The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fr...

Oyerexpression of the asnA gene from dseherichia coti K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. cofi. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the totRl soluble protei" in the E. coti cell, was readily purified to app...

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by trans...

knowing the nucleotide sequence of the cholera toxin operon, we designed oligonucleotide primers for its-pcr amplification from local clinical isolates of v. cholerae. the resulting amplification product was cloned in a common puc18 vector. subsequently, a part of this operon encoding the cholera toxin bsubunit (ctb) was reamplified and cloned between the bamh1 and ecor1 sites of the same vecto...

We have developed an autonomously replicating Xylella fastidiosa (Xf)/E. coli plasmid that efficiently transforms Xf; unfortunately this plasmid was not stably maintained in Xf cells en planta or without antibiotic selection in vitro. Another plasmid, containing an Xf native plasmid, a Kan cassette and cloned in pUC18 was also constructed and shown to be unstable without antibiotic selection. 1...

The unidirectional digestion of DNA, first cut with two different restriction enzymes generating one 3'-protrusion and than with exonuclease III and Sl-nuclease allows a very defined removal of DNA for sequencing (1,2) or mapping of biological functions. The DNA is cloned into the polylinker of e.g. pUC18 or mpl8 (2), double digested with SstI/SmaI or PstI/XbaI, phenol extracted, ethanol precip...

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (P≤0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (P≤0.05) and mean plant length was significantly greater (...

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the (32)P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991...

Complementation analysis is a powerful technique in the characterization of genes and their products. When studying cloned genes the analysis is facilitated if compatible vectors are available into which restriction fragments can be cloned. Most multicopy cloning vectors currently in use are derivatives of pBR322 (1), with ampicillin (Ap) resistance markers and the ColEl origin of replication. ...