X2JEK1 is the native 176-residue CG34171-PC product, not the 292-residue CG34171-PB product A8DZ15. The short product lacks the C-terminal region containing the S1 catalytic serine position. The longer product retains that region but has phenylalanine at its aligned catalytic-serine position. These are different molecular defects; a residue substitution in the long isoform must not be assigned to an absent position in the short isoform.
The frozen FlyBase record maps PC/FBpp0310560/NP_001286041/AHN54555 to X2JEK1 and PB/FBpp0293603/NP_001097175/ABV53692 to A8DZ15. It records two distinct polypeptides and an alternative translation stop involving multiphasic reading frames. No RNA assembly or novel isoform was inferred here.
Direct comparison finds an identical 167-residue N-terminal prefix, followed by a 9-residue X2JEK1 tail versus 125 remaining A8DZ15 residues. The target UniProt S1-domain call spans 38–161 (124 residues); the long isoform's spans 38–265 (228 residues). The alignment includes all target residues from 38 to 176, so the alternative tail was not discarded to manufacture a missing catalytic site.
Active-site coordinates are read from the frozen UniProt reference JSON records, not assigned from sequence-number memory. Both MAFFT L-INS-i (--localpair --maxiterate 1000) and G-INS-i (--globalpair --maxiterate 1000) give the following mapping:
| Catalytic position | Bovine trypsin P00760 | Toad OVCH2 Q66TN7 domain 1 | Long CG34171 A8DZ15 | Short CG34171 X2JEK1 | Q66TN7 domain 2 control |
|---|---|---|---|---|---|
| Histidine | H63 | H90 | H72 | H72 | N633 |
| Aspartate | D107 | D140 | D124 | D124 | D682 |
| Serine | S200 | S238 | F222 | absent aligned region | S773 |
X2JEK1 preserves H72 and D124 but lacks the aligned catalytic-serine region. A8DZ15 has F222 at the serine-equivalent position. The retained H/D pair alone cannot support an intact canonical serine-protease catalytic mechanism.
The two active reference domains provide a positive control, agreeing on H/D/S positions. The second Q66TN7 domain provides a contrast: the same procedure detects loss of the catalytic histidine despite retention of the serine position. This is a sequence-based control; no new enzymatic assays were performed.
The current short isoform sequence does not support the predicted intrinsic serine-type endopeptidase activity. Its missing region is more extensive than a single pseudoenzyme residue substitution. The longer isoform supplies useful evidence that the locus is a serine-protease homolog with catalytic-site divergence, but its longer domain cannot be borrowed to restore the target's missing sequence.
Neither alternative protein's folding, stable expression, biological binding partners, nor noncatalytic process participation was measured here. In particular, lack of intrinsic protease activity does not rule out participation in a proteolytic cascade as a cofactor. No target-specific evidence establishes that possibility either.
The ProtNLM response records Q66TN7 as its phmmer donor but supplies no hit coordinates. Q66TN7 has two S1 domains; the metadata do not identify which domain produced the hit. The alignments here are independent comparisons, not reconstructed ProtNLM alignments. Current target identity is established; the exact sequence delivered to the historical predictor is not reconstructed.
Run just --justfile genes/DROME/CG34171/CG34171-bioinformatics/justfile from the repository root. analyze.py reads the selected frozen UniProt JSON plus A8DZ15, P00760 and Q66TN7 reference JSON, extracts annotated S1 domains, runs both MAFFT modes and maps every annotated reference active site by alignment column. Direct results are results.json, with FASTA inputs and both alignments in this directory.
Software: MAFFT v7.526 (2024/Apr/26); Biopython 1.85, pinned through the script's uv metadata; Python 3. Inputs are local immutable snapshots retrieved 2026-09-08 from UniProt and FlyBase. The target JSON is an exact single-record extraction from the frozen fly cohort. Reference accession URLs can be regenerated by replacing X2JEK1 in the UniProt URL with the stated reference accession.