Fig. 1

Genomics of the bcd locus. (A) Original bcd Northern filter from [23], rescanned and adjusted to denote the 1.6 kb smbcd transcript that was not visible in the original publication due to printing constraints. smbcd is considerably less abundant than lgbcd but is expressed much broader during development. Stages and intervals where polyA⁺ RNAs were collected are indicated at the bottom. (B) Top, domain structure of large Bicoid with annotated domains; bottom, domain structure of small Bicoid. Notably, Smbcd lacks the DNA-binding activity of the homeodomain (bright blue) and the COOH-terminal part of the self-inhibitory domain (SID; yellow), compare in Fig. 1C. (C) Sequence of the SID domain from different insects (from top: Lonchoptera lutea, Musca domestica, Lucilia sericata, Calliphora vicina, Drosophila virilis, Drosophila sechellia, Drosophila simulans, Drosophila melanogaster) aligned, with 100% identities annotated by asterisks below. Blue dashed line denotes borders of exon 1 and exon 2, green dashed box denotes core part of the SID. The amino acid sequence of Smbcd, where exon 1 and 4 (in red) are joined, is indicated below. AAAAA denotes mutagenized amino acids, and the resulting changes in Bcd activity are indicated in percentages compared to wild-type Bcd [30]. The fusion of exon 1 to exon 4 to create smbcd has been documented in Drosophila melanogaster, all Drosophilidae and Lucilia sericata so far. (D) Molecular map of the bcd locus, illustrating genome organization, strengths, and mapped molecular lesions of bcd mutants, extent of the homology to bcd pseudogene CR14578, and outline of the CRISPR-mediated deletion with HDR. The bcd gene exhibits alternative splicing, generating five transcripts, two of which - large bicoid (lgbcd) and small bicoid (smbcd) - are displayed. In the latter, exons 2 and 3 are omitted. Three other seemingly minor alternative splice events were also reported (FlyBase). Mutants with mapped molecular lesions [34] fall into three classes, classification according to [17]: strong (red) and intermediately strong (orange) alleles reveal lesions, primarily as point mutations (asterisks) around the homeodomain (light blue). A MiMIC allele results in a truncated protein due to integration into the first intron. Three alleles are weak ones (green) and show lesions outside the homeodomain. Allele names are indicated above the lesion as FlyBase name, followed by the original name from [17]. The 3 L-region in the heterochromatin harbors a large stretch of bcd homologous sequences with almost 100% DNA sequence identity, annotated as an inactive pseudogene CR14578 (green). The extent of the CRISPR-mediated deletion of the genomic bcd region and subsequent replacement with an eyeless-DsRed cassette is shown in pink