Lee et al. 10.1073/pnas.0611008104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Figure 13
SI Figure 14
SI Figure 15
SI Figure 16
SI Figure 17
SI Figure 18




SI Figure 6

Fig. 6. cDNA and predicted amino acid sequences of zfGHRH. The numbers on the left are the positions of the nucleotide sequences. The UTR sequences are in lowercase. The coding sequences are in uppercase. The mature peptide sequence is highlighted in bold letter. Potential peptide cleavage sites are in open boxes. Stop codon is represented by an asterisks. The zfGHRH cDNA encompasses a 107-bp 5' UTR, a 339-bp ORF encoding a 113-aa protein, and a 101-bp 3' UTR. The predicted prepro-zfGHRH has a putative 23-aa signal peptide and potential peptide processing sites R and GKR at position 34 and 62-64, respectively, suggesting that the 27-aa zfGHRH is located between 35 and 61.





SI Figure 7

Fig. 7. cDNA and predicted amino acid sequences of gfGHRH. The numbers on the left are the positions of the nucleotide sequences. The UTR sequences are in lowercase. The coding sequences are in uppercase. The mature peptide sequence is highlighted in bold letter. Potential peptide cleavage sites are in open boxes. Stop codon is represented by an asterisks. The sizes of the 5' UTR, ORF and 3' UTR are 112, 420, and 97 bp, respectively, and, within the ORF, the predicted GHRH sequences between the putative cleavage sites (R and GKR, position 62 and 90-92) are identical to those of zfGHRH. As both fish GHRHs are followed immediately by GKR, they are likely a-amidated at the C termini.





SI Figure 8

Fig. 8. cDNA and predicted amino acid sequences of xGHRH. The numbers on the left are the positions of the nucleotide sequences. The UTR sequences are in lowercase. The coding sequences are in uppercase. The mature peptide sequence is highlighted in bold letter. Potential peptide cleavage sites are in open boxes. Stop codon is represented by an asterisks. Xenopus laevis GHRH cDNA is 790 bp in length with a 112-bp 5' UTR, a 420-bp ORF, and a 258-bp 3' UTR. Within the 140-aa xGHRH precursor protein, the N-terminal processing site (R at position 67) is conserved while the C-terminal site (GKT at position 95-97 or KK at position 104-105) is unclear. The mature xGHRH peptide may be 27- or 28-aa-long, depending on whether G at 95 is processed to the C-terminal amide, or 36-aa-long (68 to 103, processing site KK at position 104-105).





SI Figure 9

Fig. 9. Alignment of GHRH precursor proteins. The alignment was generated by using the default settings of the VectorNTI 10 (Invitrogen) with AlignX program. Identical and conserved residues are highlighted in red and blue, respectively. Putative GHRH peptides are in the open box. Regions found only in Xenopus and goldfish are underlined by arrows.





SI Figure 10

Fig. 10. Percentage similarity and identity of gfGHRH with other GHRHs. Chromosomal locations of GHRHs in species not yet determined are labeled as unknown. In the alignment, conserved and identical residues for GHRH and PRP are highlighted in blue and red, respectively. Residues that are conserved in PRP are labeled in green. The % identity was calculated by comparing with gfGHRH. Sf, scaffold number; Ch, chromosome number; -, sequences isolated in this report; *, sequences predicted from the genome database.





SI Figure 11

Fig. 11. cDNA and predicted amino acid sequences of zebrafish GHRH-R. The seven transmembrane domains are underlined. The RLTK motif is boxed. The conserved cysteine residues at the N-terminal are circled. The putative signal peptide is in bold letter. The stop codon is represented by an asterisk. The N-glycosylation site is marked in a black box.





SI Figure 12

Fig. 12. cDNA and predicted amino acid sequences of goldfish GHRH-R. The seven transmembrane domains are underlined. The RLTK motif is boxed. The conserved cysteine residues at the N-terminal are circled. The putative signal peptide is in bold letter. The stop codon is represented by an asterisk. The N-glycosylation site is marked in a black box.





SI Figure 13

Fig. 13. Percentage similarity and identity of gfGHRH-R with other GHRH receptors, other goldfish receptors in the same gene family, and other class IIB receptors in human.





SI Figure 14

Fig. 14. Amino acid alignment of GHRH-Rs and PRP-Rs. The alignment was generated by using the default settings of the VectorNTI 10 (Invitrogen) with AlignX program. Conserved residues for both GHRH-R and PRP-R are highlighted in blue. Residues that are conserved only among GHRH-Rs are in red. Residues conserved in fish and chicken GHRH-Rs are in green. Putative transmembrane domains (TM1 to TM7) are in open blue boxes. *, Conserved cysteine residues.





SI Figure 15

Fig. 15. Diagrammatic representation of GHRH and PRP-PACAP gene structures in various vertebrates. The boxes represent exons. The solid lines represent introns. Different domains of the precursors are shown in the legend.





SI Figure 16

Fig. 16. Predicted, sequence is predicted from genome database; None, no sequence is available; Ch, chromosome number; Sf, scaffold number; A, automatic prediction from genome project.





SI Figure 17

Fig. 17. Primer sequences used in PCR cloning of GHRH and GHRH-R cDNAs





SI Figure 18

Fig. 18. PCR primers for real-time analysis of goldfish GHRH and GHRH-R

References for SI Figs. 6-18

1. Gubler U, Monahan JJ, Lomedico PT, Bhatt RS, Collier KJ, Hoffman BJ, Bohlen P, Esch F, Ling N, Zeytin F, et al. (1983) Proc Natl Acad Sci USA 80:4311-4314.

2. Frohman MA, Downs TR, Chomczynski P, Frohman LA (1989) Mol Endocrinol 3:1529-1536.

3. Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM (1985) Nature 314:464-467.

4. Bohlen P, Esch F, Brazeau P, Ling N, Guillemin R (1983) Biochem Biophys Res Commun 116:726-734.

5. Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB, Guillemin R (1984) Biochem Biophys Res Commun 125:606-614.

6. Kimura C, Ohkubo S, Ogi K, Hosoya M, Itoh Y, Onda H, Miyata A, Jiang L, Dahl RR, Stibbs HH, et al. (1990) Biochem Biophys Res Commun 166:81-89.

7. Okazaki K, Itoh Y, Ogi K, Ohkubo S, Onda H (1995) Peptides 16:12951299.

8. Ogi K, Kimura C, Onda H, Arimura A, Fujino M (1990) Biochem Biophys Res Commun 173:1271-1279.

9. Alexandre D, Vaudry H, Jegou S, Anouar Y (2000) J Comp Neurol 421:234-246.

10. Wang CY, Wang Y, Li J, Leung FC (2006) Poult Sci 85:569-576.

11. Hu Z, Lelievre V, Tam J, Cheng JW, Fuenzalida G, Zhou X, Waschek JA (2000) Endocrinology 141:3366-3376.

12. Vaughan JM, Rivier J, Spiess J, Peng C, Chang JP, Peter RE, Vale W (1992) Neuroendocrinology 56:539-549.

13. Parker DB, Coe IR, Dixon GH, Sherwood NM (1993) Eur J Biochem 215:439-448.

14. McRory JE, Parker DB, Ngamvongchon S, Sherwood NM (1995) Mol Cell Endocrinol 108:169-177.

15. Wang Y, Wong AO, Ge W (2003) Endocrinology 144:4799-4810.

16. Mathieu M, Ciarlo M, Trucco N, Griffero F, Damonte G, Salis A, Vallarino M (2004) Brain Res Dev Brain Res 151:169-185.

17. Mayo KE (1992) Mol Endocrinol 6:1734-1744.

18. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA,Mayo KE (1993) Nat Genet 4:227-232.

19. Horikawa R, Gaylinn BD, Lyons CE, Jr, Thorner MO (2001) Endocrinology 142:2660-2668.

20. Toogood AA, Harvey S, Thorner MO, Gaylinn BD (2006) Endocrinology 147:1838-1846.

21. Porter TE, Ellestad LE, Fay A, Stewart JL, Bossis I (2006) Endocrinology 147:2535-2543.

22. Cardoso JC, Power DM, Elgar G, Clark MS (2003) DNA Seq 14:129-133.

23. Chan KW, Yu KL, Rivier J, Chow BK (1998) Neuroendocrinology 68:44-56.

24. Fradinger EA, Tello JA, Rivier JE, Sherwood NM (2005) Mol Cell Endocrinol 231:49-63.

25. Krueckl SL, Sherwood NM (2001) Direct submission

26. McRory J, Sherwood NM (1997) Endocrinology 138:2380-2390.

27. Mayo KE, Cerelli GM, Rosenfeld MG, Evans RM (1985) Nature 314:464-467.

28. Frohman MA, Downs TR, Chomczynski P, Frohman LA (1989) Mol Endocrinol 3:1529-1536.

29. Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB, Guillemin R (1984) Biochem Biophys Res Commun 125:606-614.

30. Gubler U, Monahan JJ, Lomedico PT, Bhatt RS, Collier KJ, Hoffman BJ, Bohlen P, Esch F, Ling N, Zeytin F, et al. (1983) Proc Natl Acad Sci USA 80:4311-4314.

31. Wang CY, Wang Y, Li J, Leung FC (2006) Poult Sci 85:569-576.

32. Hu Z, Lelievre V, Tam J, Cheng JW, Fuenzalida G, Zhou X, Waschek JA (2000) Endocrinology 141:3366-3376.

33. Okazaki K, Itoh Y, Ogi K, Ohkubo S, Onda H (1995) Peptides 16:1295-1299.

34. Kimura C, Ohkubo S, Ogi K, Hosoya M, Itoh Y, Onda H, Miyata A, Jiang L, Dahl RR, Stibbs HH, et al. (1990) Biochem Biophys Res Commun 166:81-89.

35. Toogood AA, Harvey S, Thorner MO, Gaylinn BD (2006) Endocrinology 147:1838-1846.

36. Porter TE, Ellestad LE, Fay A, Stewart JL, Bossis I (2006) Endocrinology 147:2535-2543.

37. Horikawa R, Gaylinn BD, Lyons CE, Jr, Thorner MO (2001) Endocrinology 142,2660-2668.

38. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA, Mayo KE (1993) Nat Genet 4:227-232.

39. Mayo KE (1992) Mol Endocrinol 6:1734-1744.

40. Chan KW, Yu KL, Rivier J, Chow BK Direct Submission Accession number: AC15699.

41. Chow BK, Yuen TT, Chan KW (1997) Gen Comp Endocrinol 105,176- 185.

42. Tse DL, Pang RT, Wong AO, Chan SM, Vaudry, H, Chow BK (2002) Endocrinology 143:1327-1336.

43. Ogi K, Miyamoto Y, Masuda Y, Habata Y, Hosoya M, Ohtaki T, Masuo Y, Onda, H, Fujino M (1993) Biochem Biophys Res Commun 196:1511-1521.

44. Gagnon AW, Aiyar, N, Elshourbagy NA (1994) Cell Signal 6:321-333.

45. Svoboda M, Tastenoy M, Van Rampelbergh J, Goossens JF, De Neef P, Waelbroeck, M, Robberecht P (1994) Biochem Biophys Res Commun 205:1617-1624.

46. Patel DR, Kong, Y, Sreedharan SP (1995) Mol Pharmacol 47:467-473.

47. Volz A, Goke R, Lankat-Buttgereit B, Fehmann HC, Bode HP, Goke B (1995) FEBS Lett 373,23-29.

48. Dillon JS, Tanizawa Y, Wheeler MB, Leng XH, Ligon BB, Rabin DU, Yoo-Warren H, Permutt MA, Boyd AE, III (1993) Endocrinology 133,1907-1910.

49. Munroe DG, Gupta AK, Kooshesh F, Vyas TB, Rizkalla G, Wang H, Demchyshyn L, Yang ZJ, Kamboj RK, Chen H, et al. (1999) Proc Natl Acad Sci USA 96:1569-1573.

50. MacNeil DJ, Occi JL, Hey PJ, Strader CD, Graziano MP (1994) Biochem Biophys Res Commun 198:328-334.

51. Kee F, Ng SS, Vaudry H, Pang RT, Lau EH, Chan SM, Chow BK (2005) Gen Comp Endocrinol 140:41-51.

52. Wolfe KH (2001) Nat Rev Genet 2:333-341.

53. Kumar, S, Hedges SB (1998) Nature 392:917-920.