We propose a gene model for the D. busckii ortholog of the D. melanogaster glycogen synthase ( Glys ) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_017848664.1 (Locus ID LOC108603968 ) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly of D. busckii ( GCA_001277935.1 ). This model is based on RNA-Seq data from D. busckii ( PRJNA274996 - Zhou and Bachtrog 2015) and Glys in D. melanogaster using FlyBase release FB2023_03 ( GCA_000001215.4 ; Larkin et al., 2021; Gramates et al., 2022; Jenkins et al., 2022).

Glycogen synthase ( Glys ; aka. GS, GlyS ) is a gene within the Insulin-signaling pathway in Drosophila and encodes a glycosyltransferase that catalyzes linkage of glucose monomers into glycogen. Glys activity is regulated allosterically by glucose 6-phosphate and phosphorylation/dephosphorylation allowing for control of cellular glycogen levels (Plyte et al., 1992; Roach et al., 2012). Null Glys mutants exhibit growth defects and reduced larval viability in Drosophila (Yamada et al., 2019).

Synteny

The reference gene, Glys, occurs on chromosome 3R in D. melanogaster and is flanked upstream by Guanylyl cyclase at 88E ( Gyc88E ) and Myofilin ( Mf ) and downstream by CG3987 and CG3984 . The tblastn search of D. melanogaster Glys-PA (query) against the D. busckii (GenBank Accession: GCA_001277935.1 ) Genome Assembly (database) placed the putative ortholog of Glys within scaffold CP012526 at locus LOC108603968 ( XP_017848664.1 )— with an E-value of 0.0 and a percent identity of 96.91%. Furthermore, the putative ortholog is flanked upstream by LOC108601478 ( XP_017844866.1 ) and LOC108603374 ( XP_017847625.1 ), which correspond to Gyc88E and Mf in D. melanogaster (E-value: 0.0 and 0.0; identity: 76.27% and 92.03%, respectively, as determined by blastp ; Figure 1A ; Altschul et al., 1990). The putative ortholog of Glys is flanked downstream by LOC108601696 ( XP_017845097.1 ) and LOC108601697 ( XP_017845098.2 ), which correspond to CG3987 and CG3984 in D. melanogaster (E-value: 2e-94 and 5e-58; identity: 45.06% and 35.43%, respectively, as determined by blastp ). The putative ortholog assignment for Glys in D. busckii is supported by the following evidence: The genes surrounding the Glys 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 LOC108603968 is the correct ortholog of Glys in D. busckii ( Figure 1A ).

Protein Model

Glys in D. busckii has two unique protein-coding isoforms Glys-PA, and Glys-PB (identical to Glys-PC, and Glys-PD; Figure 1B ). mRNA isoforms Glys-RB , Glys-RC , and Glys-RD contain four CDSs. mRNA isoform Glys-RA contains five CDSs. Relative to the ortholog in D. melanogaster , the RNA CDS number and protein isoform count are conserved . The sequence of Glys-PA in D. busckii has 94.64% identity (E-value: 0.0) with the protein-coding isoform Glys-PA in D. melanogaster , as determined by blastp ( Figure 1C ). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK065208 , BK065209 , BK065210 , BK065211 ). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).

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.