Interallelic complementation among five lethal mutations in lola. (A) Schematic representation of the genomic organization of the lola locus. The entire locus consists of 32 exons; gray, white, and black boxes indicate 5′ variable, constant, and 3′ variable exons, respectively. Transcription starts at either of the 5′ variable exons, and 3′ variable exons are alternatively spliced to the constant exons, generating variants encoding 20 Lola isoforms with distinct C-terminal domains containing Zn finger motifs (Ohsako et al. 2003). (B) Locations of point mutations in EMS-induced mutant alleles lola^ORE120, lola^ORC46, lola^ORE50, lola^ORC4, and lola^ORE119. Only the exons relevant to this study are indicated (C5–8, constant exons; V10–11, V21–22, V23, and V32, variable exons for isoforms B, K, L, and T, respectively). The arrow at the left end indicates the transcription start site and direction. (C) Relative viability (%) of F1 flies of the indicated genotype. The values were calculated based on the number of flies against expected ones, which was half of the number of flies with CyO chromosome, a balancer for all lola mutant alleles. Bars indicate the limits of 95% confidence.

RFLP analysis of interallelic trans-splicing events. The C5–8/V32 variant cDNAs were RT–PCR-amplified from total RNA fraction of D. melanogaster strains Oregon R (OR) and yellow white (YW), and their hybrids (F1), and analyzed by 1.5% agarose gel electrophoresis after restriction digestion with FspI and BstZ17I. (Lane 1) M indicates 1 kb ladder markers. (Lane 2) O indicates OR (Lane 3) Y denotes YW. (Lane 4) O+Y indicates equal amounts of OR and YW RNAs mixed before the RT–PCR. (Lane 5) O/Y F1 denotes OR/YW F1. (Lane 6) U indicates undigested RT–PCR products from OR. Structures of C5–8/V32 variant cDNAs are illustrated to the right. The segments derived from OR and YW are indicated in the rectangle and ellipsoid with designations of O and Y, respectively. The thick vertical bars represent the allele-specific restriction sites. The fragment lengths obtained after restriction digestion are indicated in base pairs (bp).

Detection of V32 exon-specific core promoter element. (A) Nucleotide sequence comparison around the V32-specific core promoter element between D. melanogaster (mel) and D. pseudoobscura (pse). The transcription start site was estimated based on the 5′ RACE, and defined as +1. The putative Inr and DPE elements are underlined and boxed, respectively. Asterisks indicate identical nucleotides. (B) Reporter assay for V32-specific promoter activity. Structures of constructs containing intron 30–luciferase fusions are schematically shown to the left. The putative transcription initiation site and direction are indicated by the bent arrow. The open box indicates the putative DPE element. The reporter constructs were transfected into Drosophila S2 cells, and luciferase activities were determined. The act–Rluc construct was cotransfected as an internal control. Results represent the mean ± S.E. for three independent experiments.