Compensating for over-production inhibition of the Hsmar1 transposon in
Hsmar1
Overproduction inhibition
Papillation assay
SETMAR
Transposable elements
Transposase
Journal
Mobile DNA
ISSN: 1759-8753
Titre abrégé: Mob DNA
Pays: England
ID NLM: 101519891
Informations de publication
Date de publication:
2020
2020
Historique:
received:
06
08
2019
accepted:
01
01
2020
entrez:
16
1
2020
pubmed:
16
1
2020
medline:
16
1
2020
Statut:
epublish
Résumé
Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.
Sections du résumé
BACKGROUND
BACKGROUND
Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less
RESULTS
RESULTS
We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the
CONCLUSIONS
CONCLUSIONS
We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.
Identifiants
pubmed: 31938044
doi: 10.1186/s13100-020-0200-5
pii: 200
pmc: PMC6954556
doi:
Types de publication
Journal Article
Langues
eng
Pagination
5Informations de copyright
© The Author(s). 2020.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare that they have no competing interests.
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