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Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica.

ABSTRACT: DNA rearrangements, including insertions, deletions, and inversions, control gene expression in numerous prokaryotic and eukaryotic systems, ranging from phase variation of surface antigens in pathogenic bacteria to generation of Ig diversity in human B cells. We report here that precise excision of the mobile element IS492 from one site on the Pseudoalteromonas atlantica chromosome directly correlates with phase variation of peripheral extracellular polysaccharide ((p)EPS) production from OFF (epsG::IS492) to ON (epsG(+)). In a previously undescribed application of quantitative PCR, we determined that the frequency of this transposase-dependent precise excision is remarkably high, ranging from 10(-3) to 10(-2) per cell per generation. High-frequency excision resulting in nonmutagenic repair of donor DNA is extremely unusual for classical transposable elements. Interestingly, high-frequency precise excision of IS492 does not occur at four different insertion sites on the P. atlantica chromosome, despite identity in the IS492 nucleotide sequences and 5- to 7-bp flanking DNA. The genome sequence revealed that epsG-associated IS492 is the only element inserted within a gene. Quantitative RT-PCR assays for externally derived transposase transcripts from each IS492 copy showed that IS492 at epsG has higher levels of host-initiated transcription through the element, suggesting that transcription per se or an increase in transposase (mooV) expression is responsible for the effect of chromosomal position on element excision. MooV levels and excision activity for IS492 inserted in forward and reverse orientations relative to plac and pT7 in Escherichia coli support that external transcription of mooV boosts transposase to a critical level required for detectable excision.

SUBMITTER: Higgins BP

PROVIDER: S-EPMC1794265 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica.

Higgins Brian P BP Carpenter Chandra D CD Karls Anna C AC

Proceedings of the National Academy of Sciences of the United States of America 20070130 6

DNA rearrangements, including insertions, deletions, and inversions, control gene expression in numerous prokaryotic and eukaryotic systems, ranging from phase variation of surface antigens in pathogenic bacteria to generation of Ig diversity in human B cells. We report here that precise excision of the mobile element IS492 from one site on the Pseudoalteromonas atlantica chromosome directly correlates with phase variation of peripheral extracellular polysaccharide ((p)EPS) production from OFF ( ...[more]

PMID: 17264213

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Project description:Histotripsy produces tissue fractionation through dense energetic bubble clouds generated by short, high-pressure, ultrasound pulses. When using pulses shorter than 2 cycles, the generation of these energetic bubble clouds only depends on where the peak negative pressure (P-) exceeds the intrinsic threshold of the medium (26 to 30 MPa in soft tissue with high water content). This paper investigates a strategic method for precise lesion generation in which a low-frequency pump pulse is applied to enable a sub-threshold high-frequency probe pulse to exceed the intrinsic threshold. This pump-probe method of controlling a supra-threshold volume can be called dual-beam histotripsy. A 20-element dual-frequency (500-kHz and 3-MHz elements confocally aligned) array transducer was used to generate dual-beam histotripsy pulses in red blood cell phantoms and porcine hepatic tissue specimens. The results showed that when sub-intrinsic-threshold pump (500-kHz) and probe (3-MHz) pulses were applied together, dense bubble clouds (and resulting lesions) were only generated when their peak negative pressures combined constructively to exceed the intrinsic threshold. The smallest reproducible lesion varied with the relative amplitude between the pump and probe pulses, and, with a higher proportion of the probe pulse, smaller lesions could be generated. When the propagation direction of the probe pulse relative to the pump pulse was altered, the shape of the produced lesion changed based on the region that exceeded intrinsic threshold. Because the low-frequency pump pulse is more immune to attenuation and aberrations, and the high-frequency probe pulse can provide precision in lesion formation, this dual-beam histotripsy approach would be very useful in situations in which precise lesion formation is required through a highly attenuative and aberrative medium, such as transcranial therapy. This is particularly true if a small low-attenuation acoustic window is available for the high-frequency probe transducer.

Dataset Information

Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica.

Publications

Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica.

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