| Autor: |
Huisman, Olivier, Kleckner, Nancy |
| Zdroj: |
Genetics; June 1987, Vol. 116 Issue: 2 p185-189, 5p |
| Abstrakt: |
We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10transposition in Escherichia coli. This test involves a new Tn10derivative called the "mini-lacZ-kanRfusion hopper" or mini-Tn10-LK for short. This element does not direct expression of β-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, transposition of this element into the chromosome of E. coli lacZ-bacteria leads to productive fusions in which the lacZgene within the transposon is expressed from external chromosomal signals. Such fusions are readily detectable on MacConkey lactose indicator plates as red (Lac+) papillae inside of white (LacZ-) colonies. The length of time required to see red papillae appearing in a white colony sensitively and accurately reflects the transposition frequency of the mini-transposon within the colonies. Differences in times for color formation are sensitive enough that 10-fold differences in transposition frequency can readily be detected. This papillation assay can be used to identify mutant clones in which the frequency of Tn10transposition is either increased or decreased. We have successfully used the assay to identify mutations in the terminal sequences of Tn10; mutations in the Tn10transposase gene or the bacterial host can be isolated just as easily. This screen should be readily adaptable to transposable elements other than Tn10. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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