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  • Author: K Tomizawa 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)
Restricted access

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)
Free access

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector

Y Kato, I Sato, T Ihara, K Tomizawa, J Mori, M Geshi, T Nagai, K Okuda, T Kato, and S Ueda

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

DOI:
https://doi.org/10.1677/jme.0.0200055
Volume 20: Issue 1,
Pages:
55–65 (11 total)