Biomedical subjects

I Bernales

Publications and source records attributed to I Bernales.

3 recordsLinked to original sources

PubMed2001

Single-stranded DNA intermediates in IS91 rolling-circle transposition.

IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of phiX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.

Conjugation, Genetic↗

PubMed1999

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions.

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.

Chloramphenicol Resistance↗

PubMed1994

Differential roles of the transposon termini in IS91 transposition.

Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.

Base Sequence↗