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 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
.
ananassae
(NCBI:txid7217) is part of the
melanogaster
species group within the subgenus
Sophophora
of the genus
Drosophila
(Sturtevant 1939; Bock and Wheeler 1972). It was first described by Doleschall (1858).
D. ananassae
is circumtropical (Markow and O'Grady 2005; https://www.taxodros.uzh.ch, accessed 1 Feb 2023), and often associated with human settlement (Singh 2010). It has been extensively studied as a model for its cytogenetic and genetic characteristics, and in experimental evolution (Kikkawa 1938; Singh and Yadav 2015).” (Lawson et al., 2024).
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We propose a gene model for the
D. ananassae
ortholog of the
D. melanogaster
Thor
(
Thor
)
gene. The genomic region of the ortholog corresponds to the uncharacterized protein
LOC6498052
(RefSeq accession
XP_001961972.1
) in the dana_caf1 Genome Assembly of
D. ananassae
(GenBank Accession:
GCA_000005115.1
, Drosophila 12 Genomes Consortium et al., 2007). This model is based on RNA-Seq data from
D. ananassae
(Graveley et al., 2011;
SRP006203
,
SRP007906
;
PRJNA257286
,
PRJNA388952
)
and
Thor
in
D. melanogaster
using FlyBase release FB2022_04, (
GCA_000001215.4
; Larkin et al., 2021; Gramates et al., 2022; Jenkins et al., 2022).
Thor
(
Thor
; also known as
4E-BP
), a core component of the insulin signaling pathway, encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of
mTor
(Bernal and Kimbrell 2000; Marr II et al., 2007). The
Drosophila
forkhead transcription factor (
dFOXO
) activates
Thor
transcription and contributes to translation regulation, response to environmental stress, and cell growth regulation (Tettweiler et al., 2005; Miron et al., 2001).
Thor
is an effector of
PI(3)K
/
Akt
signaling and cell growth in
Drosophila
(Miron et al., 2001) and participates in host immune defense by connecting a translational regulator with innate immunity (Bernal and Kimbrell 2000).
Synteny
The reference gene,
Thor
,
is found on
chromosome 2L in
D. melanogaster
and is flanked upstream by
CG31776
and Polypeptide N-Acetylgalactosaminyltransferase 4
(Pgant4)
and downstream by
CG15414
and timeless
(tim)
. The
tblastn
search of
D. melanogaster
Thor-PA (query) against the
D. ananassae
(GenBank Accession:
GCA_000005115.1
) Genome Assembly (database) placed the putative ortholog of
Thor
within scaffold_12916 (
CH902620.1
) at locus
LOC6498052
(
XP_001961972.1
)— with an E-value of 1e-78 and a percent identity of 93.16%. Furthermore, the putative ortholog is flanked upstream by
LOC6498050
(
XP_001961970.1
) and
LOC6498051
(XP_
001961971.1
), which correspond to
CG31776
and
Pgant4
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 47.45% and 74.40%, respectively, as determined by
blastp
;
Figure 1A,
Altschul et al., 1990). The putative ortholog of
Thor
is flanked downstream by
LOC6497492
(
XP_044572519.1
) and
LOC6497491
(
XP_032307461.1
), which correspond to
CG15414
and
tim
in
D. melanogaster
(E-value: 2e-108 and 0.0; identity: 83.33% and 85.86%, respectively, as determined by
blastp
). The putative ortholog assignment for
Thor
in
D. ananassae
is supported by the following evidence: The genes surrounding the
Thor
ortholog are orthologous to the genes at the same locus in
D. melanogaster
and local synteny is completely conserved, verified by e-values and percent identities, so we conclude that
LOC6498052
is the correct ortholog of
Thor
in
D. ananassae
(
Figure 1A
).
Protein Model
Thor
in
D. ananassae
has two mRNA transcripts (Thor-RA, Thor-RB), which encode identical protein-coding isoforms (Thor-PA; Thor-PB;
Figure 1B
). These isoforms (Thor-PA, Thor-PB) contain two CDSs. Relative to the ortholog in
D. melanogaster
, the CDS number is conserved. The sequence of
Thor-PA
in
D. ananassae
has 93.16% identity (E-value: 1e-78) with the
protein-coding isoform
Thor-PA
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). Consistent with the
blastp
search result which shows 93.16% identity between
D. melanogaster
Thor-PA and the
D. ananassae
protein model as well as the low sensitivity parameters used to generate the dot plot (i.e., word size = 3; neighborhood threshold = 11), the dot plot of the two protein sequences contain multiple small gaps along the diagonal (
Figure 1C
). The dots on either side of the diagonal at the end of CDS one in the dot pot (Box X,
Figure 1C
) indicate the presence of two identical copies of the sequence TPGGT, as shown in the two boxes labeled B in the protein alignment (Box X,
Figure 1D
).
Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession
BK064668
,
BK064669
). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
Special characteristics of the protein model
TPGGT Repeat:
The dots on either side of the diagonal at the end of CDS one in the dot pot (
Figure 1C
) indicates the presence of two identical copies of the sequence TPGGT, as shown in the two boxes labeled X1 and X2 in the protein alignment (
Figure 1D
). This small repeat is conserved in many
Drosophila
species as shown by the black boxes denoted X1, X2i and X2ii, with
D. ananassae
highlighted in the yellow box, Y (
Figure 1E
and
Figure 1F
).
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/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).