Cloning and Expression Pattern of a Second

Abstract

Complementary DNAs (cDNAs) encoding [Trp7Leu8]GnRH (sGnRH) and [His5Trp7Tyr8]GnRH (cGnRH-II) peptides have been isolated from the brain of goldfish (X. W. Lin and R. E. Peter, 1996, Gen. Comp. Endocrinol. 101, 282-296). In the present study we report the isolation of a second cDNA encoding cGnRH-II peptide in the brain of goldfish using reverse transcription (RT) and rapid amplification of cDNA ends. There is an overall 79.7% nucleotide sequence similarity between the two cGnRH-II cDNAs, with 65.3, 91.2, and 76.3% similarity between the 5'-untranslated regions, coding regions, and 3'-untranslated regions, respectively, of the two cGnRH-II cDNAs. Comparison of the two cGnRH-II precursors shows 87.2% amino acid similarity. The presence of two cGnRH-II genes was confirmed by the sequence analysis of the introns between exon II and exon III of the two cGnRH-II genes. Results indicate that the intron of the two cGnRH-II genes shows a high divergence in size and sequence, but contains the same splice junction. Expression of the two cGnRH-II mRNAs was detected by RT-polymerase chain reaction assay and Southern blot analysis in all five grossly dissected brain areas, olfactory bulbs and tracts, telencephalon, hypothalamus, optic tectum-thalamus, and posterior brain. However, there was a difference in apparent intensity of hybridization signal for the two cGnRH-II mRNAs in all brain areas, suggesting a difference of expression levels. sGnRH mRNA was detected in the olfactory bulbs, telencephalon, and hypothalamus, but not in midbrain and posterior brain areas. The present finding of duplicate cDNAs and genes for cGnRH-II in goldfish is in agreement with the recent tetraploidization in this species.