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We have determined the nucleotide sequence of IS1162, a new insertion sequence (IS) isolated from Pseudomonas fluorescens (Pf) strain ST. This IS element is present in two copies on the pEG plasmid harboured by Pf ST and in a single copy on the chromosome, adjacent to the styrene catabolic genes. IS1162 is 2634 bp in length with 12-bp terminal inverted repeats (IR), and could encode four proteins...
An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or drop-out , according to its position in the cloned fragment. In...
A derivative of the IS903 transposon (Tn) is described that is capable of creating lacZ gene fusions upon transposition. It should find wide use as a tool for Tn mutagenesis in bacteria since it can be used both to generate mutants and to examine gene expression. The transposase-encoding gene (tnp) is located outside the Tn in the vector, thus Tn insertions into a genome are stably maintained in...
The mechanism of activation of metastasis-associated mts1 gene transcription in the mouse myelomonocytic leukaemia WEHI-3 cell line is described. Northern blot analysis showed that WEHI-3 cells expressed two types of mts1-specific mRNA: standard (550 nt) and additional (about 800 nt). The steady-state expression level of the 800-nt mRNA was 10-fold higher than that of the 550-nt mRNA. A cDNA clone...
We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that...
We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance...
A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on...
The Enterococcus faecalis transposon Tn917 is functional in a broad range of bacteria, including both Gram-positive and Gram-negative species. We cloned Tn917-LTV3, a derivative carrying a promoterless lacZ (β-galactosidase gene), into the thermosensitive shuttle replicon pG + host4 and assayed for chromosomal insertions in group A streptococcus (GAS). Tn917 transposed into the GAS chromosome...
The mini-Tn5 derivative transposon Tn5493 transposes at a frequency as high as 3% in the Gram-positive bacterium Streptomyces lividans. The use of a thiostrepton-resistance gene and a temperature-sensitive delivery system carrying the transposase gene allows an easy selection for stable transposition events. Insertions into the S. lividans genome seem to be fairly random, as shown by Southern blot...
The retrovirus-like element Ty3 of Saccharomyces cerevisae integrates into the yeast genomic DNA in a position specific manner. Ty3 integrates within 1-2 base pairs of the site of transcription initiation by RNA polymerase III. The human tRNA 3Lys gene was used as a target for transposition in a plasmid-based assay to determine whether Ty3 integration can be targeted to a human...
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