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[Preprint]. 2026 Aug 11:2026.08.11.744233. [Version 1] doi: 10.64898/2026.08.11.744233

Gene model for the ortholog of DENR in Drosophila pseudoobscura

Megan E Lawson 1, Kylee Sanow 2, Mihai Fratian 3, Madelyn Matura 3, Riley Scanlon 4, Madeline Richard 4, Monica Nakhla 4, Chinmay P Rele 1, Jeffrey S Thompson 3, Geoffrey D Findlay 4, Kellie S O’Rourke 5,Ω
PMCID: PMC13640732  PMID: 42845617

Abstract

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Metadata: Bioinformatics, Genomics, Drosophila, Genotype Data, New Finding

Introduction

“Computational gene predictions in non-model organisms often can be improved by careful manual annotation and curation, allowing for more accurate analyses of gene and genome evolution (Mudge and Harrow 2016; Tello-Ruiz et al., 2019). The Genomics Education Partnership (thegep.org) uses web-based tools to allow undergraduates to participate in course-based research by generating manual annotations of genes in non-model species (Rele et al., 2023). These models of orthologous genes across species, such as the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community. The particular gene ortholog described here, Density regulated protein (DENR) in D. pseudoobscura, was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila.” (Myers et al., 2024).

“The IIS pathway is a highly conserved signaling pathway in animals and is central to mediating organismal responses to nutrients (Hietakangas and Cohen 2009; Grewal 2009)” (Myers et al., 2024). “DENR was first discovered in a human teratocarcinoma cell line because its concentration in cells increased with cell density (Deyo et al., 1998). Subsequent bioinformatic and biochemical analyses showed that the protein is conserved across eukaryotes and functions in non-canonical translation initiation (Fleischer et al., 2006; Skabkin et al., 2010). D. melanogaster flies homozygous for a null, knockout allele of the gene encoding DENR (FBgn0030802), die as pharate adults, showing a larval-like epidermis and reduced proliferation of histoblast cells (Schleich et al., 2014). Subsequent experiments using both RNAi in S2 cells and the knockout allele in larvae showed that DENR is required, along with its interacting partner MCT-1, for the proper expression regulation of a subset of transcripts required for cell cycle progression and growth. In particular, the loss of DENR reduces expression of the insulin receptor and makes larvae less sensitive to insulin signaling (Schleich et al., 2014), thus implicating DENR in the regulation of the insulin signaling pathway.” (Laskowski et al., 2024).

“D. pseudoobscura is part of the pseudoobscura species subgroup within the obscura species group in the subgenus Sophophora of the genus Drosophila (Sturtevant 1942; Buzzati-Traverso and Scossiroli 1955). It was first described by Frolowa and Astaurow (1929). The pseudoobscura species subgroup is endemic to the western hemisphere, where D. pseudoobscura is distributed throughout Western North America, Mexico, and Central America (Markow and O’Grady 2005). An additional population of D. pseudoobscura, found near Bogota, Colombia, is partially reproductively isolated from the North and Central American populations (Prakash & Merritt 1972). D. pseudoobscura is found primarily in chaparral and temperate forests. D. pseudoobscura has been studied extensively in the context of ecological and behavioral genetics, speciation, and genome evolution (Powell 1997).” (Lawson et al., 2025).

We propose a gene model for the D. pseudoobscura ortholog of the D. melanogaster Density regulated protein (DENR) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_001354336.1 (Locus ID LOC4814209) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly of D. pseudoobscura (GenBank Accession: GCA_000001765.2). This model is based on RNA-Seq data from D. pseudoobscura (Chen et al. 2014; SRP006203) and DENR in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Gonzalez et al., 2021), which is available at https://gander.wustl.edu.

Results

Synteny

The target gene, DENR, occurs on chromosome X in D. melanogaster and is flanked upstream by CG4880 and CG13002 and downstream by RNA polymerase III subunit I (Polr3I) and Nitrogen permease regulator-like 2 (Nprl2). The tblastn search of D. melanogaster DENR-PA (query) against the D. pseudoobscura (GenBank Accession: GCA_000001765.2) Genome Assembly (database) placed the putative ortholog of DENR within scaffold CH379066 (CH379066.3) at locus LOC4814209 (XP_001354336.1), with an e-value of 2e-56 and a percent identity of 61.49%. The putative ortholog is flanked upstream by LOC4814194 (XP_001354337.1) and LOC4814197 (XP_001354338.3), which correspond to Nprl2 and CG4872 in D. melanogaster (e-values: 0.0 and 1e-162; percent identities: 92.23% and 60.97%, respectively, as determined by blastp; Figure 1A, Altschul et al., 1990). The putative ortholog of DENR is flanked downstream by LOC4814215 (XP_001354335.2) and LOC4814191 (XP_001354334.2), which correspond to Polr3I and CG4880 in D. melanogaster (e-values: 6e-81 and 2e-73; percent identities: 68.39% and 43.38%, respectively, as determined by blastp) (Figure 1A). The putative ortholog assignment for DENR in D. pseudoobscura is supported by the following evidence: synteny of the genomic neighborhood is partially conserved, and additionally while the locations of Nprl2 and CG4880 are not syntenic, they both remain in the genomic neighborhood of DENR. All blast results used to determine orthology indicate very high quality matches.

Figure 1:

Figure 1:

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(A) Synteny comparison of the genomic neighborhoods for DENR in Drosophila melanogaster and D. pseudoobscura. Thin underlying arrows indicate the DNA strand within which the target gene–DENR–is located in D. melanogaster (top) and D. pseudoobscura (bottom). Thin arrows pointing to the left indicate that DENR is on the negative (−) strand in D. melanogaster and D. pseudoobscura. The wide gene arrows pointing in the same direction as DENR are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of DENR are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. pseudoobscura indicate orthology to the corresponding gene in D. melanogaster, black gene arrows indicate non-orthology, and gray gene arrows indicate that a gene is present in both neighborhoods but in different locations relative to the target gene. Gene symbols given in the D. pseudoobscura gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. pseudoobscura. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al., 2014). The coding-regions of DENR in D. pseudoobscura are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. pseudoobscura), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. pseudoobsura), and Splice Junctions Predicted by regtools using D. pseudoobscura RNA-Seq (Chen et al., 2014; SRP006203). Each splice junction is supported by 100–499 reads, which is indicated in pink. (C) Dot Plot of DENR-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. pseudoobscura (y-axis). Amino acid number is indicated along the left and bottom; CDS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. The gap in the dot plot region corresponding to the first CDS of DENR-PA results from decreased sequence identity in this region. (D) Protein alignment of DENR-PA in D. melanogaster (top row) vs. the orthologous peptide in D. pseudoobscura (bottom row). The alternating colored rectangles represent adjacent CDS. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemical properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. This protein alignment shows that while there is decreased sequence similarity within the beginning portion of the first CDS of DENR-PA in D. melanogaster and D. pseudoobscura, there is still high conservation of the peptide sequence and the biochemical properties of this region of the peptide.

Protein Model

DENR in D. pseudoobscura has three CDSs within the genome sequence. The single unique protein sequence (DENR-PA and DENR-PB) is translated from two mRNA isoforms that differ in their UTRs (DENR-RA and DENR-RB; Figure 1B). Relative to the ortholog in D. melanogaster, the CDS number and protein isoform count are conserved, as DENR-RA and DENR-RB are also identical with three coding CDSs in D. melanogaster. The sequence of DENR-PA in D. pseudoobscura has 89.5% identity with the protein-coding isoform DENR-PA in D. melanogaster, as determined by blastp (Figure 1C). Reduced sequence identity was found in the first CDS, but the protein alignment (Figure 1D) indicates that this region still has very high biochemical similarity across the two species. Coordinates of this curated gene model (DENR-PA, DENR-PB) are stored by NCBI at GenBank/BankIt (accession BK064553, BK064554, respectively). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).

Methods

Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2023).

Supplementary Material

Supplement 1
media-1.zip (1.2KB, zip)

Supplemental Files

1. Zip file containing FASTA, PEP, GFF files for the gene model

Acknowledgements

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.

Funding

This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
media-1.zip (1.2KB, zip)

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