Figure 1
varicose encodes a MAGUK protein. (A) Exon-intron structure of the vari locus (scale bar according to FlyBase). Two different transcripts could be isolated: the vari-long transcript contains exons 1, 2, 3 to 10 with the translational start residing in exon 1; vari-short consists of exons 3a, 3 to 10 with the translational start site in exon 4 (dark grey boxes: exons common to both transcripts, light grey boxes: exons specific to vari-long, white boxes: UTR). Mobilisation of the P(XP)-element d10880 yielded the vari-short-specific mutant allele variMD109, which removes exon 3a and adjacent 3'-intronic DNA (shaded box). Primer pairs vari-long-5/3 and vari-short-5/3 were used to detect the corresponding vari transcripts in the wild-type (D) and different vari alleles (see Fig. 5). (B) Structure and size comparison of the MAGUK proteins Vari-long and Vari-short with their human homologs hsMPP6_c (GenBank accession number EAW93810), hsMPP6_a (GenBank accession number EAW93808) and hsMPP2_b (GenBank accession number EAW51656), respectively. The percentages of amino acid identities of the domains with respect to the corresponding domains of Vari-long are shown. (C) Northern blot of 5 μg poly(A+) RNA from staged wild-type embryos (1 = 0–4 h, 2 = 4–12 h, 3 = 12–24 h) hybridised with a probe that detects both vari-long and vari-short transcripts. (D) RT-PCR on total RNA from wild-type embryos (> 8 h old) with primer pairs vari-long-5/3 and vari-short-5/3 detects single vari-long- and vari-short-transcripts. (E) Western blot analysis of protein lysates from staged wild-type embryos and embryos of different genotypes (> 8 h old), probed with an anti-Vari antibody that recognises both Vari-long and Vari-short (1 = wt, 0–4 h, 2 = wt, 4–12 h, 3 = wt, 12–24 h, 4 = daG32>UAS Flag-vari-long, 5 = daG32>UAS vari-RNAi, 6 = Df(2L)DS6). Overexpressed Flag-Vari-long in lane 4 is slightly bigger than endogenous Vari-long due to the N-terminal Flag-tag. The protein amount loaded per lane equals 5 embryos with the exception of lysates from daG32>UAS Flag-vari-long embryos (equals 0.5 embryos). The lysate from Df(2L)DS6 embryos serves as a negative control and an unspecific, cross-reacting band (asterisk) as a loading control.