Description
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This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP;
thegep.org
) for Course-based Undergraduate Research Experience (CURE). The following information in this box may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster genes in the insulin signaling pathway.
"In this GEP CURE protocol students use web-based tools to manually annotate genes in non-model
Drosophila
species based on orthology to genes in the well-annotated model organism fruitfly
Drosophila melanogaster
. The GEP 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). Computational-based gene predictions in any organism are often 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). 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.” (Myers et al., 2024).
“The particular gene ortholog described here 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
. The Insulin/insulin-like growth factor signaling pathway (IIS) 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).
“
D.
mojavensis
is part of the
mulleri complex
in the
repleta
species group within the subgenus
Drosophila
of the genus
Drosophila
(Wasserman 1992; Durando et al., 2000)
.
It was first described by Patterson (Patterson and Crow 1940).
D. mojavensis
specializes on rotting cactus as its host and is found in the Mojave and Sonoran Deserts of the southwestern United States and northwestern Mexico including the Baja Peninsula, as well as on the channel-islands off the coast of California (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).” (Congleton et al., 2023).
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We propose a gene model for the
D. mojavensis
ortholog of the
D. melanogaster
tango
(
tgo
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein
XP_002000334.1
(Locus ID
LOC6574279
) in the May 2011 (Agencourt dmoj_caf1/DmojCAF1) Genome Assembly of
D. mojavensis
(
GCA_000005175.1
- Drosophila 12 Genomes Consortium et al., 2007). This model is based on RNA-Seq data from
D. mojavensis
(
SRP006203
- Chen et al., 2014)
and
tgo
in
D. melanogaster
using FlyBase release FB2023_03 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022; Jenkins et al., 2022).
The
Drosophila
tango
(
CG11987
, FBgn0264075,
tgo
) gene encodes a bHLH-PAS protein that controls tracheal and CNS midline development (Sonnenfeld et al., 1997). Tango can form heterodimers with Single-minded and Trachealess, which bind a specific DNA enhancer element (Sonnenfeld et al., 1997), and
tango
mutants reveal defects in CNS midline and tracheal. In addition, the bHLH-PAS proteins Similar (Sima) and Tango are homologous to HIF-α and HIF-β, thus contributing to a conserved transcriptional response to hypoxia in
Drosophila melanogaster
(Lavista-Llanos et al., 2002). Insulin activates a HIF-dependent transcriptional response, which is mediated by the TOR and PI3K-AKT pathways. Sima's nuclear localization is increased when dAKT and dPDK1 are overexpressed, which mimics a hypoxic treatment (Dekanty et al., 2005).
Synteny
The reference gene,
tgo
, occurs on
chromosome 3R in
D. melanogaster
and is flanked upstream by
neuralized
(
neur
) and
hyrax
(
hyx
) and downstream by
CG11986
and
Splicing factor 3b subunit 5
(
Sf3b5
). The
tblastn
search of
D. melanogaster
tgo-PA (query) against the
D. mojavensis
(GenBank Accession:
GCA_000005175.1
Genome Assembly (database) placed the putative ortholog of
tgo
within scaffold 6540 (
CH933806.1
) at locus
LOC6574279
(
XP_002000334.1
)— with an E-value of 0.0 and a percent identity of 82.45%. Furthermore, the putative ortholog is flanked upstream by
LOC6574278
(
XP_002000333.3
) and
LOC6574277
(
XP_015022843.1
), which correspond to
neur
and
hyx
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 79.51% and 90.59%, respectively, as determined by
blastp
;
Figure 1A
; Altschul et al., 1990). The putative ortholog of
tgo
is flanked downstream by
LOC6574280
(
XP_002000335.2
) and
LOC6574281
(
XP_002000336.1
), which correspond to
CG11986
and
Sf3b5
in
D. melanogaster
(E-value: 0.0 and 8e-62; identity: 71.70% and 98.82%, respectively, as determined by
blastp
). The putative ortholog assignment for
tgo
in
D. mojavensis
is supported by the following evidence: The genes surrounding the
tgo
ortholog are orthologous to the genes at the same locus in
D. melanogaster
and local synteny is completely conserved, supported by E-values and percent identities, so we conclude that
LOC6574279
is the correct ortholog of
tgo
in
D. mojavensis
(
Figure 1A
).
Protein Model
tgo
in
D. mojavensis
has one CDS within the genome sequence that is present in the two mRNA isoforms that differ in their UTRs (tgo-RA and tgo-RB), which are then translated into one unique protein-coding sequence (tgo-PA/tgo-PB;
Figure 1B
). Relative to the ortholog in
D. melanogaster
, CDS and number of unique protein isoforms within the gene span are conserved. The sequence of
tgo-PA
in
D. mojavensis
has 77.58% identity (E-value: 0.0) with the protein isoform
tgo-PA
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). The protein sequences of tgo-PA in both species contain three glutamine tandem repeats and several insertions or deletions between the alignments, as is shown in the black box in
Figure 1C
. Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession
BK065220
,
BK065221
)
. 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). 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/dmelgenerecord/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.