Supplemental Table 1. Amino acid sequences of synthetic kisspeptins
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1 Supplemental Data Supplemental Table 1. Amino acid sequences of synthetic kisspeptins Kisspeptins Symbol Sequence Human kisspeptin-10 H-10 Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH 2 Rodent/Xenopus 1a kisspeptin-10 R/X1a-10 Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH 2 Zebrafish 1 kisspeptin-10 Z1-10 Tyr-Asn-Leu-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH 2 Zebrafish 1 kisspeptin-15 Z1-15 Gln-Asn-Val-Ala-Tyr-Tyr-Asn-Leu-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH 2 Zebrafish 1 pyroglu-kisspeptin-15 Z1-p15 pglu-asn-val-ala-tyr-tyr-asn-leu-asn-ser-phe-gly-leu-arg-tyr-nh 2 Zebrafish 2 kisspeptin-10 Z2-10 Phe-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH 2 Zebrafish 2 kisspeptin-12 Z2-12 Ser-Lys-Phe-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH 2 Xenopus 1a kisspeptin-14 X1a-14 Asp-Leu-Ser-Thr-Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH 2 Xenopus 1b pyroglu-kisspeptin-15 X1b-p15 pglu-ala-gln-val-gly-tyr-asn-val-asn-ser-phe-gly-leu-arg-phe-nh 2 Xenopus 2 kisspeptin-10 X2-10 Phe-Asn-Phe-Asn-Pro-Phe-Gly-Leu-Arg-Phe-NH 2 Xenopus 2 kisspeptin-12 X2-12 Ser-Lys-Phe-Asn-Phe-Asn-Pro-Phe-Gly-Leu-Arg-Phe-NH 2 Xenopus 2 kisspeptin-12y X2-12Y Ser-Lys-Phe-Asn-Phe-Asn-Pro-Phe-Gly-Leu-Arg-Tyr-NH 2
2 Supplemental Table 2. Oligonucleotide sequences of primers Applications Oligonucleotide sequence Full-length cdna cloning F: 5 ATATAACATCTACAATTCAGCAATATG 3 Zebrafish KiSS-2 R: 5 ATAACCGGCAAAGTGTGCCT 3 F: 5 TGTCCAGTTTGTGTCTGTTC 3 X. tropicalis KiSS-1a R: 5 GCTTGGAACTACCAGACCTT 3 F: 5 CTGGTAAGTCAGGGAACCAT 3 X. tropicalis KiSS-1b R: 5 TAGCTGGGAGCTATTCCACG 3 F: 5 GCTGCTCCTCCTCCTTCT3 X. tropicalis KiSS-2 R: 5 GTTTTGGGGCTCAGCAGGAT 3 F1: 5 TTATGCCGCAGAAAGAAATC 3 X. laevis KiSS-1 F2: 5 CCTAGCCGATCTATTTCTGC 3 R1: 5 CACCTTGGAATTTTCTGACC 3 R2: 5 TTCCAGTTGTAGGTAGAGAG 3 R3: 5 ATCTTTTTCTCTTTCCACCAA 3 F1: 5 TCCTCACGCTGCTCATTTAC 3 F2: 5 GTGTATTGTGCTGCTTTTGG 3 X. laevis KiSS-2 R1: 5 CCAAAAGCAGCACAATACAC 3 R2: 5 GGTCTCCTTCTGGGTAACTT 3 R3: 5 GTAAATGAGCAGCGTGAGGA 3 F1: 5 YTIWSITGYCAYYTIMGITTYAC 3 F2: 5 AARTTYAAYTTYAAYCCITTYGG 3 R1: 5 TTTGATAACCTCAGCCAGGG 3 glass lizard KiSS-2 R2: 5 GGATTATCCTTAGGGCCAGA 3 R3: 5 GAGCTTAACAACCTTCTCCC 3 F1: 5 AGGTGTGCTGTGGATCCT 3 F2: 5 GCAAGTTTAACTACAACCCG 3 medaka KiSS-2 R1: 5 GACATCAGAGGGAGCTTCAT 3 R2: 5 CACTGTACAAAAAGGCTGAC 3 R3: 5 TTCATGGCGCGCGCTCGGTG 3 F: 5 GCAGGATCCAATATGCTGAACTTGTCCTGGGGGACA AT 3 X. tropicalis GPR54-1a R: 5 CGCCTCGAGTTACTTAATCTCATTATTGGTTGACTCC 3 F: 5 AGCGGATCCGCCATGGATAACGAGTCATTGTTTTCC 3 X. tropicalis GPR54-1b R: 5 CGTCTCGAGTTA AGTGCCTGAGGACACAAA ATG C 3
3 X. tropicalis GPR54-2 F: 5 GCCGGATCCAGCATGGATAACTTGTCCCTGTTTTC 3 R: 5 GGCCTCGAGCTATGTTCCAGA AGAAACAAAGTGC 3 In situ hybridization X. laevis KiSS-1 X. laevis KiSS-2 Tissue distribution X. tropicalis KiSS-1a X. tropicalis KiSS-1b X. tropicalis KiSS-2 X. tropicalis GPR54-1a X. tropicalis GPR54-1b X. tropicalis GPR54-2 GAPDH F: 5 GCATAAGGAACGGAGTAGTG 3 R: 5 CACCTTGGAATTTTCTGACC 3 F: 5 TCCTCACGCTGCTCATTTAC 3 R: 5 CCAAAAGCAGCACAATACAC 3 F: 5 TGTCCAGTTTGTGTCTGTTC 3 R: 5 GCTTGGAACTACCAGACCTT 3 F: 5 CTGGTAAGTCAGGGAACCAT 3 R: 5 TAGCTGGGAGCTATTCCACG 3 F: 5 GCTGCTCCTCCTCCTTCT 3 R: 5 GTTTTGGGGCTCAGCAGGAT 3 F: 5 GCCATGGTATACCAGATGGG 3 R: 5 TGCCTGAGGACACAAAATGC 3 F: 5 TGGATACTGGTATGGACCTC 3 R: 5 CACGGTGACTATGTTCCAGA 3 F: 5 ATGCTCAAAAGGGTTGGACGG 3 R: 5 TTGGTTGACTCCATGGCTAC 3 F: 5 TGACCCCTTCATTGACTTGG 3 R: 5 CATGGACGGTAGTCATCAGT 3
4 Supplemental Table 3. Percent amino acid sequence identities among GPR54s Zebrafish-1 Zebrafish-2 Medaka-1 Medaka-2 XT-1a XT-1b XT-2 Bullfrog Platypus-1 Platypus-2 Human Zebrafish Zebrafish Medaka Medaka XT-1a XT-1b XT Bullfrog Platypus Sequences were retrieved as described in the supplemental Table 5. XT, X. tropicalis.
5 Supplemental Table 4. The log EC 50 values of kisspeptins for each GPR54 Human GPR54 zgpr54-1 zgpr54-2 xtgpr54-1a xtgpr54-1b xtgpr54-2 H ± ± ± ±0.13 Z ± ± ±0.11 Z ± ±0.19 Z1-p ± ± ±0.32 Z ± ± ±0.14 Z ± ± ±0.12 R/X1a ± ± ± ± ± ±0.09 X1a ± ± ±0.12 X1b-p ± ± ±0.06 X ± ±0.26 > -5 > ±0.12 X ± ± ± ± ±0.09 X2-12Y -5.30± ±0.27 > ± ±0.10 The kisspeptins sequences are shown in supplemental Table 1. Experimental procedures are described in the legend to Figure 6. z, zebrafish; xt, X. tropicalis.
6 Supplemental Table 5. Accession numbers for kisspeptin and GPR54 genes Name Accession number or chromosome location Source human KiSS-1 NM_ GenBank mouse KiSS-1 NM_ GenBank zebrafish KiSS-1 EF GenBank medaka KiSS-1# BAAF , GenBank lamprey KiSS-1 EB and EB GenBank platypus KiSS-1# AAPN , GenBank elephant shark KiSS-1# AAVX , GenBank X. laevis KiSS-1 AB GenBank X. tropicalis KiSS-1a EU GenBank X. tropicalis KiSS-1b EU GenBank grass lizard KiSS-2 AB GenBank X. laevis KiSS-2 AB GenBank X. tropicalis KiSS-2 EU GenBank zebrafish KiSS-2 EU GenBank medaka KiSS-2 AB GenBank platypus KiSS-2# AAPN , GenBank elephant shark KiSS-2# AAVX , GenBank lamprey KiSS-2# Contig3941, Ensembl X. Tropicalis GPR54-1a EU GenBank X. Tropicalis GPR54-1b EU GenBank X. Tropicalis GPR54-2 EU GenBank zebrafish GPR54-1 NM_ GenBank human GPR54 AY GenBank zebrafish GPR54-2 NM_ GenBank bullfrog GPR54-2 EU GenBank medaka GPR54-1# Chromosome 9: : Ensembl medaka GPR54-2# Chromosome 17: : Ensembl platypus GPR54-1# Ultra_266:390150: Ensembl platypus GPR54-1b/2# Contig_15629:1100:6789 Ensembl # indicates genes that are predicted based on genome sequences.
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