Fig. 4. Molecular map depicting the eth gene, deletions following P-element excision, and structure of the eth-egfp transgene. (A) Molecular map for the 2nd chromosome right arm (60E) region covering eth and adjacent P-element location and direction in the EP(2)1065 line. Boxes are exons for each of the genes, including orc4, eth and reg-5. The primer set used for deletion mutant screening is shown by small arrows. ATGs depict the putative translation initiation sites and directions of the genes. Asterisks indicate the stop codons in each gene. As the transcription initiation site for reg-5 is ambiguous, a thin hatched bar is used to depict the sequence of Van Gelder (Van Gelder and Krasnow, 1996) (GenBank Accession Number, U65105) and a thick hatched bar is used for the EST clones HL04722.5', LP09845.5' and SD04185.5' sequences (GenBank Accession Numbers, AA698461, AI296050 and AI532618, respectively). (B) eth deletion lines generated by imprecise P-element excision from EP(2)1065 line. Three relevant deletion mutant lines are shown. (C) eth gene structure. Boxes show exons, EcRE indicates a putative ecdysteroid responsive element (imperfect repeat of aggtca) (Park et al., 1999), ATG shows the putative translation initiation site for eth, and the star indicates location of the stop codon. ETH1, ETH2 and ETH-AP are depicted with canonical processing sites GKR, GR, KRR and GRR (Park et al., 1999). (D) The transgene pCaSpeR4[eth3egfp]. The ETH chimera with EGFP is constructed after the last canonical processing site GRR. Restriction enzyme sites used for cloning into pCaSpeR4 vector (Thummel and Pirrotta, 1991) are shown as EcoRI and XhoI.
