Expression of pAMα1 tetracycline resistance gene inCorynebacterium glutamicum: Segregation of antibiotic resistance due to intramolecular recombination

Summary AnEscherichia coli/Corynebacterium glutamicum chimeric plasmid has been constructed containing the tetracycline resistance (Tc^R) determinant from pAM1 and the kanamycin resistant (Km^R) determinant from pBD10. The paAM1 tetracycline resistance determinant is expressed inC. glutamicum although the transformed population segregates readily into tetracycline and kanamycin resistant populations. The segregation event is the result of an intramolecular recombination between the pAM1 and pUB110 regions of the chimera.     

Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis

Summary Marker rescue in plasmid transformation of competent cells of different rec mutants of B. subtilis was studied. In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions). Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.Abbreviations Km ^R Kanamycin-resistant - Cm ^R Chloramphenicol-resistant     

Plasmid dna transformation of Lactobacillus strains by electropermeabilization

Summary Two thermophilic strains of Lactobacillus were transformed by electroporation; L.fermentum with a maximum of frequency of 1×10⁵/ug of plasmid vector pPSC₂₀DNA and 1.4×10³/ug pSA₃DNA. L.helveticus showed a very low frequency of transformation, from 9 to 26 transformants/ug DNA in all the experiments carried out with both the vectors. While L.fermentum transformants were very stable, in L.helveticus the acquired plasmid was lost after 30–50 generations.     

Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco

Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Km^r) phenotype. The gene targeting frequency was 1×10^–5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×10⁸ protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Km^r calli were selected. Screening the Km^r calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Km^r calli in which gene targeting had not occurred we analysed plants regenerated from 24 Km^r calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.     

31P-15N One-Bond Couplings of Diastereoisomeric Adenosine Cyclic 3′,5′-Phosphoramidates

Abstract

¹J(³¹P¹⁵N) coupling constants of R _p and S _p adeno-sine cyclic 3′,5′-phosphoramidates (1), -N-methylphosphor-amidates (2) and -N,N-dimethylphosphoramidates (3) increase in the order of 1<2<3 and obey the Stec rule (J(R _p)< J(S _p)). A possible interpretation of coupling constant differences based on differences in substituent electronegativities and variation in hybridization at nitrogen atom, is suggested.     

Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants

Summary The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Km^r) and pUB110dB (1.5 MDa, Km^r), were obtained from pTB913 (2.9 MDa, Km^r, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Km^r, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids.     

First enantioselective hydrolysis of n-alkyl sec-alkyl carbonates by porcine pancreatic lipase

Summary n-Alkyl sec-alkyl carbonates were enantioselectively hydrolyzed by porcine pancreatic lipase to give optically active (R)-sec-alkanols. (R)-1-Phenylethanol with an optical purity of >99%ee was obtained by the resolving method.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Baeyer-Villiger oxidation of bicyclic ketones by Cylindrocarpon destructans ATCC 11011

Baeyer-Villiger oxidation of bicyclic ketones1a,1b and4 using whole cell suspensions of the fungusCylindrocarpon destructans was found to proceed quantitatively and in case of substrate (±)-1b a moderate enantioselectivity was observed leading to (1S,6R)-1b and (1R,6S)-2b with 28% and 27% e.e., respectively.     

Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns

 In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced plant promoters. For the efficient production and culture of transgenic hairy root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hairy roots. An average of 0.72 independent kanamycin-resistant (Km^R) roots were obtained per leaf piece. However, a much lower frequency of reporter gene activation was obtained than expected from experiments with the same vectors in Agrobacterium tumefaciens: of more than 700 independent Km^R hairy roots tested, only 8 were β-glucuronidase (GUS) positive. DNA hybridization was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridization patterns. In a parallel analysis of A. thaliana plants transformed with the same vectors using A. tumefaciens, relatively simple T-DNA integration patterns were obtained. The low occurrence of GUS-positive hairy root lines in our experiments could be explained by the multiple T-DNA copies, especially in inverted array, that result in high frequencies of gene inactivation. Received: 11 August 1998 / Revision received: 17 February 1999 / Accepted: 18 March 1999     

Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Km^r) transconjugants, a majority of which were gentamicin sensitive (Gm^s). A small proportion (about 0.8%) of the Km^r Gm^s clones were either auxotrophic or failed to catabolize galacturonate (Gtu⁻). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Km^r Gm^r transconjugant, but not in Km^r Gm^sE. chrysanthemi strains carrying Tn5-induced mutations. In Hfr crosses, Km^r (Tn5) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Km^s, except for Leu⁺ and Arg⁺ recombinants which were 30 to 50% Km^s. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met⁺, Leu⁺, or His⁺ clones, were Km^s. We conclude from both genetic and physical data that Tn5 transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.     

A continuous thermophilic cellulose fermentation in an upflow reactor by aClostridium thermocellum-containing mixed culture

Summary A continuous thermophilic cellulose fermentation by aCl. thermocellum-containing mixed culture was carried out in an upflow reactor for a period of 100 days. The cellulose conversion rate was finally 0.35 g.1^–1.h^–1. Evidence that the fermentation process was influenced by both pH and dilution rate was given by the changes of concentration of the main fermentation products, acetic acid and ethanol. The role of cellodextrins and glucose as reactive intermediates in the process of cellulose breakdown was established.     

An Impaired Uptake of Glucosamine in Tunicamycin Resistant Chinese Hamster Ovary Cells

Tunicamycin resistant mutants (TM^R) were isolated and characterized from Chinese hamster ovary cells. One feature of this TM^R mutants was a marked decrease in incorporation of radioactive glucosamine, both into membrane glycoproteins and G protein of vesicular stomatitis virus.

The cellular uptake and incorporation into acid insoluble materials of various radioactive substances, including glucosamine, galactosamine, mannose, 2-deoxyglucose and leucine, was examined for the purpose of determination whether the reduced incorporation of radioactive glucosamine into glycoproteins was due to a defect in the glycosylation step or decreased uptake of glucosamine by cells.

While incorporation of glucosamine and 2-deoxyglucose into acid insoluble fractions was reduced strikingly in the mutants, the incorporation of mannose and leucine were the same as in the parent cells.

The uptake of glucosamine in TM^R cells was lower than that in the wild type cells, and the Km value for glucosamine uptake differed between the mutants and wild type cells. There was no obvious difference in the uptake of 2-deoxyglucose and mannose.     

Transformation of the astaxanthin-producing yeast Phaffia rhodozyma

Summary This paper describes the genetic transformation of the astaxanthin-producing yeast Phaffia rhodozyma with the cloning vector pGH-1. The plasmid replicates autonomously in this yeast, and the selection of transformants was possible by using both, the URA3 marker from Saccharomyces cerevisiae, and the kanamycin resistance (Km^R) determinant from the bacterial transposon Tn903.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the     

Synthesis of (R - and (S)-1-[[2-Hydroxy-1-(aminomethyl)ethoxy]methyl]-5- benzyluracil,Potent Inhibitors of Uridine Phosphorylase

Abstract

Optically pure (R)- and (S)-1-[[2-hydroxy-1-(aminomethyl) ethoxy]methyl]-5-benzyluracil [(R)-AHPBU and (S)-AHPBU, respectively], two potent uridine phosphorylase inhibitors, have been synthesized via multi-step syntheses starting from independent chiral compounds. The activity of (R)-AHPBU, (S)-AHPBU, and (R,S-AHPBU which have been previously synthesized, on the inhibition of uridine phosphorylase from Sarcoma-180 cells has been studied and compared. The K. values for (R,S)-, (R)- and (S)-AHPBU were determined to be 15·2.3, 17·2.7 and 16·2.0 nM, respectively. This indicates that (R) and (S) optical enantiomers have the same affinity for binding to uridine phosphorylase. These acyclic pyrimidine amino nucleoside analogues represent a new class of potent uridine phosphorylase inhibitors, which has a bulky hydrophobic substituent at the 5-position in the uracil base, yet has remarkably high water solubility.     

Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control

Molecular tools for Gram-positive bacteria such as Mycobacterium are less well-developed than those for Gram-negatives such as Escherichiacoli. This has slowed the molecular-genetic characterisation of Mycobacterium spp, which is unfortunate, since this genus has high medical, environmental and industrial significance. Here, we developed a new Mycobacterium shuttle vector (pMycoFos, 12.5 kb, Km^R) which combines desirable features of several previous vectors (controllable copy number in E. coli, inducible gene expression in Mycobacterium) and provides a new multiple cloning site compatible with large inserts of high-GC content DNA. Copy number control in E. coli was confirmed by the increased Km^R of cultures after arabinose induction and the greater DNA yield of vector from arabinose-induced cultures. Measurement of beta-galactosidase activity in pMycoFos clones carrying the lacZ gene showed that in Mycobacterium smegmatis mc²-155, expression was inducible by acetamide, but in E. coli EPI300, the expression level was primarily determined by the vector copy number. Examination of protein profiles on SDS-PAGE gels confirmed the beta-galactosidase assay results. Construction of a fosmid library with the new vector confirmed that it could carry large DNA inserts. The new vector enabled the stable cloning and expression of an ethene monooxygenase gene cluster, which had eluded previous attempts at heterologous expression.     

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km^r) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km^r transformants.     

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.