Fig 1.
C4 yellow Disintegrase transposon.
Shown in yellow is the yellow gene, which includes its promoter, 5’ UTR and 3’ UTR. Downstream of this is the nanos promoter with its 5’ and 3’ UTRs (green). Inserted between the nanos 5’ and 3’ UTRs is the Disintegrase sequence (light orange) fused to a nuclear localization sequence (nls in red). The 3’ and 5’ P inverted repeats are labeled in dark orange.
Fig 2.
The P{RLY} transposon and the strategy for testing for CPE and making an attP landing site.
The top shows the part of the pRLY plasmid between the P-element ends (orange rectangles). The mini-w gene is flanked by M2 (light purple rectangle) and scs (purple rectangle) insulator sequences. M2 is also flanked by FRT sites (light green rectangles) so it can be excised by FLP recombinase to test for CPE (middle of figure). The insulators and mini-w are flanked by attR and attL sites. The phiC31 Dint enzyme can excise these sequences, leaving an attP site (bottom of figure). Downstream is a transcription unit made of enhancers from the y gene upstream of a y+ cDNA to serve as a marker after mini-w is removed. There are loxP sites (green rectangles) flanking the y+ transgene so it can be removed by CRE recombinase.
Fig 3.
Eyes of 2 to 3 day old heterozygous females were photographed before (M2) and after (ΔM2) flipping out the M2 insulator upstream of the mini-w gene to test for CPE. Heterozygotes were used to avoid any confounding pairing effects, although CPE is equally apparent in homozygotes. The fly lines shown were used to make attP sites. Note that the 3W.3 line shows reverse CPE, suggesting the M2 insulator is blocking a silencer rather than an enhancer.
Table 1.
Fly lines with attP sites and their CPE potential.
Fig 4.
(A) Integration of the attB plasmid into the attP docking site results in the test insulator and downstream mini-w gene being near genomic DNA. The scs insulator and rest of the plasmid (not shown, indicated by the broken line) is between the mini-w and y+ transgenes. One plasmid is shown, although three plasmids were used for integration: one lacking a test insulator, one with a monomer of the M insulator fragment from scs’, and one with the M2 dimer. (B) CPE is evident after integration into the attP-H.X line without an insulator upstream of mini-w. This CPE is partially blocked by the M insulator and is more effectively blocked when M is dimerized into the M2 insulator. (C, D) CPE that can be blocked by M2 is also evident after integration into the attP-I.2 and attP-M.X lines.