A vector for recombinant DNA in Staphylococcus aureus

Staphylococcal plasmids pS194 and pSC194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant DNA, since they each carry one single recipient site for endonuclease EcoRI. Hybrid DNA does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by EcoRI. A chloramphenicol marker present in pSC194 was used for positive hybrid selection. Hybrid plasmids generated by joining pSC194 with one or more of the four EcoRI fragments of the large (18.1-10(6) daltons) staphylococcal plasmid pI258 were constructed and permitted us to develop a physical map for pI258.     

Single amino acid mutations block a late step in the folding of beta-lactamase from Staphylococcus aureus

Two single amino acid mutant proteins of beta-lactamase PC1 from Staphylococcus aureus, P2 Thr40----Ile and P54 Asp146----Asn, have been investigated using urea-gradient polyacrylamide gel electrophoresis, circular dichroism and sedimentation velocity. Investigation of the folded states of the mutants has shown that compared to wild-type PC1 they are slightly more expanded, and have reduced aromatic circular dichroism, but the same content of secondary structure as PC1. The mutants exhibit fast refolding kinetics to the folded state, in contrast to PC1, which refolds only slowly. We conclude from these results that the folded mutants are in a state close to but distinct from the native state of PC1 and have certain properties in common with the compact intermediate in the folding of beta-lactamase. Therefore, these single amino acid substitutions result in a folding pathway blocked at a point located after collapse of the already folded structural units into a globular shape, and close to the final reshuffling step that leads to the native state of the wild-type enzyme.     

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ($\text{w}\text{v}$) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.     

Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans.

Immunoreactive somatostatin is released from islets of Langerhans, isolated from rat pancreas by collagenase digestion, when incubated in an $\text{in vitro}$ system. The rate of somatostatin secretion is independent of extracellular glucose concentration, but is stimulated by addition of 8-Br-cyclic AMP or theophylline.     

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

Construction and analysis of recombinant lambda phages containing mitochondrial DNA fragments.

Rat mtDNA has a molecular length of about 16 kilobase (kb) pairs and is cleaved into seven fragments by restriction endonuclease EcoRI. These fragments were cloned in Escherichia coli K-12 host using lambda gtWES.lambda B' (lambda gtWES.lambda B, for short, in this paper) as a vector. Recombinant DNAs containing one or a few fragments of the mtDNA were transfected to CaCl2-treated E. coli, and the plaques containing specific recombinant phages were selected. DNA amplified in the recombinanat phage lambda gt.mt was shown to contain the same restriction endonuclease cleavage sites as those found in the mtDNA. Present results permitted the DNA sequencing of any portion of the mitochondrial genome.     

Partial characterization of a small, multiple-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant

An organism classified as Streptomyces espinosus was found to carry an approx. 9.2-kb plasmid. This plasmid, designated pUC6, has a copy number of 30-40 per host genome equivalent. Plasmid pUC1061, a copy-number mutant of pUC6, was isolated after in vitro deletion of an approx. 2.0-kb XhoI restriction fragment. Plasmid pUC1061 has a copy number of 500-600. Plasmid pUC1061 appears to be incompatible with pUC6 and will transform a pUC6-containing culture at a frequency of approx. 1%. The sizes, restriction maps and copy numbers of plasmids pUC6 and pUC1061 indicate these may be valuable vectors for gene cloning Streptomyces.     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.     

Restriction map of bacteriophage SP02: ordering the SacI fragments and identification of the DNA termini

A plasmid that is able to replicate in both Escherichia coli and Streptococcus sanguis has been constructed by the in vitro joining of the pACYC184 (Cm^r Tc^r) and pVA749 (Em^r) replicons. This plasmid, designated pVA838, is 9.2 kb in size and expresses Em^r in both E. coli and S. sanguis. Its Cm^r marker is expressed only in E. coli and may be inactivated by addition of DNA inserts at its internal EcoRI or PvuII sites. The pVA838 molecule also contains unique SalI, SphI, BamHI, NruI and XbaI cleavage sites suitable for molecular cloning. pVA838 may be amplified in E. coli but not in S. sanguis. We have used the pVA838 plasmid as a shuttle vector to clone streptococcai plasmid fragments in E. coli. Such chimeras isolated from E. coli were readily introduced into S. sanguis by transformation.     

Bioprospecting in potato fields in the Central Andean Highlands: Screening of rhizobacteria for plant growth-promoting properties

The Central Andean Highlands are the center of origin of the potato plant (Solanum tuberosum). Ages of mutualism between potato plants and soil bacteria in this region support the hypothesis that Andean soils harbor interesting plant growth-promoting (PGP) bacteria. Therefore, the aim of this study was to isolate rhizobacteria from Andean ecosystems, and to identify those with PGP properties. A total of 585 bacterial isolates were obtained from eight potato fields in the Andes and they were screened for suppression of Phytophthora infestans and Rhizoctonia solani. Antagonistic mechanisms were determined and antagonistic isolates were further tested for phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and production of NH₃- and indole-3-acetic acid (IAA). PGP was studied in healthy and R. solani diseased plantlets under growth room conditions. Performance was compared to the commercial strain B. subtilis FZB24^® WG. Isolates were dereplicated with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), and identified with 16S rRNA gene sequencing and multi locus sequence analysis (MLSA). A total of 10% of the isolates were effective antagonists, of which many were able to solubilize phosphate, and produce IAA, ACC deaminase, NH₃ and hydrogen cyanide (HCN). During growth room experiments, 23 antagonistic isolates were associated with plant growth-promotion and/or disease suppression. Ten isolates had a statistically significant impact on test parameters compared to the uninoculated control. Three isolates significantly promoted plant growth in healthy plantlets compared to the commercial strain, and seven isolates outperformed the commercial strain in in vitro R. solani diseased plantlets.     

Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs

A method is described for the isolation and purification of covalently closed circular (ccc) DNA from yeast (Saccharomyces cerevisiae). Spheroplasts are lysed at pH 12.45 which denatures linear but not ccc DNA. Next, the lysate is taken through a gentle high-salt-phenol extraction to remove single-stranded DNA. The ccc DNA, recovered by ethanol precipitation, can be further studied by agarose gel electrophoresis, can be cut with restriction endonucleases and can be used to transform Escherichia coli. This method efficiently purifies large (approx. 190 kb) and small (approx. 1.5 kb, TRP1-RI Circle) circular DNAs and thus has general applicability for isolation and purification of plasmids from yeast.     

Cloning and expression of an extracellular-agarase gene from Streptomyces coelicolor A3(2) in Streptomyces lividans 66

An extracellular agarase gene was cloned from Streptomyces coelicolor A3(2) strain M130 into S. lividans 66 using the multicopy plasmid vector pIJ702. Various deletion derivatives of the initial clone (pMT605) were obtained by in vitro and in vivo methods. This allowed the gene to be localised to a 1.9-kb segment of DNA. The agarase enzyme was overproduced (up to 500 times) and exported efficiently into the medium. The agarase protein was identified as a 28-kDal band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); in the case of one derivative, pMT608, this band accounted for nearly 50% of the total extracellular protein. Differences in agarase production between the deletion derivatives correlated well with plasmid stability.     

Membrane-active antimicrobial peptide identified in Rana arvalis by targeted DNA sequencing

Antimicrobial peptides (AMPs) are naturally produced, gene encoded molecules with a direct antimicrobial activity against pathogens, often also showing other immune-related properties. Anuran skin secretions are rich in bioactive peptides, including AMPs, and we have reported a novel targeted sequencing approach to identify novel AMPs simultaneously in different frog species, from small quantities of skin tissue. Over a hundred full-length peptides were identified from specimens belonging to five different Ranidae frog species, out of which 29 were novel sequences. Six of these were selected for synthesis and testing against a panel of Gram-negative and Gram-positive bacteria. One peptide, identified in Rana arvalis, proved to be a potent and broad-spectrum antimicrobial, active against ATCC bacterial strains and a multi-drug resistant clinical isolate. CD spectroscopy suggests it has a helical conformation, while surface plasmon resonance (SPR) that it may self-aggregate/oligomerize at the membrane surface. It was found to disrupt the bacterial membrane at sub-MIC, MIC and above-MIC concentrations, as observed by flow cytometry and/or visualized by atomic force microscopy (AFM). Only a limited toxicity was observed towards peripheral blood mononuclear cells (PBMC) with a more pronounced effect observed against the MEC-1 cell line.     

Design and synthesis of new antitumor agents with the 1,7-epoxycyclononane framework. Study of their anticancer action mechanism by a model compound

This article describes the design, synthesis and biological evaluation of a new family of antitumor agents having the 1,7-epoxycyclononane framework. We have developed a versatile synthetic methodology that allows the preparation of a chemical library with structural diversity and in good yield. The synthetic methodology has been scaled up to the multigram level and can be developed in an enantioselective fashion. The study in vitro of a model compound, in front of the cancer cell lines HL-60 and MCF-7, showed a growth inhibitory effect better than that of cisplatin. The observation of cancer cells by fluorescence microscopy showed the presence of apoptotic bodies and a degradation of microtubules. The study of cell cycle and mechanism of death of cancer cells by flow cytometry indicates that the cell cycle arrested at the G₀/G₁ phase and that the cells died by apoptosis preferably over necrosis. A high percentage of apoptotic cells at the subG₀/G₁ level was observed. This indicates that our model compound does not behave as an antimitotic agent like nocodazole, used as a reference, which arrests the cell cycle at G₂/M phase. The interaction of anticancer agents with DNA molecules was evaluated by atomic force microscopy, circular dichroism and electrophoresis on agarose gel. The results indicate that the model compound has not DNA as a target molecule. The in silico study of the model compound showed a potential good oral bioavailability.