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1.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  4.—

F igure 4.—. From: The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.

The insertion-site preferences of hermes. (A) A sequence logo was generated for the 26 insertions identified from YHL912 containing the donor hermes–kanMX6 (pHL2577) and Rep81X Tpase (pHL2578). Nucleotide preferences within the target-site duplication are shown for each of the eight positions. The most preferred nucleotide is shown at the top of each column and the least preferred at the bottom. The height of each nucleotide correlates with its frequency. (B) Insertion sites for 26 strains were mapped relative to their position in or near an ORF. Positions in the ORF were determined by calculating the distance as a percentage within each individual ORF. Positions in the 5′ or 3′ region were determined on the basis of whether the insertion was closest to the 5′- or the 3′-end of an ORF.

Adam G. Evertts, et al. Genetics. 2007 December;177(4):2519-2523.
2.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  1.—

F igure 1.—. From: The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.

The donor and expression plasmids for hermes transposition in S. pombe. (A) Six expression plasmids were built to control and vary the expression of the hermes transposase. Variation is achieved by using three strengths of the nmt1 promoter as well as a T317A mutant transposase. The XhoI–XmaI fragment containing the transposase was 1844 bp. (B) The donor plasmids provide the source of transposon DNA flanked by the TIRs. Here the kanMX6 cassette was cloned between the TIRs, giving cells with insertions resistance to the drug G418. The XhoI–EagI fragment containing kanMX6 and the TIRs was 2782 bp. (C) Another version of the donor plasmid was constructed using nat, a gene that gives cells with insertions resistance to nourseothricin. The XhoI–EagI fragment containing nat was 2548 bp. (D) This donor plasmid contains a p15A bacterial origin of replication, allowing for insertions to be readily isolated in bacteria. The XhoI–EagI fragment containing p15A and kanMX6 was 3333 bp.

Adam G. Evertts, et al. Genetics. 2007 December;177(4):2519-2523.
3.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  5.—

F igure 5.—. From: The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.

Quantitative measures of insertion rates. (A) Levels of transposition of three transformants of YHL912 containing the REP81X-Tpase plasmid (pHL2578) and the hermes–kan donor (pHL2577). The percentage of cells with chromosomal insertions is shown on the y-axis and the total number of cell generations on the x-axis. Growth was carried out in a series of four liquid cultures, each starting at OD 0.05, which were diluted when the OD was ∼5.0. One culture of transformant 1 reached OD 7.6. The number of colonies on a YES + FOA + G418 plate divided by the number of colonies on an EMM + FOA plate gave the transposition frequency. The control strain with an empty pREP3 expression plasmid (pHL423) shows no transposition. (B) Transposition rates per generation were calculated by determining the slope of the line between the third and final data points for each transformant.

Adam G. Evertts, et al. Genetics. 2007 December;177(4):2519-2523.
4.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  3.—

F igure 3.—. From: The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.

The DNA blots of strains with insertions of hermes. (A) Independent patches of YHL912 containing the donor hermes–kanMX6 (pHL2577) were induced for the expression of hermes transposase with Rep3X Tpase (pHL2574), Rep41X Tpase (pHL2575), or Rep81X Tpase (pHL2578). Genomic DNA from FOA/G418-resistant strains was digested with EcoRI and separated by agarose gel and then transferred to a nitrocellulose membrane and probed with a radiolabeled neo fragment. Individual bands indicate insertions of the hermes transposon. The expression vector used to generate the strains is shown at the top of each blot. Lanes labeled “C” contained DNA from the parental strain YHL912. (B) The method for inverse PCR used to identify the position of the insertions is shown. Two micrograms of genomic DNA was digested with EcoRI, phenol extracted, and diluted to 1 ng/μl. The dilution was ligated using T4 DNA ligase at 18° overnight. The resulting material was ethanol precipitated and 100 ng of circularized DNA was amplified by PCR. The arrows indicate the PCR primers used to amplify the genomic DNA adjacent to hermes right (HL1430, 5′-GCCTCGACATCATCTGCCC out from kanMX6 and HL1431, 5′-CTCTAGCGGTGATCTTAACATC out from hermes right). The junction with hermes left was amplified using a primer in hermes left (HL1893, 5′-ACCCGAGTGTCGATGAATCAATGAA) and one in the flanking genomic DNA as identified by the inverse PCR.

Adam G. Evertts, et al. Genetics. 2007 December;177(4):2519-2523.
5.
F <span style="font-variant: small-caps" class="small-caps">igure</span>  2.—

F igure 2.—. From: The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe.

The expression and activity of hermes transposase in S. pombe. (A) The immunoblot of extracts showed the expression of YHL912 containing the transposase-expressing plasmids REP3X-Tpase (pHL2574), REP41X-Tpase (pHL2575), and REP81X-Tpase (pHL2578) compared to the empty vector Rep3 (pHL423). A whole-cell protein extract was obtained using a bead-beater protocol. Cells were grown in EMM minus leucine to a final OD of 1.0 and 40 OD units of cells were pelleted. After protein extraction, 12 μg of protein was subjected to electrophoresis on a 10–20% Tris–glycine gel (Invitrogen, San Diego) and transferred to a membrane. The membrane was probed overnight at room temperature with an antitransposase antibody at a concentration of 1:1000. The antibody was polyclonal and was raised in rabbit (X. Li, and N. Craig, unpublished results). The band-labeled background was independent of transposon expression. (B) This patch assay qualitatively measures the levels of transposition. Cells were initially grown as patches on EMM −ura −leu −B1 for 2 days to induce transposase expression. These patches were then replica printed to plates containing EMM −leu + FOA + B1 to select for cells lacking the donor plasmid. Patches from these plates were printed to plates with YES + FOA + G418 to detect cells with insertions. (Left) Strains with the donor plasmid hermes–kanMX6 (pHL2577). (Right) Strains with the donor plasmid hermes–kanMX6-p15A (pHL2641). Each row has two patches of YHL912 containing the expression plasmids Rep81X Tpase (pHL2578), Rep3X mutant Tpase (pHL2623), Rep41X mutant Tpase (pHL2624), and Rep81X mutant Tpase (pHL2625) (left) and Rep3X Tpase (pHL2574), Rep41X Tpase (pHL2575), Rep81X Tpase (pHL2578), Rep3X mutant Tpase (pHL2623), Rep41X mutant Tpase (pHL2624), and Rep81X mutant Tpase (pHL2625) (right). The control strains were YHL912 with hermes–kanMX6 (pHL2577) and the empty expression vector pHL423.

Adam G. Evertts, et al. Genetics. 2007 December;177(4):2519-2523.

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