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  • Author: K. Gen x
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Molecular cloning of cDNAs encoding two gonadotrophin β subunits (GTH-Iβ and -IIβ) from the masu salmon, Oncorhynchus masou: rapid divergence of the GTH-Iβ gene

Y Kato, K Gen, O Maruyama, K Tomizawa, and T Kato

ABSTRACT

Two types of cDNA (GTH-Iβ and -IIβ) encoding the β subunit of masu salmon (Oncorhynchus masou) gonadotrophin were cloned using the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequence of GTH-Iβ cDNA was 469 bp long, encoding 137 amino acids, and GTH-IIβ cDNA was 476 bp long, encoding 142 amino acids. These two masu salmon β subunit types showed low homologies of 52% (nucleotide sequence) and 33% (amino acid sequence). The evolutionary interval between masu and chum salmon was estimated to be 5·65 and 1·43 million years by comparing GTH-Iβ and GTH-IIβ respectively. These time values are markedly inconsistent with the evolutionary time (30 million years) estimated from fossil records and an isozyme study. Southern blot analyses showed that the Iβ gene restriction fragment lengths differed among five teleosts, whereas, with one exception, the IIβ gene showed well conserved patterns. Therefore, the GTH-Iβ gene may have diverged at a faster rate than the GTH-IIβ gene.

DOI:
https://doi.org/10.1677/jme.0.0110275
Volume 11: Issue 3,
Pages:
275–282 (8 total)
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Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries

N. Takahashi, S. Kikuyama, K. Gen, O. Maruyama, and Y. Kato

ABSTRACT

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1·2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.

DOI:
https://doi.org/10.1677/jme.0.0090283
Volume 9: Issue 3,
Pages:
283–289 (7 total)
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Molecular cloning of cDNAs encoding two types of gonadotrophin α subunit from the masu salmon, Oncorhynchus masou: construction of specific oligonucleotides for the α1 and α2 subunits

K Gen, O Maruyama, T Kato, K Tomizawa, K Wakabayashi, and Y Kato

ABSTRACT

Two types of cDNA (GTHα1 and -α2) encoding the α subunits of masu salmon (Oncorhynchus masou) gonadotrophin were cloned by the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequences showed that the GTHα1 cDNA was 380 bp long, encoding 119 amino acids, and that GTHα2 cDNA was 365 bp long, encoding 114 amino acids. The masu salmon α subunit types had a few differences between the sequences, with homologies of 80% (nucleotide sequence) and 72% (amino acid sequence). The structural difference between the α1 and α2 subunits was predicted using hydropathic analysis. The evolutionary interval between masu and chum salmon was estimated to be 4·0 and 2·3 million years by comparing their GTHα1 and -α2 subunits respectively. These time values are roughly consistent with the evolutionary time interval (3·0 million years) estimated from fossil records and an isozyme study. Specific synthetic oligonucleotide probes were constructed and used for genomic Southern blot analyses. The restriction fragment sizes of the GTHα1 and -α2 genes were similar, and when their patterns were compared with those from four other teleosts, each species showed a different pattern from the others, but no difference between their respective α1 and α2 genes. Therefore, the structural features of the GTHα1 and -α2 genes may have diverged in a similar manner in these five teleosts.

DOI:
https://doi.org/10.1677/jme.0.0110265
Volume 11: Issue 3,
Pages:
265–273 (9 total)
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Molecular cloning of the cDNAs encoding pituitary glycoprotein hormone α- and gonadotropin IIβ-subunits of the Japanese eel, Anguilla japonica, and increase in their mRNAs during ovarian development induced by injection of chum salmon pituitary homogenate

M Nagae, T Todo, K Gen, Y Kato, G Young, S Adachi, and K Yamauchi

ABSTRACT

cDNAs encoding the glycoprotein hormone α- and gonadotropin (GTH) IIβ-subunits of Japanese eel (Anguilla japonica) pituitary were cloned using the polymerase chain reaction. The nucleotide sequence of the glycoprotein hormone α-subunit cDNA was 364 base pairs (bp) long, encoding 117 amino acids, and that of the GTH IIβ-subunit cDNA was 433 bp long, encoding 140 amino acids. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts, indicating that the structure of GTH subunits has been conserved during the evolution of teleosts. Changes in the expression of these subunit genes during ovarian development induced artificially by the injection of chum salmon pituitary homogenate were examined using Northern blot analysis. Glycoprotein hormone α-subunit mRNA increased almost linearly during ovarian development, whereas GTH IIβ-subunit mRNA was detected only at the late vitellogenic and migratory nucleus stages. These data indicate that eel GTH II is synthesized mainly at the late vitellogenic and migratory nucleus stages, and suggest that GTH II plays an important role in final oocyte maturation of Japanese eel. Changes in the expression of glycoprotein hormone α- and GTH IIβ-subunits mRNA correlate with the serum estradiol-17β (E2) and testosterone profile during ovarian development. The increase in mRNA of both subunits is probably due to positive feedback of E2 and testosterone produced by ovarian follicles in response to the GTH contained in chum salmon pituitary homogenate.

DOI:
https://doi.org/10.1677/jme.0.0160171
Volume 16: Issue 2,
Pages:
171–181 (11 total)