Fig. 3. The hdl allele is the first mutant allele of toy. (A) hdl is not allelic to ey, but located between ey and D-Pax2. A complete complementation analysis was carried out among the mutant alleles indicated below the loci of ey, 102CDh, toy, D-Pax2, 102CDg, and the deficiencies; the extent of each deficiency is indicated by horizontal lines below these loci. The hdl allele complements, and maps distal to, two mutant alleles of the l(4)102CDh locus, which are also located between ey and D-Pax2 and one of which bears a synonym identical to that of an allele of the l(4)8 locus according to the original nomenclature (Hochman et al., 1964). hdl also complements the l(4)102CDg3 allele whose previous synonymous name, l(4)19, is identical to another allele of the l(4)8 locus according to the original nomenclature. Since we have mapped l(4)102CDg3 to the most distal portion of the right arm of chromosome 4, it is probably identical to the l(4)19 mutation of the revised nomenclature, which has been mapped to this region (Hochman, 1971). The broken line of Df(4)spa³⁰ indicates incomplete complementation with the 102CDg locus. The deficiencies Df(4)spa³⁰, Df(4)spa⁴⁷, Df(4)spa⁶⁶ (Fu et al., 1998), Df(4)spa^m18 and Df(4)spa^m100 have been obtained in two EMS-induced mutagenesis screens for D-Pax2 mutants, while Df(4)BA uncovering ey was a gift from K. Basler (Brunner, 1997). The inverse sequence of the three loci toy, D-Pax2 and 102CDg in the right telomeric region of chromosome 4 is excluded because Df(4)G, which complements both ey and hdl but uncovers D-Pax2, is a telomeric deficiency (Hochman, 1971). The map is consistent with a previously published map (Locke et al., 2000), but not with that currently available at FlyBase, which erroneously localizes toy distal to D-Pax2. The map is further consistent with the gene order as determined by in situ hybridization to polytene chromosomes (Fu and Noll, 1997; Czerny et al., 1999). (B) The hdl mutation is a deletion of the 3' portion of the toy transcript. The extent of genomic fragments isolated as clones from a wild-type (HDL.1 and HDL.4) and a homozygous hdl genomic library (l(4)8.3) in DASH II are shown with respect to an EcoRI restriction map below, derived from the genomic sequence provided by FlyBase. The genomic region deleted by the toy^hdl mutation is indicated above the restriction map and includes 5,863 bp, extending from nucleotide 1,855 of intron 5 to nucleotide 356 of the last exon, that are replaced by the five base pairs 5'-ATATC-3'. The exon-intron map shown below the genomic restriction map was determined by comparison of the genomic sequence with those of several toy cDNAs, isolated from an embryonic and an eye-disc cDNA library, and of products of a 5'-RACE with poly(A)⁺ RNA from 0- to 20-hour-old embryos raised at 25°C. Protein coding portions of the exons are indicated in black, untranslated leader and trailer in white. Vertical arrows mark alternative 3' ends as determined by sequencing of toy cDNAs and 3'-RACE products. They are preceded by a canonical poly(A) addition signal AATAAA with the exception of the first poly(A) addition site, which is preceded by CATAAA. Restriction sites: A, AccI; B, BamHI; R, EcoRI; S, SalI. (C) The toy^hdl deficiency produces a truncated Toy^hdl protein. The wild-type Toy protein of 543 amino acids, including a paired-domain P and prd-type homeodomain H (Czerny et al., 1999), is shown schematically above the truncated Toy^hdl protein generated by the toy^hdl deficiency. The truncated protein consists of 343 amino acids and includes the N-terminal paired-domain, 46 amino acids of the homeodomain, and, if intron 5 is not spliced out, a 33 amino acid C-terminal portion encoded by the 5' end of intron 5 whose first amino acid, Val, is identical to the 47th amino acid of the homeodomain. If intron 5 sequences are removed by splicing to a cryptic 3' acceptor site close to the toy^hdl deficiency breakpoint in exon 9, the C-terminal tail of the truncated Toy^hdl protein (black) is shorter. The positions of introns are indicated by arrowheads.