Steady-state levels of fem-3 mRNAs. (A) Masculinization of the germline (Mog) is dependent on the temperature and the genetic background. Germlines analyzed are heterozygous for fem-3(gf) and homozygous for ccr-4, rnp-8, or larp-1. (n) Number of animals tested. Mutant phenotypes increase in severity with temperature. 15°C is permissive, 20°C intermediate, and 25°C restrictive temperature. (B) Analysis of fem-3(q95)/+ extracts by semiquantitative PCR and Southern blotting. Genomic (gDNA) and reverse-transcribed (cDNA) products are visible. gDNA allows monitoring of amplification efficiency. (C) fem-3(gf) alleles. Left: Mutations in the fem-3 3′ UTR analyzed in this study. fem-3(q95) deletes the entire PME. Right: Compilation of fem-3 mRNA steady-state levels in fem-3(gf)/fem-3(+) heterozygotes as obtained by different techniques. The proportion of each transcript is indicated as a percentage of total fem-3 mRNA. The data from three to four independent experiments are shown, including standard deviations: fem-3(q95,gf) was analyzed by real-time PCR and Southern blotting; fem-3(q96,gf) was tested by sequencing; fem-3(q23,gf) was assayed by restriction fragment polymorphism analysis and by sequencing. (D) In the absence of ccr-4 and rnp-8, fem-3(q23) mRNA levels are slightly increased relative to wild type. Removal of larp-1 does not modify the amount of fem-3(q23) mRNA when compared to wild type. The probe used for the blot was directed against a fragment that is common for both alleles. Right: Quantification of the Southern blot by phosphoimager analysis. The proportion of fem-3(q23) and fem-3(+) mRNAs is indicated as a percentage of total fem-3 mRNA. Three independent experiments are compiled. (E,F) FBF-1 binds weakly to the fem-3(q23) mRNA. The results from three independent yeast three-hybrid assays on plates (E) and in solution (F) are shown. The interactions were tested on wild-type and mutant PME sequences.
