Expression of salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II precursor messenger ribonucleic acids in the brain and ovary of goldfish.
The complementary DNAs (cDNA) encoding the [Trp7Leu8]gonadotropin-releasing hormone (salmon GnRH; sGnRH) precursor and the [His5Trp7Tyr8]GnRH (chicken GnRH-II; cGnRH-II) precursor of the goldfish brain were isolated and sequenced using reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH precursor cDNA consists of 540 bp, including an open reading frame of 282 bp, and the cGnRH-II precursor cDNA consists of 682 bp, including an open reading frame of 258 bp. The 94 amino acid-long goldfish sGnRH precursor and 86 amino acid-long goldfish cGnRH-II precursor have the same molecular architecture as GnRH precursors identified to date in other vertebrate species. Using two sets of primers designed to be sense and antisense to the goldfish brain sGnRH precursor cDNA sequence, reverse transcription-polymerase chain reaction (RT-PCR) amplification of total RNA from brain and ovary at gonadal recrudescent, mature ( = prespawning), and postovulatory stages resulted in two predicted sizes of PCR products. The intensities of staining signals of ethidium bromide were similar between brain and ovary samples. The same RT-PCRs were carried out with two sets of primers for cGnRH-II precursor cDNA, resulting in two PCR products of predicted size; however, the ethidium bromide staining signals are much weaker for products amplified from ovarian cDNA than that from brain cDNA. Restriction enzyme analysis verified the expected RT-PCR products. Sequence analysis of ovarian sGnRH precursor cDNA generated by RACE of total RNA from recrudescent ovarian tissue revealed the identical sequence to that of the brain sGnRH cDNA. Northern blot analysis detected a single mRNA transcript of approximately 650 bases for the sGnRH precursor in both the brain and ovary, and 750 bases for the cGnRH-II precursor in the brain. These results demonstrate that two forms of GnRH precursor (sGnRH and cGnRH-II) mRNA are expressed in goldfish brain tissue and that the sGnRH transcript and a low level of cGnRH-II transcript are also expressed in the goldfish ovary.
Lin
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Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Peter
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002645:Chickens / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D005260:Female; D006054:Goldfish / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012474:Salmon / Q000378:metabolism*
D017931:DNA Primers; D012322:RNA Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone; C047717:LHRH, His(5)-Trp(7)-Tyr(8)-