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
.
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
Pi3K21B
(
Pi3K21B
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_ 001965389.2 (Locus ID
LOC6503336
) in the May 2011 (Agencourt dana_caf1/DanaCAF1; Drosophila 12 Genomes Consortium et al., 2007) Genome Assembly of
D. ananassae
(
GCA_000005115.1
). This model is based on RNA-Seq data from
D. ananassae
(
SRP006203
,
SRP007906
,
PRJNA257286
,
PRJNA388952
; Graveley et al., 2011)
and
Pi3K21B
in
D. melanogaster
using FlyBase release FB2023_03 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022; Jenkins et al., 2022).
The gene product of the
Pi3K21B
gene (FBgn0020622), p60, was identified through affinity purification based on binding to a phosphorylated peptide in a heterodimer with p110, the Pi3K92E gene product (Weinkove et al., 1997). Additional experiments indicated that the p60:p110 complex was present in all life cycle stages in
Drosophila
, and demonstrated protein and lipid phosphatase activity of the complex
in vitro
(Weinkove et al., 1997). Null alleles were generated in
Drosophila
through P-element mobilization, and homozygous null animals display reduced larval growth with death in the third instar or early pupal stage of development (Weinkove et al., 1999). Further work indicated that the p60 adaptor protein was required for PI3K activity in the insulin signaling pathway, and was involved in determining both cellular and organismal size and growth (Britton et al., 2002; Oldham et al., 2002).
Synteny
The reference gene,
Pi3K21B
, occurs on chromosome 2L in
D. melanogaster
and is flanked upstream by
U2 small nuclear riboprotein auxiliary factor 38
(
U2af38
) and
Stress induced phosphoprotein 1
(
Stip1
) and downstream by
Phospholipase C at 21C
(
Plc21C
) and
Equilibrative nucleoside transporter 1
(
Ent1
). There are five genes nested within
Plc21C
:
CG11912
,
CG11911
,
CG33127
,
CG43755
, and
CG31921
. The
tblastn
search of
D. melanogaster
Pi3K21B
-PB (query) against the
D. ananassae
(GenBank Accession:
GCA_000005115.1
) Genome Assembly (database) placed the putative ortholog of
Pi3K21B
within scaffold_12943 at locus
LOC6503336
(
XP_001965389.2
)— with an E-value of 3e-117 and a percent identity of 66.67%. Furthermore, the putative ortholog is flanked upstream by
LOC6507521
(
XP_001965391.1
) and
LOC6503334
(
XP_001965390.1
), which correspond to
U2af38
and
Stip1
in
D. melanogaster
(E-value: 1e-175 and 0.0; identity: 93.89% and 90.00%, respectively, as determined by
blastp
;
Figure 1A
; Altschul et al., 1990). The putative ortholog of
Pi3K21B
is flanked downstream by
LOC6503337
(
XP_032311151.1
) and
LOC6507456
(
XP_001965382.1
), which correspond to
Plc21C
and
Ent1
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 92.53% and 85.03%, respectively, as determined by
blastp
). There are five genes nested within
Plc21C
:
LOC6507510
(
XP_001965388.1
),
LOC6507500
(
XP_001965387.1
),
LOC26514683
(
XP_014760654.1
),
LOC6507489
(
XP_014760615.1
), and
LOC6507467
(
XP_001965384.1
). These correspond to
CG11912
,
CG11911
,
CG33127
,
CG43755
, and
CG31921
in
D. melanogaster
(E-value: 1e-111, 9e-169, 5e-163, 1e-128, 3e-153; percent identity: 58.58%, 77.98%, 81.99%, 57.06%, 42.24%). The putative ortholog assignment for
Pi3K21B
in
D. ananassae
is supported by the following evidence: The genes surrounding the
Pi3K21B
ortholog are orthologous to the genes at the same locus in
D. melanogaster
and local synteny is completely conserved, supported by results generated from
blastp
, so we conclude that
LOC6503336
is the correct ortholog of
Pi3K21B
in
D. ananassae
(
Figure 1A
).
Protein Model
Pi3K21B
in
D. ananassae
has four protein-coding isoforms (
Pi3K21B
-PB,
Pi3K21B
-PC,
Pi3K21B
-PD,
Pi3K21B
-PE;
Figure 1B
).
Pi3K21B
-PB has an identical coding sequence to
Pi3K21B
-PE, and
Pi3K21B
-PC has an identical coding sequence to
Pi3K21B
-PD. In
D. melanogaster
, mRNA isoforms
Pi3K21B
-RB and
Pi3K21B
-RE have two protein-coding CDSs, and
Pi3K21B
-RC and
Pi3K21B
-RD have one protein-coding CDS. Relative to the ortholog in
D. melanogaster
, the protein isoform count is conserved, but mRNA isoforms
Pi3K21B
-RC and
Pi3K21B
-RD have two CDSs in
D. melanogaster
. The sequence of
Pi3K21B
-PB
in
D. ananassae
has 73.41% identity (E-value: 0.0) with the protein-coding isoform
Pi3K21B
-PB
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession
BK064621
). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
Special characteristics of the protein model
In
D. melanogaster
,
Pi3K21B
-RC and
Pi3K21B
-RD have one CDS, but in
D. ananassae
, they have two CDSs (
Figure 1D
). The presence of this intron is supported by the RNA-Seq of Adult Males, Transcripts and Coding Regions Predicted by TransDecoder, and a predicted splice junction with a score of 76. Therefore, we conclude that this CDS split in two as a result of the evolutionary distance between
D. melanogaster
and
D. ananassae
.
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).