PubMed Health⌕ Search

Biomedical subjects

M V Mendiola

Publications and source records attributed to M V Mendiola.

9 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↗
PubMed1996

Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum.

The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described. They all contain the pACYC184 origin of replication enabling them to replicate in E. coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E. coli and mycobacteria. They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E. coli. A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis.

Base Sequence↗
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↗
PubMed1992

Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain.

A high degree of IS6110 restriction fragment length polymorphism (RFLP) is observed amongst the different strains of the Mycobacterium tuberculosis complex. The sequences of the IS6110 flanking regions from a M. tuberculosis strain harbouring four IS6110 copies were determined. Duplication of 3-4 nucleotides was found at the extremities of the four IS6110 copies, suggesting that IS6110 RFLP is due to transposition of the IS element. One of the copies of IS6110 analysed in the study was shown to be located at the same site in the genome of M. tuberculosis as the single copy present in an M. bovis BCG strain.

Base Sequence↗
PubMed1992

DNA sequence of IS91 and identification of the transposase gene.

IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.

Amino Acid Sequence↗
PubMed1989

Specificity of insertion of IS91, an insertion sequence present in alpha-haemolysin plasmids of Escherichia coli.

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT. IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.

Bacterial Proteins↗
PubMed1987

Transposition of IS91 does not generate a target duplication.

We determined the DNA sequences surrounding the junctions of IS91 in two insertion derivatives: pSU234 (pACYC184::IS91) and pSU240 (pBR322::IS91). The termini of IS91 consist of two imperfect inverted repeats eight base pairs long. Their sequence is 5'-TCGAGTAGG. . . CCTATCGA-3'. Insertion of IS91 did not generate direct repetitions in the target DNAs.

Base Sequence↗