Screen for SIMs. dp cn bw/dp cn bw; III/III males, isogenic for chromosomes II, marked with dp cn and bw, and III, were fed EMS and mated to T(2;3)ap^Xa/II; Cbx Ubx gl³ virgin females. Single T(2;3)ap^Xa/(dp cn bw)*; III* F₁ males bearing mutagenized (*) autosomes were then mated in vials to Su(z)2¹/CyO; III/III virgin females. The vials were subsequently scored for the absence Su(z)2¹/(dp cn bw)*; III/III* F₂ progeny, indicating that at least one of the mutagenized autosomes may carry a SIM. Because T(2;3)ap^Xa causes a dominant notched wing phenotype, and the CyO balancer causes a dominant curly wing phenotype, vials lacking Su(z)2¹/(dp cn bw)*; III/III* F₂ progeny were identified by the absence of flies with normal (non-notched, straight) wings. Note that use of T(2;3)ap^Xa allowed for the simultaneous testing of both autosomes because it is a translocation between chromosomes II and III.

ClustalX alignment of Su(z)2, Psc, and L(3)73Ah from Drosophila with Bmi-1 and Mel-18 from both Homo sapiens and Mus musculus. The alignment generated by ClustalX is focused on the region of the proteins located between the RF and HTH domains and begins with the last conserved cysteine residue in the RF. Amino acid positions are indicated on the left. Amino acids highlighted in red are identical (equivalent to “*” in ClustalX) among the proteins indicated, while strongly conserved amino acids (equivalent to “:” in ClustalX) are highlighted in yellow. More weakly conserved amino acids (equivalent to “.” in ClustalX) are not highlighted. The CSR1 core sequence is underlined twice, with the Su(z)2^s20 mutation indicated above. The Su(z)2¹ missense mutation is shown to the right of CSR1. Residues that correspond to α-helical regions 3 and 4 in the Bmi-1/Ring1B structure are shown. The CSR2 core sequence is underlined once, with the Su(z)2^s21 mutation indicated above.

Disorder analysis of Psc. See Figure 5 for explanation of format.

Comparison of Drosophila Su(z)2, Psc, and l(3)73Ah with human Bmi-1 and Mel-18. The HR and CTR are labeled. The RF is in red and the HTH is in blue. Regions enriched for specific amino acids are in black and labeled with the relevant residue. The figure is drawn to scale.

Analysis of Su(z)2 alleles. (A) Structure of the wild-type Su(z)2 locus and insertions and deletions associated with Su(z)2 mutations. Psc (not shown) is located to the left. Exons 1–6 are shown as numbered rectangles. The RF is in red, and the HTH is in blue (exon 4–5 junction). Noncoding sequence is in gray. The TATA notation shown upstream of exon 1 is the putative TATA box identified 8 bp upstream of the Su(z)2^1.b7 breakpoint. R, EcoRI; N, NotI. (B) Frameshift, nonsense, and missense mutations associated with Su(z)2 mutations. Nonsense mutations are shown above the protein and are divided into two classes: those associated with a frameshift followed by a stop codon (FS*, top) and those associated with only a stop codon (*, bottom). Missense mutations are shown below the protein. The RF is shown in an exploded view, with bases that are altered by Su(z)2 mutations shown in black. Zn⁺⁺-coordinating residues are indicated. Su(z)2¹ (green), Su(z)2^4-34 (orange), Su(z)2^h29 (purple), and Su(z)2^s95 (blue) are complex and have been color coded to highlight the multiple mutations that they contain. Su(z)2¹ contains an L120F missense mutation and a K284* nonsense mutation. Both Su(z)2^4-31 and Su(z)2^4-34 contain an 8-bp deletion in exon 5 (not shown) that generates a three-amino-acid frameshift ending in a K238* nonsense codon (B). Su(z)2^4-34 differs from Su(z)2^4-31 in that it also contains an insertion (A) that has been localized to a 1.6-kb ClaI/BamHI fragment in the distal half of intron 1. Su(z)2^h29 contains a G-to-A transition that disrupts a 5′ acceptor site for exon 4 (A) and is predicted to result in a frameshift ending in a T186* nonsense codon (B). Su(z)2^s95 contains a roo or roo-like (A) and a Q370* nonsense codon (B). *, Su(z)2^1.b7, a derivative of Su(z)2¹, carries a deletion (A), a 4-bp insertion (TTCT, not shown) at the site of the deletion, and the K284* nonsense codon that is also present in Su(z)2¹ (B). We have not determined whether Su(z)2^1.b7 also contains the L120F missense mutation that is found in Su(z)2¹. Both A and B are drawn to scale.

Disorder analysis of Su(z)2. (A) DisEMBL analysis of Su(z)2 showing the predictions for loops or coil (blue), remark-465 (green), and hot loops (red). (B) Su(z)2 protein is shown at a scale matching that of the plot above. The RF is in red, and the HTH is in blue. Regions of protein disorder predicted by all three methods are shown in orange. The predicted points of truncation of the truncation alleles are shown below.