We propose a gene model for the Drosophila ananassae ortholog of the D. melanogaster Protein tyrosine phosphatase 61F ( Ptp61F ) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_014764155.1 (Locus ID LOC6492936 ) in the May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly of Drosophila ananassae ( GCA_000005115.1 , Drosophila 12 Genomes Consortium et al., 2007). This model is based on RNA-Seq data from Drosophila ananassae ( SRP006203 , SRP007906 , PRJNA257286 , PRJNA388952 ; Graveley et al., 2011) and Ptp61F in D. melanogaster using FlyBase release FB2023_03 ( GCA_000001215.4 ) (Larkin et al., 2021; Gramates et al., 2022; Jenkins et al., 2022).

The protein product of Ptp61F (PTP61) negatively regulates the Insulin/TOR pathway by dephosphorylating the insulin receptor (InR) (Wu et al., 2011; Buszard et al., 2013). PTP61Fm-mediated dephosphorylation of InR requires an interaction with the SH2/SH3 adaptor protein Dock ( dreadlocks ) while PTP61Fn dephosphorylates InR in a Dock-independent manner (Clemens et al., 1996; Buszard et al., 2013; Willoughby et al., 2017). The Ptp61F gene in Drosophila melanogaster was first isolated using rat PTP61F cDNA in a low stringency hybridization screening method (McLaughlin and Dixon 1993). Ptp61F is differentially spliced where the longer isoform (PTP61Fm) is targeted to the ER by the hydrophobic C-terminal domain, and the shorter isoform (PTP61Fn) is targeted to the nucleus by a nuclear localization signal (NLS) (McLaughlin and Dixon 1993). PTP61F plays a role in the regulation of a variety of signaling pathways through negative regulation of tyrosine kinases including Janus kinase/signal transducers and activators of transcription (JAK/STAT), Mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR), and platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) (Baeg et al., 2005; Buszard et al., 2013; Tchankouo-Nguetcheu et al., 2014; Willoughby et al., 2017).

Synteny

The reference gene, Ptp61F , occurs on chromosome 3L in D. melanogaster and is flanked upstream by rhomboid ​( rho ) and Stromalin 2 ( SA2 ) and downstream by indra and Hydroxymethylbilane synthase ( Hmbs ). There are also six genes nested within Ptp61F in D. melanogaster : CG9173 , FucTD , CG9168 , CG32320 , ru , and CG9166 . The tblastn search of D. melanogaster Ptp61F-PA (query) against the Drosophila ananassae (GenBank Accession: GCA_000005115.1 ) Genome Assembly (database) placed the putative ortholog of Ptp61F within scaffold_13337 at locus LOC6492936 ( XP_014764155.1 )— with an E-value of 7e-28 and a percent identity of 84.48%. Furthermore, the putative ortholog is flanked upstream by LOC6492935 ( XP_001956685.1 ) and LOC6507090 ( XP_001956686.1 ), which correspond to Reg-2 and CG9166 in D. melanogaster (E-value: 0.0 and 2e-140; identity: 89.62% and 83.55%, respectively, as determined by blastp ) ( Figure 1A ; Altschul et al., 1990). The putative ortholog of Ptp61F is flanked downstream by LOC6492937 (XP_ 001956688.1) and LOC6507089 (XP_ 001956689.1), which correspond to indra and Hmbs in D. melanogaster (E-value: 2e-96 and 0.0; identity: 32.20% and 86.85%, respectively, as determined by blastp ). The putative ortholog assignment for Ptp61F in D. ananassae is supported by the following evidence: The blastp and tblastn results support the presence of a Ptp61F ortholog in this location in D. ananassae although synteny is not completely conserved. In D. melanogaster , the Ptp61F-PC isoform has 6 genes nested within it, but this characteristic is not present in D. ananassae , so we hypothesize that the Ptp61F-PC isoform does not exist in this species. CG9166 is present in both genomic neighborhoods, but in slightly different locations (i.e., not nested within Ptp61F ), and the first and second downstream genes ( indra and Hmbs ) are orthologous. Therefore, we conclude that LOC6492936 is the correct ortholog of Ptp61F in D. ananassae ( Figure 1A ).

Protein Model

Ptp61F in D. melanogaster has mRNA isoforms: Ptp61F-RA, Ptp61F-RB, Ptp61F-RC, Ptp61F-RD, and Ptp61F-RE. Ptp61F-RE and Ptp61F-RD have identical coding sequences. mRNA soform Ptp61F-RA contains six CDSs, Ptp61F-RB and Ptp61F-RC have seven CDSs, and Ptp61F-RD and Ptp61F-RE have four CDSs. In D. ananassae , the isoform count is not conserved (see Special characteristics of the protein model), and we predict five total isoforms (Ptp61F-PA, Ptp61F-PB, Ptp61F-PD, Ptp61F-PE,and Ptp61F-PNF) . The sequence of Ptp61F-PA in Drosophila ananassae has 76.87% identity (E-value: 0.0) with the protein-coding isoform Ptp61F-PA in D. melanogaster , as determined by blastp ( Figure 1C ). Given that D. ananassae and D. melanogaster belong to distinct species groups, the observed degree protein divergence is well explained and consistent with that seen in other ortholog comparisons between these two species ( Drosophila 12 Genomes Consortium et al., 2007). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accessions BK064622 , BK064623 , BK064624 , BK064625 , and BK064626 ). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).

Special characteristics of the protein model

We hypothesize that the isoform Ptp61F-PC does not exist in D. ananassae . In addition, we hypothesize that there is a new isoform, Ptp61F-PNF, due to alternative splicing. Ptp61F-PC in D. melanogaster has a long first intron that has six genes nested within it, but this does not appear in D. ananassae ( Figure 1A ). A tblastn search of the amino acid sequence of the first CDS in Ptp61F-PC in D. melanogaster against the D. ananassae genome did not return any results, and there are no predicted splice junctions consistent with a long first intron for Ptp61F-PC in this species. This leads us to conclude that the Ptp61F-PC isoform is not present in D. ananassae . Finally, we determined that XM_032453921 ( LOC6492936 ) is a novel isoform, as there is not an orthologous isoform in D. melanogaster that has the same gene structure. It has seven CDSs, and its sixth CDS is longer than the corresponding CDS in isoform Ptp61F-PB ( Figure 1D ). The seventh CDS of the proposed novel isoform is only eight base pairs long (including the stop codon), as compared to 37 base pairs long for the seventh CDS of isoform Ptp61F-RB. The sixth and seventh CDSs of this novel isoform are supported by a splice junction with a score of 457 ( Figure 1D, shown in pink). These features of XM_032453921 ( LOC6492936 ) lead us to believe that new alternative splicing has occurred, resulting in a novel isoform. This novel isoform has 75.86% protein identity (E-value: 0.0) to the D. melanogaster Ptp61F-PB isoform. We propose that this isoform be named Ptp61F-PNF.

Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2023). Briefly, students use the GEP instance of the UCSC Genome Browser v.435 ( https://gander.wustl.edu ; Kent WJ et al., 2002; Navarro Gonzalez et al., 2021) to examine the genomic neighborhood of their reference IIS gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students then retrieve the protein sequence for the D. melanogaster reference gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly on the NCBI BLAST server ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ; Altschul et al., 1990) to identify potential orthologs. To validate the potential ortholog, students compare the local genomic neighborhood of their potential ortholog with the genomic neighborhood of their reference gene in D. melanogaster . This local synteny analysis includes at minimum the two upstream and downstream genes relative to their putative ortholog. They also explore other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Detailed explanation of how these lines of genomic evidenced are leveraged by students in gene model development are described in Rele et al. (2023). Genomic structure information (e.g., CDSs, intron-exon number and boundaries, number of isoforms) for the D. melanogaster reference gene is retrieved through the Gene Record Finder ( https://gander.wustl.edu/~wilson/dmelgenerecord/index.html ; Rele et al ., 2023). Approximate splice sites within the target gene are determined using tblastn using the CDSs from the D. melanogaste r reference gene. Coordinates of CDSs are then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirm the biological validity of their target gene model using the Gene Model Checker ( https://gander.wustl.edu/~wilson/genechecker/index.html ; Rele et al., 2023), which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. At least two independent models for a gene are generated by students under mentorship of their faculty course instructors. Those models are then reconciled by a third independent researcher mentored by the project leaders to produce the final model. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol (Gruys et al., 2025).