Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"\n\n
Introduction
\nThe Insulin Signaling Pathway is a highly conserved signaling pathway in animals, and is central to nutrient uptake (Hietakangas and Cohen, 2009; Grewal 2009). Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).
\nWe 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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
\n\n
Synteny
\nThe target gene, Thor, occurs 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).
\nProtein Model
\nConsistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
\n\n
Special characteristics of the protein model
\nTPGGT Repeat: The dots on either side of the diagonal at the end of exon 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).
\n","references":[{"reference":"Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). "Basic local alignment search tool." J. Mol. Biol. 215:403-410. PMID: 2231712","pubmedId":null,"doi":null},{"reference":"Bernal A, Kimbrell DA. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A. 2000 May 23;97(11):6019-24. doi: 10.1073/pnas.100391597. PMCID: PMC18551; PMID: 10811906","pubmedId":null,"doi":null},{"reference":"Bock, I.R., Wheeler, M.R. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428","pubmedId":null,"doi":null},{"reference":"Doleschall, C.L. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark, A.G., Eisen, M.B., Smith, D.R., Bergman, C.M., Oliver, B., …, MacCallum, I. (2007). Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203--218. PMID: 17994087","pubmedId":null,"doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. Exhaustive matching of the entire protein sequence database. Science. 1992 Jun 5;256(5062):1443-5. doi: 10.1126/science.1604319. PMID: 1604319.","pubmedId":null,"doi":null},{"reference":"Graveley, B. R., Brooks, A. N., Carlson, J. W., Duff, M. O., Landolin, J. M., Yang, L., Artieri, C. G., van Baren, M. J., Boley, N., Booth, B. W., Brown, J. B., Cherbas, L., Davis, C. A., Dobin, A., Li, R., Lin, W., Malone, J. H., Mattiuzzo, N. R., Miller, D., … Celniker, S. E. (2011). The developmental transcriptome of Drosophila melanogaster. Nature, 471(7339), 473–479. https://doi.org/10.1038/nature09715 PMID: 21179090","pubmedId":null,"doi":null},{"reference":"Grewal SS. (2009 )Insulin/TOR signaling in growth and homeostasis: a view from the fly world. FBrf0207364 PMID: 18992839","pubmedId":null,"doi":null},{"reference":"Hietakangas V, Cohen SM. (2009) Regulation of tissue growth through nutrient sensing. PMID: 19694515","pubmedId":null,"doi":null},{"reference":"Kent, W. J., Sugnet, C. W., Furey, T. S., Roskin, K. M., Pringle, T. H., Zahler, A. M., Haussler, D. (2002). The Human Genome Browser at UCSC. Genome Res. 12, 996-1006. PMID: 12045153","pubmedId":null,"doi":null},{"reference":"Kikkawa H. 1938 Studies on the genetics and cytology of Drosophila ananassae. Genetica20, 458–516. https://doi.org/10.1007/BF01531779","pubmedId":null,"doi":null},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, dos Santos G, Garapati PV, Goodman JL, Gramates LS, Millburn G, Strelets VB, Tabone CJ, and Thurmond J and the FlyBase Consortium (2021). FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49(D1) D899–D907. PMID: 33219682","pubmedId":null,"doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. 978-0-12-473052-6","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 2007 Jan 15;21(2):175-83. doi: 10.1101/gad.1506407. PMCID: PMC1770900; PMID: 17234883","pubmedId":null,"doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol. 2001 Jun;3(6):596-601. doi: 10.1038/35078571. PMID: 11389445","pubmedId":null,"doi":null},{"reference":"Navarro Gonzalez, J., Zweig, A. S., Speir, M. L., Schmelter, D., Rosenbloom, K. R., Raney, B. J., et al. (2021). The UCSC Genome Browser database: 2021 update. Nucleic Acids Research. 49(1), 1046-1057. PMID: 33221922","pubmedId":null,"doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik D, Kent WJ. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics. 2014 Apr 1;30(7):1003-5. PMID: 24227676","pubmedId":null,"doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521","pubmedId":null,"doi":null},{"reference":"Singh BN, Yadav JP. Status of research on Drosophila ananassae at global level. J Genet. 2015 Dec;94(4):785-92. doi: 10.1007/s12041-015-0577-y. PMID: 26690536.","pubmedId":null,"doi":null},{"reference":"Singh BN. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol. 2010 Apr;48(4):333-45. PMID: 20726331.","pubmedId":null,"doi":null},{"reference":"Sturtevant AH. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A. 1939 Mar;25(3):137-41. doi: 10.1073/pnas.25.3.137. PMCID: PMC1077728; PMID: 16577879","pubmedId":null,"doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev. 2005 Aug 15;19(16):1840-3. doi: 10.1101/gad.1311805. Epub 2005 Jul 29. PMCID: PMC1186182; PMID: 16055649","pubmedId":null,"doi":null}],"title":"
D. ananassae – Thor
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"b4417a00-31a7-4980-973a-869d60678800","decision":"revise","abstract":"Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":null,"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"\n\n
Introduction
\nThe Insulin Signaling Pathway is a highly conserved signaling pathway in animals, and is central to nutrient uptake (Hietakangas and Cohen, 2009; Grewal 2009). Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).
\nWe 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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
\n\n
Synteny
\nThe target gene, Thor, occurs 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).
\nProtein Model
\nConsistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
\n\n
Special characteristics of the protein model
\nTPGGT Repeat: The dots on either side of the diagonal at the end of exon 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).
\n","references":[{"reference":"Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). "Basic local alignment search tool." J. Mol. Biol. 215:403-410. PMID: 2231712","pubmedId":null,"doi":null},{"reference":"Bernal A, Kimbrell DA. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A. 2000 May 23;97(11):6019-24. doi: 10.1073/pnas.100391597. PMCID: PMC18551; PMID: 10811906","pubmedId":null,"doi":null},{"reference":"Bock, I.R., Wheeler, M.R. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428","pubmedId":null,"doi":null},{"reference":"Doleschall, C.L. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark, A.G., Eisen, M.B., Smith, D.R., Bergman, C.M., Oliver, B., …, MacCallum, I. (2007). Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203--218. PMID: 17994087","pubmedId":null,"doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. Exhaustive matching of the entire protein sequence database. Science. 1992 Jun 5;256(5062):1443-5. doi: 10.1126/science.1604319. PMID: 1604319.","pubmedId":null,"doi":null},{"reference":"Graveley, B. R., Brooks, A. N., Carlson, J. W., Duff, M. O., Landolin, J. M., Yang, L., Artieri, C. G., van Baren, M. J., Boley, N., Booth, B. W., Brown, J. B., Cherbas, L., Davis, C. A., Dobin, A., Li, R., Lin, W., Malone, J. H., Mattiuzzo, N. R., Miller, D., … Celniker, S. E. (2011). The developmental transcriptome of Drosophila melanogaster. Nature, 471(7339), 473–479. https://doi.org/10.1038/nature09715 PMID: 21179090","pubmedId":null,"doi":null},{"reference":"Grewal SS. (2009 )Insulin/TOR signaling in growth and homeostasis: a view from the fly world. FBrf0207364 PMID: 18992839","pubmedId":null,"doi":null},{"reference":"Hietakangas V, Cohen SM. (2009) Regulation of tissue growth through nutrient sensing. PMID: 19694515","pubmedId":null,"doi":null},{"reference":"Kent, W. J., Sugnet, C. W., Furey, T. S., Roskin, K. M., Pringle, T. H., Zahler, A. M., Haussler, D. (2002). The Human Genome Browser at UCSC. Genome Res. 12, 996-1006. PMID: 12045153","pubmedId":null,"doi":null},{"reference":"Kikkawa H. 1938 Studies on the genetics and cytology of Drosophila ananassae. Genetica20, 458–516. https://doi.org/10.1007/BF01531779","pubmedId":null,"doi":null},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, dos Santos G, Garapati PV, Goodman JL, Gramates LS, Millburn G, Strelets VB, Tabone CJ, and Thurmond J and the FlyBase Consortium (2021). FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49(D1) D899–D907. PMID: 33219682","pubmedId":null,"doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. 978-0-12-473052-6","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 2007 Jan 15;21(2):175-83. doi: 10.1101/gad.1506407. PMCID: PMC1770900; PMID: 17234883","pubmedId":null,"doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol. 2001 Jun;3(6):596-601. doi: 10.1038/35078571. PMID: 11389445","pubmedId":null,"doi":null},{"reference":"Navarro Gonzalez, J., Zweig, A. S., Speir, M. L., Schmelter, D., Rosenbloom, K. R., Raney, B. J., et al. (2021). The UCSC Genome Browser database: 2021 update. Nucleic Acids Research. 49(1), 1046-1057. PMID: 33221922","pubmedId":null,"doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik D, Kent WJ. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics. 2014 Apr 1;30(7):1003-5. PMID: 24227676","pubmedId":null,"doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521","pubmedId":null,"doi":null},{"reference":"Singh BN, Yadav JP. Status of research on Drosophila ananassae at global level. J Genet. 2015 Dec;94(4):785-92. doi: 10.1007/s12041-015-0577-y. PMID: 26690536.","pubmedId":null,"doi":null},{"reference":"Singh BN. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol. 2010 Apr;48(4):333-45. PMID: 20726331.","pubmedId":null,"doi":null},{"reference":"Sturtevant AH. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A. 1939 Mar;25(3):137-41. doi: 10.1073/pnas.25.3.137. PMCID: PMC1077728; PMID: 16577879","pubmedId":null,"doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev. 2005 Aug 15;19(16):1840-3. doi: 10.1101/gad.1311805. Epub 2005 Jul 29. PMCID: PMC1186182; PMID: 16055649","pubmedId":null,"doi":null}],"title":"
Drosophila ananassae – Thor
","reviews":[{"reviewer":{"displayName":"Rob Kulathinal"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"9704099e-79b6-4887-aae1-b84b09dff9cd","decision":"edit","abstract":"Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"Introduction
The Insulin Signaling Pathway is a highly conserved signaling pathway in animals, and is central to nutrient uptake (Hietakangas and Cohen, 2009; Grewal 2009). Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). 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 exon 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).
","references":[{"reference":"Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990). "Basic local alignment search tool." J. Mol. Biol. 215:403-410. PMID: 2231712","pubmedId":null,"doi":null},{"reference":"Bernal A, Kimbrell DA. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A. 2000 May 23;97(11):6019-24. doi: 10.1073/pnas.100391597. PMCID: PMC18551; PMID: 10811906","pubmedId":null,"doi":null},{"reference":"Bock, I.R., Wheeler, M.R. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428","pubmedId":null,"doi":null},{"reference":"Doleschall, C.L. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark, A.G., Eisen, M.B., Smith, D.R., Bergman, C.M., Oliver, B., …, MacCallum, I. (2007). Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203--218. PMID: 17994087","pubmedId":null,"doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. Exhaustive matching of the entire protein sequence database. Science. 1992 Jun 5;256(5062):1443-5. doi: 10.1126/science.1604319. PMID: 1604319.","pubmedId":null,"doi":null},{"reference":"Graveley, B. R., Brooks, A. N., Carlson, J. W., Duff, M. O., Landolin, J. M., Yang, L., Artieri, C. G., van Baren, M. J., Boley, N., Booth, B. W., Brown, J. B., Cherbas, L., Davis, C. A., Dobin, A., Li, R., Lin, W., Malone, J. H., Mattiuzzo, N. R., Miller, D., … Celniker, S. E. (2011). The developmental transcriptome of Drosophila melanogaster. Nature, 471(7339), 473–479. https://doi.org/10.1038/nature09715 PMID: 21179090","pubmedId":null,"doi":null},{"reference":"Grewal SS. (2009 )Insulin/TOR signaling in growth and homeostasis: a view from the fly world. FBrf0207364 PMID: 18992839","pubmedId":null,"doi":null},{"reference":"Hietakangas V, Cohen SM. (2009) Regulation of tissue growth through nutrient sensing. PMID: 19694515","pubmedId":null,"doi":null},{"reference":"Kent, W. J., Sugnet, C. W., Furey, T. S., Roskin, K. M., Pringle, T. H., Zahler, A. M., Haussler, D. (2002). The Human Genome Browser at UCSC. Genome Res. 12, 996-1006. PMID: 12045153","pubmedId":null,"doi":null},{"reference":"Kikkawa H. 1938 Studies on the genetics and cytology of Drosophila ananassae. Genetica20, 458–516. https://doi.org/10.1007/BF01531779","pubmedId":null,"doi":null},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, dos Santos G, Garapati PV, Goodman JL, Gramates LS, Millburn G, Strelets VB, Tabone CJ, and Thurmond J and the FlyBase Consortium (2021). FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49(D1) D899–D907. PMID: 33219682","pubmedId":null,"doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. 978-0-12-473052-6","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 2007 Jan 15;21(2):175-83. doi: 10.1101/gad.1506407. PMCID: PMC1770900; PMID: 17234883","pubmedId":null,"doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol. 2001 Jun;3(6):596-601. doi: 10.1038/35078571. PMID: 11389445","pubmedId":null,"doi":null},{"reference":"Navarro Gonzalez, J., Zweig, A. S., Speir, M. L., Schmelter, D., Rosenbloom, K. R., Raney, B. J., et al. (2021). The UCSC Genome Browser database: 2021 update. Nucleic Acids Research. 49(1), 1046-1057. PMID: 33221922","pubmedId":null,"doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik D, Kent WJ. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics. 2014 Apr 1;30(7):1003-5. PMID: 24227676","pubmedId":null,"doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521","pubmedId":null,"doi":null},{"reference":"Singh BN, Yadav JP. Status of research on Drosophila ananassae at global level. J Genet. 2015 Dec;94(4):785-92. doi: 10.1007/s12041-015-0577-y. PMID: 26690536.","pubmedId":null,"doi":null},{"reference":"Singh BN. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol. 2010 Apr;48(4):333-45. PMID: 20726331.","pubmedId":null,"doi":null},{"reference":"Sturtevant AH. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A. 1939 Mar;25(3):137-41. doi: 10.1073/pnas.25.3.137. PMCID: PMC1077728; PMID: 16577879","pubmedId":null,"doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev. 2005 Aug 15;19(16):1840-3. doi: 10.1101/gad.1311805. Epub 2005 Jul 29. PMCID: PMC1186182; PMID: 16055649","pubmedId":null,"doi":null}],"title":"Drosophila ananassae – Thor
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"e186d1e0-1bf2-406d-99f5-54dfe48af41f","decision":"edit","abstract":"Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"Information to provide project, pathway, gene, and species context. This information may be repeated in multiple Drosophila ortholog gene model microPublications submitted by the Genome Education Partnership: “Computational gene predictions in non-model organisms often can be 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). The Genomics Education Partnership (thegep.org) 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). 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. 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.” (Myers et al., 2023) “The IIS pathway 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., 2023). \"Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).\" \"D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (Markow and O’Grady 2006; 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., 2023).\" |
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). 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 exon 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).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403-10.
","pubmedId":"2231712","doi":null},{"reference":"Bernal A, Kimbrell DA. 2000. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A 97(11): 6019-24.
","pubmedId":"10811906","doi":null},{"reference":"Bock, I.R., Wheeler, M.R. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428","pubmedId":null,"doi":null},{"reference":"Doleschall, C.L. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al., MacCallum I. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203-18.
","pubmedId":"17994087","doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. 1992. Exhaustive matching of the entire protein sequence database. Science 256(5062): 1443-5.
","pubmedId":"1604319","doi":null},{"reference":"Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, et al., Celniker SE. 2011. The developmental transcriptome of Drosophila melanogaster. Nature 471(7339): 473-9.
","pubmedId":"21179090","doi":null},{"reference":"Grewal SS. 2009. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41(5): 1006-10.
","pubmedId":"18992839","doi":null},{"reference":"Hietakangas V, Cohen SM. 2009. Regulation of tissue growth through nutrient sensing. Annu Rev Genet 43: 389-410.
","pubmedId":"19694515","doi":null},{"reference":"Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The human genome browser at UCSC. Genome Res 12(6): 996-1006.
","pubmedId":"12045153","doi":null},{"reference":"Kikkawa H. 1938 Studies on the genetics and cytology of Drosophila ananassae. Genetica20, 458–516. https://doi.org/10.1007/BF01531779","pubmedId":null,"doi":null},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, et al., FlyBase Consortium. 2021. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49(D1): D899-D907.
","pubmedId":"33219682","doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. London: Academic Press. ISBN: 978-0-12-473052-6
","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. 2007. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev 21(2): 175-83.
","pubmedId":"17234883","doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3(6): 596-601.
","pubmedId":"11389445","doi":null},{"reference":"Navarro Gonzalez J, Zweig AS, Speir ML, Schmelter D, Rosenbloom KR, Raney BJ, et al., Kent WJ. 2021. The UCSC Genome Browser database: 2021 update. Nucleic Acids Res 49(D1): D1046-D1057.
","pubmedId":"33221922","doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, et al., Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7): 1003-5.
","pubmedId":"24227676","doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521
","pubmedId":null,"doi":"10.1101/2020.12.12.420521"},{"reference":"Singh BN, Yadav JP. 2015. Status of research on Drosophila ananassae at global level. J Genet 94(4): 785-92.
","pubmedId":"26690536","doi":null},{"reference":"Singh BN. 2010. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol 48(4): 333-45.
","pubmedId":"20726331","doi":null},{"reference":"Sturtevant AH. 1939. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A 25(3): 137-41.
","pubmedId":"16577879","doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"ebe4aeb3-5fc6-45d5-b149-9bdd932abba2","decision":"edit","abstract":"Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"Information to provide project, pathway, gene, and species context. This information may be repeated in multiple Drosophila ortholog gene model microPublications submitted by the Genome Education Partnership: “Computational gene predictions in non-model organisms often can be 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). The Genomics Education Partnership (thegep.org) 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). 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. 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.” (Myers et al., 2023) “The IIS pathway 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., 2023). \"Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).\" \"D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (Markow and O’Grady 2006; 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., 2023).\" |
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). 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 exon 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).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403-10.
","pubmedId":"2231712","doi":null},{"reference":"Bernal A, Kimbrell DA. 2000. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A 97(11): 6019-24.
","pubmedId":"10811906","doi":null},{"reference":"Bock, I.R., Wheeler, M.R. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428
","pubmedId":null,"doi":null},{"reference":"Doleschall, C.L. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091
","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al., MacCallum I. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203-18.
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","pubmedId":"18992839","doi":null},{"reference":"Hietakangas V, Cohen SM. 2009. Regulation of tissue growth through nutrient sensing. Annu Rev Genet 43: 389-410.
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","pubmedId":"33219682","doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. London: Academic Press. ISBN: 978-0-12-473052-6
","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. 2007. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev 21(2): 175-83.
","pubmedId":"17234883","doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3(6): 596-601.
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","pubmedId":null,"doi":"10.1101/2020.12.12.420521"},{"reference":"Singh BN, Yadav JP. 2015. Status of research on Drosophila ananassae at global level. J Genet 94(4): 785-92.
","pubmedId":"26690536","doi":null},{"reference":"Singh BN. 2010. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol 48(4): 333-45.
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","pubmedId":"16577879","doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"bc0bba75-1a41-40a4-850b-eccbbd189638","decision":"revise","abstract":"Gene model for the ortholog of Thor (Thor) in the dana_caf1 Genome Assembly (GenBank AccessionGCA_000005115.1) of D. ananassae .
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent exons. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of exon one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first exon of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first exon of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second exon of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second exon of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","methods":"Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2020).
","reagents":"","patternDescription":"Information to provide project, pathway, gene, and species context. This information may be repeated in multiple Drosophila ortholog gene model microPublications submitted by the Genome Education Partnership: “Computational gene predictions in non-model organisms often can be 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). The Genomics Education Partnership (thegep.org) 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). 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. 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.” (Myers et al., 2023) “The IIS pathway 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., 2023). \"Thor (aka d4E-BP in Drosophila) encodes a eukaryotic translation initiation factor 4E binding protein that is controlled by the product of tor (Bernal and Kimbrell, 2000; Marr 2nd MT 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 and connects a translational regulator with innate immunity (Bernal and Kimbrell, 2000).\" \"D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (Markow and O’Grady 2006; 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., 2023).\" |
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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). D. ananassae 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 Doeschall (1858). D. ananassae is circumtropical (https://www.taxodros.uzh.ch; Markow and O’Grady, 2006), 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 & Yadav 2015). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al., 2002; Navarro Gonzalez et al., 2021), which is available at https://gander.wustl.edu.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding exons. Relative to the ortholog in D. melanogaster, the coding-exon 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). The dots on either side of the diagonal at the end of exon 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 BKXXXXXX). 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 exon 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).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403-10.
","pubmedId":"2231712","doi":null},{"reference":"Bernal A, Kimbrell DA. 2000. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A 97(11): 6019-24.
","pubmedId":"10811906","doi":null},{"reference":"Bock, IR, Wheeler, MR. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428
","pubmedId":null,"doi":null},{"reference":"Doleschall, CL. (1858). Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091
","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al., MacCallum I. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203-18.
","pubmedId":"17994087","doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. 1992. Exhaustive matching of the entire protein sequence database. Science 256(5062): 1443-5.
","pubmedId":"1604319","doi":null},{"reference":"Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, et al., Celniker SE. 2011. The developmental transcriptome of Drosophila melanogaster. Nature 471(7339): 473-9.
","pubmedId":"21179090","doi":null},{"reference":"Grewal SS. 2009. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41(5): 1006-10.
","pubmedId":"18992839","doi":null},{"reference":"Hietakangas V, Cohen SM. 2009. Regulation of tissue growth through nutrient sensing. Annu Rev Genet 43: 389-410.
","pubmedId":"19694515","doi":null},{"reference":"Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The human genome browser at UCSC. Genome Res 12(6): 996-1006.
","pubmedId":"12045153","doi":null},{"reference":"Kikkawa H. 1938. Studies on the genetics and cytology of Drosophila ananassae. Genetica 20: 458-516.
","pubmedId":null,"doi":"10.1007/BF01531779"},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, et al., FlyBase Consortium. 2021. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49(D1): D899-D907.
","pubmedId":"33219682","doi":null},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. London: Academic Press. ISBN: 978-0-12-473052-6
","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. 2007. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev 21(2): 175-83.
","pubmedId":"17234883","doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3(6): 596-601.
","pubmedId":"11389445","doi":null},{"reference":"Navarro Gonzalez J, Zweig AS, Speir ML, Schmelter D, Rosenbloom KR, Raney BJ, et al., Kent WJ. 2021. The UCSC Genome Browser database: 2021 update. Nucleic Acids Res 49(D1): D1046-D1057.
","pubmedId":"33221922","doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, et al., Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7): 1003-5.
","pubmedId":"24227676","doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521
","pubmedId":null,"doi":"10.1101/2020.12.12.420521"},{"reference":"Singh BN, Yadav JP. 2015. Status of research on Drosophila ananassae at global level. J Genet 94(4): 785-92.
","pubmedId":"26690536","doi":null},{"reference":"Singh BN. 2010. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol 48(4): 333-45.
","pubmedId":"20726331","doi":null},{"reference":"Sturtevant AH. 1939. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A 25(3): 137-41.
","pubmedId":"16577879","doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"6e7bf5ae-62b3-4d77-96af-1311e0bdaccd","decision":"edit","abstract":"Gene model for the ortholog of Thor (Thor) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession: GCA_000005115.1 ) of Drosophila ananassae. This ortholog 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 using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"cprele@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; CDSS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent CDSs. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of CDS one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first CDS of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first CDS of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second CDS of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second CDS of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"","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. ananassae (GCA_000005115.1- Graveley et al., 2011) 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. melanogaster 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.
","reagents":"","patternDescription":"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., submitted). |
Introduction
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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).
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 tor (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).
Species details can be found in the description above.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding 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). 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).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403-10.
","pubmedId":"2231712","doi":null},{"reference":"Bernal A, Kimbrell DA. 2000. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A 97(11): 6019-24.
","pubmedId":"10811906","doi":null},{"reference":"Bock, IR, Wheeler, MR. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1--102. FBrf0024428
","pubmedId":null,"doi":null},{"reference":"Doleschall, CL. 1858. Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73--128. FBrf0000091
","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al., MacCallum I. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203-18.
","pubmedId":"17994087","doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. 1992. Exhaustive matching of the entire protein sequence database. Science 256(5062): 1443-5.
","pubmedId":"1604319","doi":null},{"reference":"Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, et al., Celniker SE. 2011. The developmental transcriptome of Drosophila melanogaster. Nature 471(7339): 473-9.
","pubmedId":"21179090","doi":null},{"reference":"Grewal SS. 2009. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41(5): 1006-10.
","pubmedId":"18992839","doi":null},{"reference":"Hietakangas V, Cohen SM. 2009. Regulation of tissue growth through nutrient sensing. Annu Rev Genet 43: 389-410.
","pubmedId":"19694515","doi":null},{"reference":"Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The human genome browser at UCSC. Genome Res 12(6): 996-1006.
","pubmedId":"12045153","doi":null},{"reference":"Kikkawa H. 1938. Studies on the genetics and cytology of Drosophila ananassae. Genetica 20: 458-516.
","pubmedId":null,"doi":"10.1007/BF01531779"},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, et al., FlyBase Consortium. 2021. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49(D1): D899-D907.
","pubmedId":"33219682","doi":null},{"reference":"Lawson ME, McAbee M, Lucas RA, Tanner S, Wittke-Thompson J, Pelletier TA, Ozsoy Z, Sterne-Marr R, Rele CP (2024) Gene model for the ortholog of Ilp5 in Drosophila ananassae, microPublication Biology, submitted
","pubmedId":"","doi":""},{"reference":"Markow, T.A. and O’Grady, P. (2005) Drosophila: A guide to species identification and use. London: Academic Press. ISBN: 978-0-12-473052-6
","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. 2007. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev 21(2): 175-83.
","pubmedId":"17234883","doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3(6): 596-601.
","pubmedId":"11389445","doi":null},{"reference":"Navarro Gonzalez J, Zweig AS, Speir ML, Schmelter D, Rosenbloom KR, Raney BJ, et al., Kent WJ. 2021. The UCSC Genome Browser database: 2021 update. Nucleic Acids Res 49(D1): D1046-D1057.
","pubmedId":"33221922","doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, et al., Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7): 1003-5.
","pubmedId":"24227676","doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521
","pubmedId":null,"doi":"10.1101/2020.12.12.420521"},{"reference":"Singh BN, Yadav JP. 2015. Status of research on Drosophila ananassae at global level. J Genet 94(4): 785-92.
","pubmedId":"26690536","doi":null},{"reference":"Singh BN. 2010. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol 48(4): 333-45.
","pubmedId":"20726331","doi":null},{"reference":"Sturtevant AH. 1939. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A 25(3): 137-41.
","pubmedId":"16577879","doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"4baecad0-2f9b-4095-a22b-0611c3e00408","decision":"revise","abstract":"Gene model for the ortholog of Thor (Thor) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession: GCA_000005115.1 ) of Drosophila ananassae. This ortholog 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 using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project.","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"js383@evansville.edu","firstName":"Joyce","lastName":"Stamm","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-8894-1897"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"gep@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[{"description":"GFF, FASTA, PEP","doi":null,"resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D.melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; CDSS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent CDSs. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of CDS one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first CDS of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first CDS of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second CDS of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second CDS of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"Genomic neighborhood and gene model for Thor in Drosophila 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 target gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly (D. ananassae (GCA_000005115.1- Graveley et al., 2011) 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. melanogaster 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.
","reagents":"","patternDescription":"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., submitted). |
Introduction
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- Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). 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).
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 tor (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).
Species details can be found in the description above.
Synteny
The target gene, Thor, occurs 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
Consistent with the blastp search result which shows 93.16% identity between D. melanogaster Thor-PA and the D. ananassae gene 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. Thor in D. ananassae has two identical protein-coding isoforms (Thor-PA; Thor-PB; Figure 1B). Isoforms (Thor-PA, Thor-PB) contain two protein-coding 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). 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).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403-10.
","pubmedId":"2231712","doi":null},{"reference":"Bernal A, Kimbrell DA. 2000. Drosophila Thor participates in host immune defense and connects a translational regulator with innate immunity. Proc Natl Acad Sci U S A 97(11): 6019-24.
","pubmedId":"10811906","doi":null},{"reference":"Bock IR, Wheeler MR. (1972). The Drosophila melanogaster species group. Univ. Texas Publs Stud. Genet. 7(7213): 1-102. FBrf0024428
","pubmedId":null,"doi":null},{"reference":"Doleschall CL. 1858. Derde bijdrage tot de kennis der Dipteren fauna van nederlandsch indie. Natuurk. Tijd. Ned.-Indie 17: 73-128. FBrf0000091
","pubmedId":null,"doi":null},{"reference":"Drosophila 12 Genomes Consortium, Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al., MacCallum I. 2007. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450(7167): 203-18.
","pubmedId":"17994087","doi":null},{"reference":"Gonnet GH, Cohen MA, Benner SA. 1992. Exhaustive matching of the entire protein sequence database. Science 256(5062): 1443-5.
","pubmedId":"1604319","doi":null},{"reference":"Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, et al., Celniker SE. 2011. The developmental transcriptome of Drosophila melanogaster. Nature 471(7339): 473-9.
","pubmedId":"21179090","doi":null},{"reference":"Grewal SS. 2009. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol 41(5): 1006-10.
","pubmedId":"18992839","doi":null},{"reference":"Hietakangas V, Cohen SM. 2009. Regulation of tissue growth through nutrient sensing. Annu Rev Genet 43: 389-410.
","pubmedId":"19694515","doi":null},{"reference":"Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The human genome browser at UCSC. Genome Res 12(6): 996-1006.
","pubmedId":"12045153","doi":null},{"reference":"Kikkawa H. 1938. Studies on the genetics and cytology of Drosophila ananassae. Genetica 20: 458-516.
","pubmedId":null,"doi":"10.1007/BF01531779"},{"reference":"Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, et al., FlyBase Consortium. 2021. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49(D1): D899-D907.
","pubmedId":"33219682","doi":null},{"reference":"Lawson ME, McAbee M, Lucas RA, Tanner S, Wittke-Thompson J, Pelletier TA, et al., Reed LK. 2024. Gene model for the ortholog of Ilp5 in Drosophila ananassae. MicroPubl Biol 2024.
","pubmedId":"39717145","doi":""},{"reference":"Markow TA and O’Grady P. (2005) Drosophila: A guide to species identification and use. London: Academic Press. ISBN: 978-0-12-473052-6
","pubmedId":null,"doi":null},{"reference":"Marr MT 2nd, D'Alessio JA, Puig O, Tjian R. 2007. IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev 21(2): 175-83.
","pubmedId":"17234883","doi":null},{"reference":"Miron M, Verdú J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3(6): 596-601.
","pubmedId":"11389445","doi":null},{"reference":"Navarro Gonzalez J, Zweig AS, Speir ML, Schmelter D, Rosenbloom KR, Raney BJ, et al., Kent WJ. 2021. The UCSC Genome Browser database: 2021 update. Nucleic Acids Res 49(D1): D1046-D1057.
","pubmedId":"33221922","doi":null},{"reference":"Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, et al., Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7): 1003-5.
","pubmedId":"24227676","doi":null},{"reference":"Rele CP, Sandlin KM, Leung W, Reed LK. (2020). Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol. bioRxiv 2020.12.10.420521 doi: 10.1101/2020.12.12.420521
","pubmedId":null,"doi":"10.1101/2020.12.12.420521"},{"reference":"Singh BN, Yadav JP. 2015. Status of research on Drosophila ananassae at global level. J Genet 94(4): 785-92.
","pubmedId":"26690536","doi":null},{"reference":"Singh BN. 2010. Drosophila ananassae: a good model species for genetical, behavioural and evolutionary studies. Indian J Exp Biol 48(4): 333-45.
","pubmedId":"20726331","doi":null},{"reference":"Sturtevant AH. 1939. On the Subdivision of the Genus Drosophila. Proc Natl Acad Sci U S A 25(3): 137-41.
","pubmedId":"16577879","doi":null},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[{"reviewer":{"displayName":"Justin DiAngelo"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":null}]},{"id":"1c534734-979f-465c-a75d-886647fbf3fb","decision":"accept","abstract":"Here is presented a gene model for the ortholog of Thor (Thor) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession: GCA_000005115.1) of Drosophila ananassae. This ortholog 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 using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models.
","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"gep@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["conceptualization","fundingAcquisition","project","methodology","supervision","writing_reviewEditing"],"email":"lreed1@ua.edu","firstName":"Laura K.","lastName":"Reed","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4381-494X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"GFF, FASTA, PEP","doi":"10.22002/7z36m-eek13","resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D. melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; CDSs number is indicated along the top and right, and CDSs are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent CDSs. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of CDS one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first CDS of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first CDS of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second CDS of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second CDS of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"Genomic neighborhood and gene model for Thor in Drosophila 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. melanogaster 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).
","reagents":"","patternDescription":"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). |
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).
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","pubmedId":"31658277","doi":""},{"reference":"Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. 2005. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19(16): 1840-3.
","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
","reviews":[],"curatorReviews":[{"curator":{"displayName":"FlyBase Curators"},"openAcknowledgement":false,"submitted":"1783058806714"}]},{"id":"bf2b2eb6-b007-415d-9358-87811d2098ea","decision":"publish","abstract":"Here is presented a gene model for the ortholog of Thor (Thor) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession: GCA_000005115.1) of Drosophila ananassae. This ortholog 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 using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.
","acknowledgements":"This publication is dedicated to the memory of Joshua Williams. We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models.
","authors":[{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation"],"email":"mgruys@ua.edu","firstName":"Madeline L.","lastName":"Gruys","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3527-2458"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["formalAnalysis","validation","writing_originalDraft","writing_reviewEditing"],"email":"jdasgupta@crimson.ua.edu","firstName":"Jhilam","lastName":"Dasgupta","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0001-8639-7798"},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"joshuawilliams@stfrancis.edu","firstName":"Joshua","lastName":"Williams","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of Evansville, Evansville, IN USA"],"departments":[""],"credit":["formalAnalysis","writing_reviewEditing"],"email":"em238@evansville.edu","firstName":"Emile","lastName":"Moura Coelho da Silva","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":""},{"affiliations":["University of St. Francis, Joliet, IL USA"],"departments":[""],"credit":["supervision","writing_reviewEditing"],"email":"jwittkethompson@stfrancis.edu","firstName":"Jacqueline","lastName":"Wittke-Thompson","submittingAuthor":null,"correspondingAuthor":null,"equalContribution":null,"WBId":null,"orcid":"0000-0002-0828-9596"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["dataCuration","formalAnalysis","methodology","project","software","supervision","validation","visualization","writing_reviewEditing"],"email":"gep@ua.edu","firstName":"Chinmay P.","lastName":"Rele","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":null,"WBId":null,"orcid":"0000-0002-3473-9319"},{"affiliations":["The University of Alabama, Tuscaloosa, AL USA"],"departments":[""],"credit":["conceptualization","fundingAcquisition","project","methodology","supervision","writing_reviewEditing"],"email":"lreed1@ua.edu","firstName":"Laura K.","lastName":"Reed","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4381-494X"}],"awards":[{"awardId":"1915544","funderName":"National Science Foundation (United States)","awardRecipient":"LK Reed"},{"awardId":"R25GM130517","funderName":"National Institutes of Health (United States)","awardRecipient":"LK Reed"}],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"GFF, FASTA, PEP","doi":"10.22002/7z36m-eek13","resourceType":"Model","name":"DanaCAF1_Thor.zip","url":"https://portal.micropublication.org/uploads/2f4991a6e159d6c0f3f1446fd530f693.zip"}],"funding":"This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/d064553b1f895dfc4e85a38ee43c2d56.png"},"imageCaption":"(A) Synteny comparison of the genomic neighborhoods for Thor in Drosophila melanogaster and D. ananassae. Thin underlying arrows indicate the DNA strand within which the target gene–Thor–is located in D. melanogaster (top) and D. ananassae (bottom). Thin arrows pointing to the right indicate that Thor is on the positive (+) strand in both D. ananassae and D. melanogaster. The wide gene arrows pointing in the same direction as Thor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Thor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. ananassae indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al. 2014). The coding-regions of Thor in D. ananassae are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. ananassae), and Splice Junctions Predicted by regtools using D. ananassae RNA-Seq (Graveley et al., 2011; SRP006203, SRP007906; PRJNA257286, PRJNA388952). The splice junction shown in red (JUNC00011951) has a read-depth of 4677. (C) Dot Plot of Thor-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; CDSs number is indicated along the top and right, and CDSs are also highlighted with alternating colors. The black box X highlights a repeating amino acid sequence in that region. (D) Protein Alignment Thor-PA in D. melanogaster and the orthologous peptide in D. ananassae. The alternating colored rectangles represent adjacent CDSs. The symbols in the match line denote the level of similarity between the aligned residues. An asterisk (*) indicates that the aligned residues are identical. A colon (:) indicates the aligned residues have highly similar chemical properties—roughly equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix (Gonnet et al., 1992). A period (.) indicates that the aligned residues have weakly similar chemically properties—roughly equivalent to scoring > 0 and ≤ 0.5 in the Gonnet PAM 250 matrix. A space indicates a gap or mismatch when the aligned residues have a complete lack of similarity—roughly equivalent to scoring ≤ 0 in the Gonnet PAM 250 matrix. The amino acid sequence shows there is a small repeat at the end of CDS one (TPGGT) as shown in the 2 boxes, corresponding to Box X in the dot plot. (E) End of first CDS of Thor in D. melanogaster displaying conservation of TPGGT repeats. Evidence tracks include base positions shown in black at the top of the browser, amino acid sequence (grey blocks), FlyBase Protein-Coding Genes (blue), ROAST Alignment and Conservation (36 RefSeq Drosophila Genomes) (burgundy), Basewise Conservation of 36 Drosophila Genomes generated by phastCons and phyloP shown in green and dark blue, respectively; ROAST Alignments of 36 Drosophila species with species listed on the left in dark blue and amino acid sequence in pale blue. Boxes in black denoted X1 and X2i highlight the conservation of the amino acid repeat (TPGGT) at the end of the first CDS of Thor across 36 Drosophila species. The yellow highlighted box labeled Y on the left encloses the sequence in D. ananassae, with X2i encompassing only a portion of the TPGGT repeat, (TPGG). (F) Beginning of second CDS of Thor in D. melanogaster displaying conservation of amino acid sequence. Evidence tracks are identical to those in Figure 1E. The box in black denoted X2ii highlights the conservation of the amino acid repeat (TPGGT) at the beginning of the second CDS of Thor in D. melanogaster, featuring the remaining amino acids of the repeat (T). The continuation of the yellow highlighted box Y encloses the sequence in D. ananassae.
","imageTitle":"Genomic neighborhood and gene model for Thor in Drosophila 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. melanogaster 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).
","reagents":"","patternDescription":"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). |
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).
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","pubmedId":"16055649","doi":null}],"title":"Gene model for the ortholog of Thor in Drosophila ananassae
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