Evidence that F'' lac replicates asynchronously during the cell cycle of Escherichia coli B/r.

Summary The replication of F lac was studied in exponentially growing cultures of E. coli B/r. The cells were pulse induced for the synthesis of -galactosidase and their DNA pulse labelled with ³H thymidine. The cells were then separated into age classes by centrifugation through a sucrose gradient in a zonal rotor. Plasmid replication was measured in each age fraction by three methods: the rate at which -galactosidase could be induced, the amount of label incorporated into CCC plasmid DNA which had been separated from chromosomal DNA on agarose gels, and the amount of label incorporated into plasmid DNA which had been separated from chromosomal DNA by ultracentrifugation through CsCl-EtBr gradients. All these methods gave the same result, that replication of F lac occurs in cells of all ages and is not confined to a part of the cell cycle.     

Indigoidine and other bacterial pigments related to 3,3′-bipyridyl

Zusammenfassung Die extracelluläre Abscheidung eines unlöslichen blauen Pigments (Indigoidin) wurde zuerst bei Pseudomonas indigofera beobachtet. Historisch wird auf die verschiedenen Benennungen dieses Bakteriums eingegangen. Beschrieben wird die Darstellung blauer Farbstoffe aus Kulturen verschiedener Bakterien. Die von Corynebacterium insidiosum, Arthrobacter atrocyaneus und Arthrobacter polychromogenes gebildeten Pigmente sind identisch mit Indigoidin von P. indigofera. Die Identität wird bewiesen durch physikalische und chemische Vergleiche der Pigmente und ihrer Derivate. Der Name Indigoidin, der früher nur für das Pigment von P. indigofera verwendet wurde, wird nun unabhängig von der Herkunft des Pigments benützt.Indigoidin (I), C₁₀H₈N₄O₄, ist 5,5-Diamino-4,4-dihydroxy-3,3-diazadiphenochinon-(2,2). Durch Erhitzen mit 6 n HCl entsteht daraus ein Hydrolyseprodukt (III), C₁₀H₆N₂O₆, das als 4,5,4,5-Tetrahydroxy-3,3-diazadiphenochinon-(2,2) erkannt wurde. Dieses Hydrolyseprodukt (III) bildet ein Monokaliumsalz (VII), das identisch ist mit dem grünen Pigment, das Arthrobacter crystallopoietes bei Zusatz von Pyridon-(2) bildet. Über Synthesen des Indigoidins (I) und seines Hydrolyseprodukts (III), die von 3,3-Bipyridyl, von Citrazinsäure oder 5-Amino-pyridon-(2) ausgehen, wird an anderer Stelle berichtet.Beschrieben wird die Darstellung folgender Indigoidin-Derivate: 5,5-Diacetamino-4,4-dihydroxy-3,3-diazadiphenochinon-(2,2) (II), C₁₄H₁₂N₄O₆; 4,4-Dihydroxy-5,5-diacetoxy-3,3-diazadiphenochinon-(2,2) (IV), C₁₄H₁₀N₂O₈; 2,5,6,2.5.6-Hexaacetoxy-3,3-bipyridyl (VI), C₂₂H₂₀N₂O₁₂ und 4,4-Dihydroxy-5,5-dimethoxy-3,3-diazadiphenochinon-(2,2) (V), C₁₂H₁₀N₂O₆.     

Directed integration of an F'' plasmid by integrative suppression: isolation of plaque forming lambda transducing phage for the dnaC gene.

Summary A new approach for isolation of a plaque forming specialized transducing phage is described. It consists of directed transposition of an F plasmid into the gal region of a dnaAts galE ^- Escherichia coli strain by integrative suppression and deletion of the chlD region in order to shorten the distance between the marker of interest on the F and the prophage serving to prepare an LFT¹ lysate.An F danC ⁺ thr ⁺ plasmid was used here and dthr and ddnaC phages were isolated. In addition, pdnaC was obtained from a double lysogen for ddnaC and b2.     

Escherichia coli mutants temperature-sensitive for DNA synthesis

Summary A series of mutants of E. coli temperature-sensitive for DNA synthesis has been studied. The temperature-sensitive DNA mutations map in seven distinct genetic loci most of which have not been previously reported. Mutations in dnaA and in dnaC affect the initiation of DNA replication; those at the remaining loci affect chain elongation. A temperature-sensitive Flac is shown to suppress a group A mutant with somewhat less efficiency than other F factors previously reported by others. The gene products rendered temperaturesensitive by the mutations have not been identified for any of the loci.     

Protection of foreign DNA against host-controlled restriction in bacterial cells

Summary Foreign Flac plasmid DNA which is introduced into potentially restricting E. coli recipient cells can be protected from restriction by preinfecting the recipient cells with UV-inactivated T3 or T7 bacteriophages which express the ocr gene function. The recipient cells survive and are able to replicate themselves as well as the newly acquired plasmid.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

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.     

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.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects

Summary The sequences of a collection of 261 spontaneous lacI^- mutants recovered in a PolA^- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5-GTGG-3 sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5-GTGG-3 sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5-GTGG-3 sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.     

Chromosome mobilization and integration of F-factors in the chromosome of RecA strains of E. coli under the influence of bacteriophage Mu-1

Summary Transfer of chromosome promoted by an F⁺ or an F needs a recombinational event between episome and chromosome and is therefore very low in RecA strains (Wilkins, 1969).When F⁺ or F RecA cells are mated with F^- cells and simultaneously infected with bacteriophage Mu-1 the transfer of chromosome is greatly stimulated and seems to take place on any site of the chromosome even when homology is present between the F and the chromosome.A spot-test based on Mu-1 promoted chromosome-mobilization was developed to search for mutants of Mu-1 that had lost the ability to promote chromosome-mobilization. Two conditional lethal amber mutants falling in different complementation groups were found to have lost this property.The integration of an Ftslac ⁺ in the chromosome of a RecA strain is also strongly stimulated by Mu-1 and not site-specific. The resulting Hfr's are very stable and of the clockwise and counterclockwise types. Some of the integrated F-primes are sterile and not able to transfer chromosome or do not form F-pilli.Also the Mu-1 stimulated integration of an Fts(nadA-chlA)⁺ in the chromosome of a RecA strain which has a deletion on the chromosome from nadA-chlA was studied. It was found that when the F is integrated under the influence of Mu-1 also episomal genes carried by that F can be inactivated.     

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.     

Kidney-Specific Activity of the Bovine Uromodulin Promoter

A 10-kilobase (kb) bacteriophage bovine genomic clone containing 5.4 kb of the 5-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5 deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.     

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli

Summary The frequency of lac ^- mutations induced in an F lacI ^S plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, pro lac recipients, is 2-3 fold higher than that typical of non-mating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication.     

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1 and 2 promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.Abbreviations NPT-II neomycin phosphotransferase II - neo NPT-II gene from Tn5 - GUS ß-glucuronidase - gus GUS gene from Escherichia coli - TR 1–2 genes 1 and 2 of TR-DNA of pTiAch5 - Rif rifampicin     

Analysis of sex determination in the monogenic blowfly Chrysomya rufifacies by pole cell transplantation

Summary Sex determination in the monogenic blowfly Chrysomya rufifacies is controlled by a dominant or epistatic female sex realizer (F) having sex predetermining properties (F/f=female-producing female; f/f=male-producing female or male, respectively). To determine (1) the cell type in which the maternal effect gene F is expressed. and (2) the autonomous or nonautonomous sexual differentiation of the germ cells germ-line mosaics were constructed in C. rufifacies by pole-cell transplantations. The production of bisexual progeny by germ-line mosaics generated by transplanting pole cells between both types of female embryos shows that the F gene product is synthesized by germ-line cells themselves, not by maternal (intra- or extraovarian) somatic cells. Pole cell transplantations between male and female embryos yielded completely fertile heterosexual germ-line mosaics thus demonstrating phenotypic sex reversal of donor germ cells in a host of the opposite sex. Consequently, the sexual differentiation of a germ cell in C. rufifacies is not determined by its own genotypic constitution but is induced by the surrounding somatic cells.The male sex of F/f individuals generated by fertilization with F-bearing sperm from a heterosexual germ-line mosaic indicates that the F gene is either not expressed during spermatogenesis and early embryogenesis or is expressed too late or in not sufficient amounts to direct differentiation into the female sex. This finding is consistent with the assumption that progamic expression of F is found exclusively during oogenesis in F/f females.     

Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types

Summary Four E. coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F plasmids. Type II Fs were found to be the most prevalent F plasmid formed from all of the Hfrs, while the percentages of tra Fs increased as the stability of the Hfr increased. Two observations suggested that F formation in unstable Hfrs like Ra-2 may proceed through a type II F precursor. First, the major F products of Ra-2 are tra ⁺ type II Fs and, second, other F types (I, II) and classes (tra ⁺, tra) from Ra-2 appeared to be deletion derivatives of a larger F progenitor. By monitoring the molecular changes that occur when the Ra-2 derived type II F pWS200 is transferred from one recA host to another, we have found that all F types and classes can be generated from pWS200 in a recA-independent manner. F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated junctions of F and chromosomal DNA. A model for the formation of Fs in unstable Hfrs is postulated in which a tra ⁺ type II F primary excision product is seen to be modified, through recA-independent processes, to other F types and classes. This model differs from the current model of F formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II Fs.These studies have also shown that the formation of tra Fs is a recA-independent process that can occur from the F and Hfr states, that -mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this mini-Hfr cointegrate.     

Loss of an episomal fertility factor following the multiplication of coliphage M13

Summary The infection of an Escherichia coli F merodiploid strain by the male specific bacteriophage M13 followed by its multiplication and release, induces a conversion of the cells to the F^- phenotype. Evidence is presented to show that this conversion is associated with a) the simultaneous loss of three F determined properties, b) the loss of a molecular form of DNA characteristic of F and c) the ability of the converted cells to be conjugally reinfected with the same F factor. These findings indicate that the F episome is physically eliminated from the cells that survive M13 infection.