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

2010 ◽  
Vol 56 (3) ◽  
pp. 187-192 ◽  
Author(s):  
Takashi Saito ◽  
Taku Chibazakura ◽  
Kiwamu Takahashi ◽  
Hirofumi Yoshikawa ◽  
Yasuhiko Sekine
Keyword(s):  
Insertion Sequence ◽  
Transposition Frequency

scholarly journals High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

2005 ◽  
Vol 71 (4) ◽  
pp. 1822-1828 ◽  
Author(s):  
Yoshiyuki Ohtsubo ◽  
Hiroyuki Genka ◽  
Harunobu Komatsu ◽  
Yuji Nagata ◽  
Masataka Tsuda
Keyword(s):  
High Temperature ◽  
Insertion Sequence ◽  
Experimental Protocol ◽  
Specific Primer ◽  
Transposition Frequency ◽  
Burkholderia Multivorans ◽  
Is Elements ◽  
Insertion Elements ◽  
High Temperature Condition ◽  
Starvation Conditions

ABSTRACT An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.

scholarly journals In Vitro Analysis of ISEcp1B-Mediated Mobilization of Naturally Occurring β-Lactamase Gene blaCTX-M of Kluyvera ascorbata

2006 ◽  
Vol 50 (4) ◽  
pp. 1282-1286 ◽  
Author(s):  
Marie-Frédérique Lartigue ◽  
Laurent Poirel ◽  
Daniel Aubert ◽  
Patrice Nordmann
Keyword(s):  
Insertion Sequence ◽  
Transposition Frequency ◽  
Promoter Sequences ◽  
E Coli ◽  
Naturally Occurring ◽  
Kluyvera Ascorbata ◽  
Vitro Analysis ◽  
Lactamase Gene ◽  
In Vitro Analysis

ABSTRACT ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum β-lactamase bla CTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the bla CTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the bla CTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 μg/ml). In those cases, ISEcp1B brought promoter sequences enhancing bla CTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the bla CTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-bla CTX-M-2 occurred at (6.4 ± 0.5) × 10−7 in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40°C.

scholarly journals The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

2021 ◽  
Vol 22 (12) ◽  
pp. 6490
Author(s):  
Olga A. Postnikova ◽  
Sheetal Uppal ◽  
Weiliang Huang ◽  
Maureen A. Kane ◽  
Rafael Villasmil ◽  
...  
Keyword(s):  
Amino Acid ◽  
Insertion Sequence ◽  
Experimental Studies ◽  
Spike Protein ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
New Host ◽  
S Protein ◽  
Furin Cleavage Site ◽  
Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

scholarly journals Excision Patterns of Activator (Ac) and Dissociation (Ds) Elements in Zea mays L.: Implications for the Regulation of Transposition

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1851-1869 ◽  
Author(s):  
Manfred Heinlein
Keyword(s):  
Zea Mays ◽  
Zea Mays L ◽  
Waxy Gene ◽  
Transposition Frequency ◽  
Cis Acting ◽  
Dosage Effects ◽  
In Trans ◽  
Ds Excision ◽  
Maize Kernels ◽  
Strong Contrast

The pattern of aleurone variegation of maize kernels carrying Ac and bz-m2(DI) as reporter allele for Ac activity depends on the dosage of both Ac and Ds. Alterations of Ac dosage can abolish Ds excision at certain times and allow it to occur at other times. wx-m7 and wx-m9 are different Ac insertions in the Waxy gene which have different dosage effects on Ds excision. Kernels, heterozygous for the two Ac alleles and being either wx-m7/wx-m7/wx-m9 or wx-m9/wx-m9/wx-m7 exhibit characteristic patterns of predominantly late excisions; this is in strong contrast to the pattern of early excisions present on wx-m7/wx-m7/wx-m7 homozygotes. This observation supports the hypothesis that the Ac alleles express different amounts of transposase (TPase) during development and that above a certain level of TPase transposition is inhibited. Furthermore, experimental results suggest that the frequency of Ac-induced events is influenced by the dosage and composition of the transactivated Ds or Ac allele. Thus, transposition frequency seems not to be exclusively determined in trans by the amount of active TPase, but also by specific cis-acting properties of the TPase substrate.

scholarly journals Distribution of the ermG Gene among Bacterial Isolates from Porcine Intestinal Contents

2005 ◽  
Vol 71 (8) ◽  
pp. 4930-4934 ◽  
Author(s):  
Yanping Wang ◽  
Gui-Rong Wang ◽  
Nadja B. Shoemaker ◽  
Terence R. Whitehead ◽  
Abigail A. Salyers
Keyword(s):  
Insertion Sequence ◽  
Bacillus Sphaericus ◽  
Gram Positive ◽  
Sequence Element ◽  
Conjugative Transposon ◽  
Gram Positive Bacteria ◽  
First Finding ◽  
Intestinal Contents ◽  
Porcine Feces ◽  
Insertion Sequence Element

ABSTRACT The ermG gene was first found in the soil bacterium Bacillus sphaericus. More recently, it was found in several human intestinal Bacteroides species. We report here the first finding of ermG genes in gram-positive bacteria isolated from porcine feces and from under-barn manure pits used to store porcine wastes. The porcine ermG sequences were identical to the sequence of the B. sphaericus ermG gene except that six of the seven ermG-containing strains contained an insertion sequence element insertion in the C-terminal end of the gene. The porcine ermG genes were found in three different gram-positive genera, an indication that it is possible that the gene is being spread by horizontal gene transfer. A segment of a Bacteroides conjugative transposon that carries an ermG gene cross-hybridized with DNA from six of the seven porcine isolates, but the restriction patterns in the porcine strains were different from that of the Bacteroides conjugative transposon.

scholarly journals Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion

2008 ◽  
Vol 190 (18) ◽  
pp. 6111-6118 ◽  
Author(s):  
P. Rousseau ◽  
C. Loot ◽  
C. Turlan ◽  
S. Nolivos ◽  
M. Chandler
Keyword(s):  
Insertion Sequence ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Functional Differences ◽  
Left And Right ◽  
Ordered Assembly ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

scholarly journals Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

1993 ◽  
Vol 139 (12) ◽  
pp. 3265-3273 ◽  
Author(s):  
S. Ouahrani ◽  
S. Michaux ◽  
J. S. Widada ◽  
G. Bourg ◽  
R. Tournebize ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Insertion Sequence ◽  
Genomic Structure
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