at the suprachiasmatic nuclei (SCN), which lacked the circadian modulation of behaviour. Simultaneous deprivation of water and food in 8 voles without circadian rhythmicity during 40 experiments also did not lead to any change in the ultradian periodicity of feeding attempts.Rest deprivation was studied in 5 SCN lesioned voles, by forcing running wheel activity to continue following spontaneous running. Thus, the experimental activity bout was artificially lengthened to 2–9 h in 67 experiments. The onset of the subsequent rest episodes occurred independent of the duration of the preceding . The duration of was dependent on the preceding, experimental in a periodic fashion. The interval experimental (=lengthened +following ) was equal to one, two or three times the control (obtained on nonexperimental days). This result fits the prediction of a clock model and is in conflict with a monotonicincrease of with , as expected in a homeostatic, restorative process.It is concluded that the ultradian timing of activity in the common vole can be explained neither by homeostatic hunger or thirst mechanisms nor by homeostatic rest/activity regulation. The results strongly suggest an independent clock system generating ultradian feeding rhythms in the common vole.Abbreviations DD continuous darkness - LD light-dark regime - LL continuous light - RCA retrochiasmatic area - ARC arcuate nucleus - SCN suprachiasmatic nuclei - ultradian period - ultradian activity time - ultradian rest time     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Mycolic acid patterns of some species of Mycobacterium

Representative strains of some species of Mycobacterium were degraded by both acid and alkaline methanolysis. Two-dimensional thin-layer chromatography was used to determine the patterns of mycolic acids and other long-chain components in these methanolysates. Patterns composed of -, methoxy- and ketomycolates were found in Mycobacterium asiaticum, Mycobacterium bovix, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium marinum and Mycobacterium tuberculosis; a representative of Mycobacterium thermoresistibile also contained lower molecular weight -mycolates in addition to these three acids. In representatives or Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium nonchromogenicum, Mycobacterium novum, Mycobacterium paratuberculosis, Mycobacterium scrofulaceum, Mycobacterium terrae, Mycobacterium xenopi, and Mycobacterium sp. MNC 165 - and ketomycolates were accompanied by -carboxymycolates and 2-eicosanol and homologous alcohols which are derived from wax-ester mycolates. Mycobacterium fortuitum and Mycobacterium giae contained - and epoxymycolates and both serovars of Mycobacterium simiae had a very characteristic pattern of -, - and ketomycolic acids. Comparison with data for other mycobacteria showed the chemotaxonomic significance of these mycolic acid patterns.Abbreviations TLC thin-layer chromatography - TBDMS t-butyldimethylsilyl Supplementary data: Copies of the chromatographic patterns of the mycolic acids from all the strains examined can be provided on request from one of the authors (D.E.M.)     

"Nick translation" in Escherichia coli rep strains deficient in DNA polymerase I activities.

Summary Using X174 replicative form (RF) DNA as an in vivo probe, we have investigated the coordinated action of the 53 exonuclease and polymerase activities of DNA polymerase I in order to understand better its physiological role. We constructed double mutants containing the rep mutation (the replication of X174 RF does not occur in rep mutants) together with a mutation affecting DNA polymerase I, either polA12 or polA546ex. Using these mutants, which are believed to be thermosensitive in the polymerase function or the 53 exonuclease function respectively, we studied the kinetics of nick translation at the permissive and non-permissive temperatures in vivo. The substrate was the X174 replicative form DNA nicked by the X174 gene A protein. E. coli rep polA546ex gave the lowest rate of nick translation, although the ability to perform nick translation, at least as measured by our assay, was still present. E. coli rep polA12 showed a similar low rate at the non-permissive temperature but a rate close to the wildtype level at the permissive temperature. Formation of the parental replicative form molecule in either strain was affected little, even at the restrictive temperature. Our results suggest that DNA polymerase I may not play a major role in ongoing DNA replication.     

Identification of an amber fragment of the beta subunit of Escherichia coli RNA polymerase: a yardstick for measuring controls on RNA polymerase subunit synthesis.

Summary An amber fragment of the subunit of Escherichia coli RNA polymerase has been recovered from strains carrying the rpoB12 amber mutation, indicating that the B12 mutation resides in the structural gene for the subunit. The fragment is readily assayed and can be used to determine the degree of expression of a single rpoB cistron in strains haploid or diploid for this region. These studies confirm that the bacterial mechanism, which can compensate for reduced translation of the message, operates by the co-ordinate induction of rpoB and rpoC. Furthermore, I show that rpo control depends upon cistron(s) located on the F factor, KLF10, whose product(s) can act negatively in trans on rpoBC expression.     

Content of RNA polymerase in haploid and merodiploid strains of Escherichia coli

Summary In cell-free extracts of E. coli merodiploids carrying F-factor with ilv-thi chromosome fragment the activity of RNA polymerase is not increased, and there is no excess of free active core-enzyme or sigma-factor. Only immunochemical analysis reveals 25% excess of RNA polymerase material in some merodiploids as compared to a haploid. However, neither the amount of + relative to total protein nor : ratio does not differ in haploid and merodiploids.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.     

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.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .