Yeast transposition assay constructs. (A) Structures of pMuta1_PAG415 and pWL89Ae. AmpR, ampicillin resistance gene; ori, E. coli replication origin; Pgal1, GAL1 promoter; CYC1 ter, terminator; CEN, centromere sequences of yeast chromosomes; ARS, autonomous replication site. Dashed lines indicate the position of nonautonomous element insertions, in the 5âUTR and coding region respectively. Black arrows indicate the positions of primers used for PCR analysis in Figure S3A. (B) Excision from coding region of ADE2. (C) Excision from 5â UTR of ADE2. (D) Reintegration. In the parental strain, pWL89A carries Muta1HIS in the coding region of ADE2. Reintegration is assayed by selecting cells that retain the HIS marker in Muta1HIS when the parental plasmid is excluded by 5-FOA treatment, which is toxic to Ura+ cells. (TIF 602 kb

Kun Liu

Susan R. Wessler

Mobile DNA

PubMed

Footprints from Muta1NA1 excision events. Arrows indicate the Muta1NA1 insertion, length and sequence of the TSD in each assay (shown above the arrows). TSD or sequences derived from TSDs are in bold, sequences derived from Muta1NA1 are underlined, the number of recovered events is on the right. (A) Footprints of Muta1NA1 excision from the ADE2 5’ UTR without donor site TSD. (B-D) Footprints of Muta1NA1 excision from the ADE2 5’ UTR with different 8 bp TSD sequences. (B) TTCAATAG; (C) CGATTCAA; (D) GGTAACTC. (E-G) Footprints of Muta1NA1 excision from the ADE2 5’ UTR with different 9 bp TSD sequences. (E) TCGATTCAA, (F) CGGTAACTC, (G) ATTCAATAG. (H-I) Footprints of Muta1NA1 excision from the ADE2 5’UTR with the transposase W401F mutation. (H) the 8bp TSD TTCAATAG was used. (I) the 9bp TSD ATTGAATAG was used. (TIF 665 kb

Kun Liu (148201)

Susan Wessler (3631237)

The Francis Crick Institute

Additional file 10: Figure S8. of Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

Springer - Publisher Connector

Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti
        

BackgroundMutator-like transposable elements (MULEs) are widespread with members in fungi, plants, and animals. Most of the research on the MULE superfamily has focused on plant MULEs where they were discovered and where some are extremely active and have significant impact on genome structure. The maize MuDR element has been widely used as a tool for both forward and reverse genetic studies because of its high transposition rate and preference for targeting genic regions. However, despite being widespread, only a few active MULEs have been identified, and only one, the rice Os3378, has demonstrated activity in a non-host organism.ResultsHere we report the identification of potentially active MULEs in the mosquito Aedes aegypti. We demonstrate that one of these, Muta1, is capable of excision and reinsertion in a yeast transposition assay. Element reinsertion generated either 8 bp or 9 bp target site duplications (TSDs) with no apparent sequence preference. Mutagenesis analysis of donor site TSDs in the yeast assay indicates that their presence is important for precise excision and enhanced transposition. Site directed mutagenesis of the putative DDE catalytic motif and other conserved residues in the transposase protein abolished transposition activity.ConclusionsCollectively, our data indicates that the Muta1 transposase of Ae. aegypti can efficiently catalyze both excision and reinsertion reactions in yeast. Mutagenesis analysis reveals that several conserved amino acids, including the DDE triad, play important roles in transposase function. In addition, donor site TSD also impacts the transposition of Muta1

Liu, Kun

Wessler, Susan R

eScholarship - University of California

English

Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

Crossref

Summary of 14 MULE families in Ae. aegypti. (DOCX 81 kb

Additional file 2: Table S1. of Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

ADE2 revertant colonies from the yeast transposition assay. (A-G) Excision activity of nonautonomous elements from the ADE2 coding region. (H) Negative control, Muta1AR excision from the ADE2 coding region is tested on plates without galactose. (I-L) Muta1AR excision from the ADE2 coding region is tested with mutant transposases. (TIF 3512 kb

Additional file 5: Figure S4. of Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

Additional file 11: Table S3. of Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

http://dx.doi.org/10.1186/s13100-016-0084-6

Functional characterization of the active Mutator-like transposable element, Muta1 from the mosquito Aedes aegypti

Abstract

Similar works

Full text

Available Versions

The Francis Crick Institute

Springer - Publisher Connector

Springer - Publisher Connector

eScholarship - University of California

Crossref

The Francis Crick Institute

The Francis Crick Institute

The Francis Crick Institute