Measurements of transposition frequency of insertion sequence IS1 by GFP hop-on assay

Saito, Takashi; Chibazakura, Taku; Takahashi, Kiwamu; Yoshikawa, Hideki; Sekine, Yoshiaki

doi:10.2323/jgam.56.187

Cited by 5 publications

(6 citation statements)

References 20 publications

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“…These estimates were typically obtained by ‘mating-out’ assays [9], papillation assays [13] and fluctuation tests. Estimates obtained by direct observation are rare [14].…”

Section: Introductionmentioning

confidence: 99%

“…Estimates of the transposition and excision rates of IS elements vary widely, ranging from 10 23 to 10 27 for transposition [9,10] and about 10 29 to 10 210 for excision [11,12]. These estimates were typically obtained by 'mating-out' assays [9], papillation assays [13] and fluctuation tests. Estimates obtained by direct observation are rare [14].…”

Section: Introductionmentioning

confidence: 99%

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Rates of transposition in Escherichia coli

et al. 2013

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The evolutionary role of transposable elements (TEs) is still highly controversial. Two key parameters, the transposition rate (u and w, for replicative and non-replicative transposition) and the excision rate (e) are fundamental to understanding their evolution and maintenance in populations. We have estimated u, w and e for six families of TEs (including eight members: IS1, IS2, IS3, IS4, IS5, IS30, IS150 and IS186) in Escherichia coli, using a mutation accumulation (MA) experiment. In this experiment, mutations accumulate essentially at the rate at which they appear, during a period of 80 500 (1610 generations × 50 lines) generations, and spontaneous transposition events can be detected. This differs from other experiments in which insertions accumulated under strong selective pressure or over a limited genomic target. We therefore provide new estimates for the spontaneous rates of transposition and excision in E. coli. We observed 25 transposition and three excision events in 50 MA lines, leading to overall rate estimates of u ∼ 1.15 × 10–5, w ∼ 4 × 10−8 and e ∼ 1.08 × 10−6 (per element, per generation). Furthermore, extensive variation between elements was found, consistent with previous knowledge of the mechanisms and regulation of transposition for the different elements.

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“…These estimates were typically obtained by ‘mating-out’ assays [9], papillation assays [13] and fluctuation tests. Estimates obtained by direct observation are rare [14].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rates of transposition in Escherichia coli

et al. 2013

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“…These mutational events are often mediated by mobile genetic elements, and their rates depend on the type of mobile element, the precise genomic location as well as the organism (Díaz-Maldonado et al, 2015;Periwal and Scaria, 2015;Steinrueck and Guet, 2017). Reported rates span a wide range (from 10 -3 to 10 -8 per cell per generation), but are typically orders of magnitude higher than rates of point mutations (Hudson et al, 2002;Saito et al, 2010;Sousa et al, 2013;Tomanek et al, 2020). Given this elevated frequency of small-and large-scale genomic rearrangements in various bacterial species, changes in local genetic context have the potential to shape bacterial phenotypes even in absence of sequence changes.…”

Section: Discussionmentioning

confidence: 99%

Local genetic context shapes the function of a gene regulatory network

Staroń

Tomasek

Carter

et al. 2021

eLife

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Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions, such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks remains a major challenge. Here, we use a well-defined synthetic gene regulatory network to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one gene regulatory network with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual transcriptional units, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of gene regulatory networks.

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“…The transposition frequency of IS Sau2 was estimated using a modified “GFP hop-on assay” method, which has been used to determine the transposition of transposons in both Bacillus subtilis and E. coli [ 19 , 20 ]. This method, making it possible to detect transposition at the single cell level, was more efficient to estimate the transposition frequency than other methods [ 19 , 20 ], such as papillation assays [ 21 , 22 ], mutation accumulation experiment [ 23 ] and mating-out assay [ 24 ]. Thus, the GFP hop-on assay was adapted to measure the transposition frequency of IS Sau2 in the presence of OrfA, OrfB or OrfAB.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a functional insertion sequence ISSau2 from Staphylococcus aureus

Wang

Xue

et al. 2018

Mobile DNA

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BackgroundISSau2 has been suggested as a member of the IS150 f subgroup in the IS3 family. It encodes a fusion transposase OrfAB produced by programmed − 1 translational frameshifting with two overlapping reading frames orfA and orfB. To better characterize ISSau2, the binding and cleaving activities of the ISSau2 transposase and its transposition frequency were studied.ResultsThe purified ISSau2 transposase OrfAB was a functional protein in vitro since it bound specifically to ISSau2 terminal inverted repeat sequences (IRs) and cleaved the transposon ends at the artificial mini-transposon pUC19-IRL-gfp-IRR. In addition, the transposition frequency of ISSau2 in vivo was approximately 1.76 ± 0.13 × 10− 3, based on a GFP hop-on assay. Furthermore, OrfB cleaved IRs with the similar catalytic activity of OrfAB, while OrfA had no catalytic activity. Finally, either OrfA or OrfB significantly reduced the transposition of ISSau2 induced by OrfAB.ConclusionWe have confirmed that ISSau2 is a member of IS150/IS3 family. The ISSau2 transposase OrfAB could bind to and cleave the specific fragments containing the terminal inverted repeat sequences and induce the transposition, suggesting that ISSau2 is at least partially functional. Meanwhile, both OrfA and OrfB inhibited the transposition by ISSau2. Our results will help understand biological roles of ISSau2 in its host S. aureus.

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Measurements of transposition frequency of insertion sequence IS1 by GFP hop-on assay

Cited by 5 publications

References 20 publications

Rates of transposition in Escherichia coli

Rates of transposition in Escherichia coli

Local genetic context shapes the function of a gene regulatory network

Characterization of a functional insertion sequence ISSau2 from Staphylococcus aureus

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