Description
|
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)
.
“Myc acts downstream of the insulin signaling pathway, with Myc protein accumulating in response to insulin through transcriptional and post-transcriptional mechanisms
(Parisi et al., 2011)
, resulting in the activation of genes involved in anabolic processes that promote cell growth
(Terakawa et al., 2022)
.
Myc
encodes a basic helix-loop-helix transcription factor in
Drosophila melanogaster
that is homologous to vertebrate
Myc
proto-oncogenes
(Gallant et al., 1996)
. In
Drosophila melanogaster
, Myc transcriptionally regulates a wide range of genes, including those that influence cell growth and metabolism
(Teleman et al., 2008; Gallant 2013)
.”
(Myers et al., 2024)
.
“
D. eugracilis
(NCBI:txid29029) is part of the
melanogaste
r species group within the subgenus
Sophophora
of the genus
Drosophila
(Pélandakis et al., 1993)
. It was first described as
Tanygastrella gracilis
by Duda (1924) and revised to
Drosophila eugracilis
by Bock and Wheeler (1972).
D. eugracilis
is found in humid tropical and subtropical forests across southeast Asia (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).”
(Morgan et al., 2022)
.
|
The model presented here is the ortholog of
Myc
in the Apr. 2013 (BCM-HGSC/Deug_2.0) assembly of
D. eugracilis
(
GCA_000236325.2
)
and corresponds to the
Gnomon Peptide ID (
XP_017065790.1
)
predicted model
in
D. eugracilis (
LOC108104317
).
This gene model is based on RNA-seq data from
D. eugracilis
(Chen et al., 2014;
PRJNA63469
) and the
Myc
(Drosophila 12 Genomes Consortium et al
.
, 2007;
GCA_000001215.4
) in
D. melanogaster
from FB2023_03 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022).
Gene and species details can be found in the description above.
Synteny
Myc
occurs on
chromosome X
in
D. melanogaster
and is flanked by
ng4
and
dnc
upstream and
CG12535
and
CG14269
downstream
.
The upstream
dnc
gene in
D. melanogaster
nests
Pig1, Sgs4,
and
CG10793
.
We determined that the putative ortholog of
Myc
is found on the AFPQ02005162.1 scaffold in
D. eugracilis
(GB2 assembly
GCA_000236325.2
) with
LOC108104317
(
XP_017065790.1
) (via
tblastn
search with an e-value of 0.0 and percent identity of 68.18%). It is flanked downstream by
LOC108104312
(
XP_017065785.1
) and
LOC108104315
(
XP_017065789.1
), which correspond to
CG12535
and
CG14269
in D. melanogaster
with e-values of 7e-72 and 6e-110 respectively, and percent identities of 55.83% and 79.47% respectively,
as determined by
blastp
(
Figure 1A, Altschul et al
., 1990).
Myc
is the first gene on the AFPQ02005162.1 scaffold in
D. eugracilis
, so there are no upstream genes to analyze for local synteny. However,
blastp
results indicated that the orthologs of genes upstream of
Myc
in
D. melanogaster
are located on scaffold KB464875.1 in
D. eugracilis
with
LOC108118138
(
XP_017086187.1
)
,
LOC108118129
(
XP_041674349.1
)
,
LOC108118132
(
XP_017086181.1
)
,
and
LOC108118131
(
XP_017086180.1
), which correspond to
ng4, dnc, Pig1,
and
CG10793
with e-values of 9e-22, 0.0, 2e-77, and 0.0, respectively, and percent identities of 82.76%, 95.42%, 58.33%, and 87.81% respectively, as determined by
blastp
(
Figure 1A, Altschul et al
., 1990). Local synteny was conserved within this part of the neighborhood in
D. eugracilis
as well, with the exception of
Sgs4
, for which an ortholog in
D. eugracilis
could not be located.
We believe this is the correct ortholog assignment for
Myc
in
D. eugracilis
because all of the
BLAST
hits had very low e-values and were the best
BLAST
result by a wide margin, and because local synteny is well-conserved throughout the genomic neighborhood.
Protein Model
Myc
in
D. eugracilis
has one unique protein coding isoform encoded by mRNAs Myc-RA and Myc-RB, which differ in their UTRs (
Figure 1B
). Myc-PA/Myc-PB contain two protein coding CDSs. This is the same relative to the ortholog in
D. melanogaster.
The sequence of
Myc
in
D. eugracilis
has 67.0% identity with
Myc
in
D. melanogaster
as determined by
blastp
(
Figure 1C
). This is more divergence between the sequence than one would expect, considering how closely related
D. melanogaster
and
D. eugracilis
are.
The coordinates of the curated gene models can be found in NCBI at GenBank/BankIt using the accessions
BK063012
and
BK063013
. These data are also available in Extended Data files below, which are archived in CaltechData.
Special characteristics of the protein model
Tandem repeat in CDS two
There is a tandem repeat present in CDS two in
Myc
, which is shown in figures C and D in black box a. Specifically, the repeat is made up of nine uninterrupted Serine amino acids present in the protein sequence.
Low sequence similarity in CDS one
There are many regions in CDS one of
Myc
that have very low conservation of their amino acid sequences between the two species. This is pictured in the green boxes (box b) in figures C and D.
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
target gene for a given isoform and run it using
tblastn
against their target
Drosophila
species genome assembly (
D. eugracilis
(
GCA_000236325.2
)
) 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. Genomic structure information (e.g., CDSs, CDS 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 this gene were generated by students under mentorship of their faculty course instructors. These models were then reconciled by a third independent researcher mentored by the project leaders to produce the final model presented here. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.