Genomic and expression analysis of transition proteins in Drosophila

Figures & data

Table 1. All NCBI open reading frame (ORF) finder sequence matches for Tpl^94D in the original 12 sequenced Drosophila species

			GI Number
Drosophila Species	Match Number	Gene Locus	Whole Nucleotide and Transcript Sequences	Protein Sequence
D. melanogaster^‡^#	Control (Tpl^94D)	CG31281	24649165	24649166
D. simulans^*^‡^#	1–2	GD20990	195573025	195573026
D. simulans^†	2–2	GD21472	195574870	195574871
D. sechellia^*^#	1–2	GM26474	195331176	195331177
D. sechellia^†	2–2	GM12829	195341320	195341321
D. yakuba^*^‡^#	1–2	GE10340	195502744	195502745
D. yakuba^†	2–2	GE23890	195503141	195503142
D. erecta^*^#	1–2	GG11172	194910675	194910676
D. erecta^†	2–2	GG24235	194857282	194857283
D. ananassae^*^‡^#	1–3	GF19889	194743971	194743972
D. ananassae^†	2–3	GF20096	194746963	194746964
D. ananassae^†	3–3	GF15002	194758514	194758515
D. pseudoobscura^*^‡^#	1–1	GA22645	198471329	198471330
D. persmilis^†	1–1	GL26871	195168587	195168588
D. willistoni^†	1–2	GK14607	195435142	195435143
D. willistoni^†	2–2	GK12423	195461023	195461024
D. mojavensis^♦	‐	‐	‐	‐
D. virilis^†	1–1	GJ16066	195385648	195385649
D. grimshawi^♦	‐	‐	‐	‐

* Denotes better than threshold

^† Denotes worse than threshold

^‡ Denotes verified through RNA-Seq analysis

^♦ Denotes no matches found for D. mojavensis and D. grimshawi

^# Denotes identified orthologs for Tpl^94D

The cut off threshold was query coverage of 40% with maximum identity score of 36 and an E-value of 7 × 10⁻⁵

Table 2. Best NCBI nucleotide BLAST sequence matches and orthologs for Tpl^94D (GI: 24649165)

			Genomic DNA Sequence			Transcript Sequence
Species Name	Gene Locus	GI Number	Maximum Identity (%)	Query Coverage (%)	E- Value Score	Maximum Identity (%)	Query Coverage (%)	E- Value Score
D. simulans^Footnote***Footnote#	GD20990	195573025	86	36	0	86	33	1e-170
D. sechellia^Footnote***Footnote#	GM26474	195331176	86	36	0	86	33	7e-168
D. yakuba^Footnote***Footnote#	GE10340	195502744	69	35	1e-82	69	32	1e-69
D. erecta^Footnote***Footnote#	GG11172	194910675	67	34	2e-61	71	31	9e-78
D. simulans^†	GD21472	195574870	100	0	0.11	—	—	—
D. sechellia^†	GM12829	195341320	100	0	0.11	—	—	—
D. yakuba^†^♦	GE23890	195503141	—	—	—	—	—	—
D. erecta^†^♦	GG24235	194857282	—	—	—	—	—	—
D. ananassae^*^#	GF22417	194766791	86	2	4e-07	86	2	3e-07
D. ananassae^†	GF19889	194743971	100	0	0.38	100	0	0.32
D. ananassae^†^♦	GF20096	194746963	—	—	—	—	—	—
D. ananassae^†	GF15002	194758514	100	7	0.031	100	0	0.32
D. pseudoobscura^*^#	GA22363	198467493	93	4	3e-08	93	5	3e-08
D. pseudoobscura^†^♦	GA22645	198471329	—	—	—	—	—	—
D. persmilis^*	GL18087	195175349	93	1	1e-07	93	1	1e-07
D. persmilis^†^♦	GL26871	195168587	—	—	—	—	—	—
D. willistoni^*	GK19855	195432301	85	6	1e-06	85	3	1e-06
D. willistoni^†^♦	GK14607	195435142	—	—	—	—	—	—
D. willistoni^†^♦	GK12423	195461023	—	—	—	—	—	—
D. mojavensis^*	GI13566	195128228	92	3	2e-05	92	2	1e-05
D. virilis^*	GJ22187	195383563	100	5	1e-06	85	3	1e-06
D. virilis^†	GJ16066	195385648	100	0	0.38	—	—	—
D. grimshawi^*	GH21505	195027639	—	—	—	83	2	1e-05

* *Denotes best matches for Drosophila species within the Drosophila melanogaster species subgroup and greater than threshold for NCBI Open Reading Frame Finder

* Denotes best match for nucleotide sequence of Tpl^94D for Drosophila species outside the Drosophila melanogaster species subgroup

^† Denotes NCBI Open Reading Frame Finder Match and match not found based on transcript sequence of Tpl^94D

^# Denotes identified orthologs for Tpl^94D

^♦ No match based on Tpl^94D's genomic and transcript sequences

Figure 1. T-Coffee alignment of Tpl^94D for melanogaster species subgroup, D. ananassae, and D. pseudoobscura. T-Coffee conserved region alignment for Tpl^94D. Key on the bottom right shows 87 consensus score for all sequence matches.

Figure 2. CLUSTAL Omega Alignment of Tpl^94D sequence matches CLUSTAL Omega alignment of the best Tpl^94D sequence matches in the sequenced 12 Drosophila species.

Table 3. NCBI protein BLAST Tpl^94D (GI: 24649166) orthologs

Species Name	Gene Locus	GI Number	Maximum Identity (%)	Query Coverage (%)	E – Value Score
D. simulans	GD20990	195573026	78	100	1e-91
D. sechellia	GM26474	195331177	78	100	7e-92
D. yakuba	GE10340	195502745	52	100	7e-56
D. erecta	GG11172	194910676	54	100	1e-65
D. ananassae^*	GF19889	194743972	46	40	3e-18
D. pseudoobscura^*	GA22645	198471330	36	65	5e-13

* Matches cannot be retrieved through traditional BLAST means due to best genomic sequences not matching their respective best protein matches.

Table 4. Amino acid analysis for Tpl^94D orthologs

Species	Gene	Cysteine	Lysine	Arginine	Serine
D. mel	Tpl^94D (164)	0.61 (1)	7.32 (12)	11.59 (19)	13.41 (22)
D. sim	GD20990 (167)	1.8 (3)	7.78 (13)	10.18 (17)	10.78 (18)
D. sec	GM26474 (167)	1.8 (3)	7.78 (13)	10.78 (18)	10.78 (18)
D. yak	GE10340 (187)	0 (0)	10.16 (19)	10.16 (19)	7.49 (14)
D. ere	GG11172 (175)	0 (0)	8.57 (15)	12 (21)	8.57 (15)
D. ana^Footnote*	GF19899 (79)	2.53 (2)	7.59 (6)	11.39 (9)	7.59 (6)
D. pse^Footnote*	GA22645 (101)	1.98 (2)	11.88 (12)	8.91 (9)	9.90 (10)

Percentage (Total number present).

^* =not confirmed through normal NCBI BLAST / ORF – RNA-Seq only.

Figure 3. CLUSTAL Omega Alignment of Tpl^94D with mammalian TP2 CLUSTAL Omega alignment of the orthologs for Tpl^94D and transition protein 2 from M. musculus, R. norvegicus, B. taurus, and H. sapiens.

Figure 4. Phyre2 Tpl^94D best sequence matches conserved DNA binding region Tertiary structure alignment of a wire frame model for the Tpl^94D orthologs. The different colors indicate each of the species indicated on the bottom right.

Table 5. Detailed analysis of conserved functional groups found in Tpl^94D orthologs

Sample Matches for D. mel Tpl^94D		D. mel Tpl^94D	D. sim GD20990 Tpl^94D	D. sec GM26474 Tpl^94D	D. yak GE10340 Tpl^94D	D. ere GG11172 Tpl^94D	D. ana GF19899 Tpl^94D	D. pse GA22645 Tpl^94D
Model Confidence (Conserved Region) >90%		94	94	94	100	100	91	89
c2e6oA	% Confidence	97.7	97.7	97.7	98.3	98.9	91.2	97.9
	% Identity	15	13	13	15	16	13	13
Info: a b c d	% Coverage	71	70	70	81	89	82	92
	Residues	15–60	15–60	15–60	9–61	7–64	6–59	2–62
c2cs1A	% Confidence	97.9	97.8	97.9	98.4	98.9	92	98
	% Identity	25	27	22	19	17	21	13
Info: e f g	% Coverage	90	68	68	87	90	89	83
	Residues	2–59	15–59	15–59	8–64	6–64	2–59	8–62
d1v64a	% Confidence	98	98	98	98.3	98.8	92.7	98.1
	% Identity	20	18	18	12	8	14	12
Info: h	% Coverage	69	68	68	76	92	65	86
	Residues	15–59	15–59	15–59	11–60	5–64	17–59	6–62
d1hmfa	% Confidence	97.9	97.6	97.6	98.4	98.9	89.4	97.9
	% Identity	19	18	17	14	14	19	20
Info: h	% Coverage	85	68	70	85	87	82	87
	Residues	5–59	15–59	15–60	5–60	5–61	6–59	5–62
c2yrqA	% Confidence	96.1	96.2	96.3	97.4	98.3	74.1	96.8
	% Identity	20	18	18	16	17	19	18
Info: e i j	% Coverage	76	75	76	76	96	81	86
	Residues	11–59	11–59	11–60	11–60	2–64	7–59	6–62

^a transcription

^b cell cycle

^c hmg box-containing protein 1

^d solution structure of the hmg box domain from human hmg-box2 transcription factor

^e dna binding protein

^f pms1 protein homolog 1

^g solution structure of the hmg domain of human dna mismatch2 repair protein

^h HMG – box

ⁱ high mobility group protein b1

^j solution structure of the tandem hmg box domain from human2 high mobility group protein b1

Table 6. Detailed analysis of functional groups found in Tpl^94D whole protein sequence matches

Sample Matches for D. mel Tpl^94D		D. mel Tpl^94D	D. sim GD20990 Tpl^94D	D. sec GM26474 Tpl^94D	D. yak GE10340 Tpl^94D	D. ere GG11172 Tpl^94D	D. ana GF19899 Tpl^94D	D. pse GA22645 Tpl^94D
Model Confidence >90%		98	98	98	82	87	81	56
c2e6oA	% Confidence	98.7	99.1	99.2	98.9	97.2	90.9	97.5
	% Identity	18	10	10	15	15	12	12
Info: a b c d	% Coverage	40	42	41	27	34	81	55
	Residues	2–69	4–75	4–74	7–59	111–172	6–70	6–62
c2cs1A	% Confidence	99	99.3	99.4	98.9	97.4	91.2	97.6
	% Identity	19	18	20	20	19	21	13
Info: e f g	% Coverage	51	50	50	31	29	72	43
	Residues	2–86	2–87	2–87	6–64	6–57	2–59	18–62
d1v64a	% Confidence	98.9	99.3	99.4	98.8	97.4	92.3	97.8
	% Identity	17	16	14	10	10	16	13
Info: h	% Coverage	39	53	49	47	33	60	54
	Residues	9–73	4–93	9–91	11–100	114–172	17–65	7–62
d1hmfa	% Confidence	98.8	99.2	99.3	98.9	97	88.3	97.3
	% Identity	19	14	17	15	17	19	20
Info: h	% Coverage	41	42	40	28	31	67	56
	Residues	8–76	5–76	8–76	5–59	1–56	6–59	5–62
c2yrqA	% Confidence	99.6	99.8	99.8	99.5	98.7	70.4	95.6
	% Identity	16	12	13	26	19	20	18
Info: e i j	% Coverage	96	97	97	97	97	60	55
	Residues	2–161	2–164	2–164	2–184	2–172	11–59	6–62

^a transcription

^b cell cycle

^c hmg box-containing protein 1

^d solution structure of the hmg box domain from human hmg-box2 transcription factor

^e dna binding protein

^f pms1 protein homolog 1

^g solution structure of the hmg domain of human dna mismatch2 repair protein

^h HMG – box

ⁱ high mobility group protein b1

^j solution structure of the tandem hmg box domain from human2 high mobility group protein b1

Table 7. Ovaries vs. testes transcriptome Cuffdiff 2.0.2 RNA-Seq analysis summary

Species - Gene ID	Ovaries (FPKM)^*	Testes (FPKM)^*	Log2 (Ovaries/ Testes)^*	P value^*	Q Value^*
D. mel – TPL94D – Iso A	0.0087	123.524	13.8006	0.0623	0.2226
D. mel – TPL94D – Iso B	0	19.0749	1.7977e+308	0.1929	0.4164
D. sim - GD20990	0	266.525	1.7977e+308	0.0001	0.0011
D. yak - GE10340	0.0120	506.227	15.3673	0.0070	0.0105
D. ana - GF19889	0.0116	78.6323	12.7224	0	0
D. pse - GA22645	0.0548	232.614	12.0528	0.0001	0.0016

* Values were rounded to the 10-thousandths decimal point as compared to File S5.

Figure 5. Phyre2 Tpl^94D orthologs, MST35Ba, MST35Bb orthologs, Dpse GA18970 Exon 1 (GA31252) best sequence matches conserved DNA binding region Tertiary structure alignment of a wire frame model for the Tpl^94D orthologs. The different colors indicate each of the species shown on the bottom right.

Figure 6. T-Coffee Alignment of Tpl^94D orthologs with MST35Ba and MS35Bb orthologs. A T-Coffee alignment of the whole protein Tpl^94D conserved region and the whole proteins of the Drosophila protamine-like proteins found in the 12 sequenced Drosophila species shows high conservation of the conserved regions with a T-Coffee consensus score of 72. D. pseudoobscura GA18970s first exon (GA31252) was used due to size length of the whole protein being 569 amino acids.

Figure 7. T-Coffee Alignment of Tpl^94D and Drosophila protamine-like protein (MST35Ba and MST35Bb) conserved regions. T-Coffee alignment of the DNA binding-HMG box conserved regions in Drosophila Tpl^94D and Drosophila protamine-like proteins (MS35Ba and MST35Bb) orthologs. The area in red indicates strong conservation. Consensus score equals 93. Also D. pseudoobscura GA18970s first exon (GA31252) contains the conserved region for the D. pseudoobscura MST35Ba/Bb ortholog.