Figure 1.

Plasmid construct for expression of hairpin ADE2 dsRNA within cells to interfere with the ADE2 message. (a) Schematic of the ADE2 hairpin construct. ADE2 sequences from nt +1 to +414 were cloned in opposite orientation flanking 100 bp of yEGFP3, downstream of the enolase promoter (ENO1p) and with the 3′ untranslated region of HWP1 (3′ HWP1) in pENO1GFP3. Arrows indicate 5′ to 3′ of ADE2. Relevant restriction endonuclease sites are shown above the construct. (b) Endogenous ADE2 mRNA levels are not affected in a strain bearing the dsRNA plasmid construct. Top panel: northern blot analysis of the wild-type strain SC5314, lane 1; parental strain (CAI4), lane 2; and EAGA (CAI4 transformed with pEAGA), lane 3; probed for ADE2 sequences with a ³²P-labelled PCR amplicon. Bottom panel: equal loading of total RNA was assessed by ethidium bromide staining of rRNA bands separated in standard formaldehyde gels used in northern blot analysis