Figure 4.

Molecular and functional characterization of mfl mutants. (a) Northern blot analysis of total RNA extracted from wild-type or mfl animals with a genomic probe including the fourth mfl intron. Female (F) and male (M) adult flies carrying the hypomorphic mfl¹ allele or first-instar larvae (L1) carrying the mfl⁰⁵ and mfl⁰⁶ alleles were analyzed. (b) Western blot analysis of extracts obtained from wild-type or mfl animals, carrying (+) or not carrying (−) a MFL coding transgene. An affinity-purified rabbit polyclonal anti-MFL antibody, kindly provided by S. Poole, was used. Both wt and mfl homozygous animals were grown under heat shock regimen (30 min/d). As shown, MFL level is reduced in all mfl mutants (lanes −) but reaches nearly the wild-type amount in mfl⁰⁵ and mfl⁰⁶ transformed larvae (lanes +) at 6 or 24 h from the heat-shock pulse. (c) Lethal phase (see Materials and Methods) of mfl mutants is compared with that of mfl⁰⁵ and mfl⁰⁶ transgenic lines in which MFL was overexpressed from the heat-inducible hsp70 promoter. While most of mfl⁰⁵ or mfl⁰⁶ homozygotes develop only until the first or the third-larval instar, respectively, 30% of mfl⁰⁵ and 80% of mfl⁰⁶ transgenic animals reach the pupal stage when grown under daily heat-shock treatment. Moreover, these transgenic animals develop synchronously with their wild-type siblings and show a normal increase in their size.