ABSTRACT

Porcine prolactin cDNA clones were screened using antiserum against ovine prolactin from a cDNA library of porcine anterior pituitary, and their nucleotide sequences were determined by the chain-termination method. The nucleotide sequence of the 5′ untranslated region and part of the signal peptide region were determined by direct RNA sequencing with reverse transcriptase. The composite sequence of 957 nucleotides showed a signal sequence of 30 amino acids and a further 199 amino acids corresponding to the mature prolactin molecule. The predicted sequence confirmed the amino acid sequence determined previously by direct protein analysis, except for one amide form at residue 122 (Gln instead of the reported Glu). Northern blot analysis showed that the length of the porcine prolactin mRNA was about 1·1 kb. The porcine prolactin amino acid sequence showed 81, 80, 64, 62, 80 and 31% homology with human, bovine, rat, mouse, chick and salmon forms respectively. The identical amino acid residues showed marked clustering in four domains, two of which are highly conserved throughout a wide range of species. The hydropathy and secondary structure of porcine prolactin were analysed and compared with those of porcine GH, which shares the same ancestral gene. The two highly conserved regions of both hormones showed similar hydrophilicity, and the predicted secondary structures indicated that these regions in each hormone form different structures with differences in extension of the hydrophilic residues outside the molecule.

ABSTRACT

The gene for the common α subunit of the porcine anterior pituitary glycoprotein hormones was cloned from a genomic library constructed in EMBL3. The nucleotide sequence of the entire coding sequence of the porcine common α-subunit gene was determined in addition to one intron and 1059 and 160 bp of the 5′-and 3′-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the common α-subunit gene is present as a single copy. The transcriptional unit of the porcine common α subunit spanned about 14kb and contained four exons interrupted by three introns of about 11.5, 1.2 and 0.4kb. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues +9/+10 and +71/+72 were highly conserved among the rat, human and bovine common α-subunit genes. In the proximal portion of the 5′-flanking region, one TATA box and one CCAAT box were present. A steroid-responsive element was not found up to 1059 bases upstream from the transcription start site. The potential AP-1 and AP-2 factor-responsive elements were present at three and one positions respectively in the 5′-flanking region. This feature suggests that hypothalamic gonadotrophin-releasing hormone stimulates the expression of the common α-subunit gene predominantly by a signal-transduction system, with the protein kinase C cascade and factors AP-1 and AP-2 as mediators. The cyclic AMP-responsive element was also present at two positions, but a single base substitution was found in each sequence compared with the consensus sequence. The porcine common α-subunit gene has a structure distinct from its counterparts, the porcine FSH-β and LH-β genes, reflecting differential control of their synthesis during gametogenesis.

ABSTRACT

The gene for the β subunit of porcine LH (LH-β) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-β in addition to 1277 and 372 bp of the 5′- and 3′-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-β gene is present as a single copy. The transcriptional unit of porcine LH-β spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues −16/−15 and +41/+42 were highly conserved in the rat, human and bovine LH-β genes. In the 5′-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5′-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-β gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-β gene was the presence of clusters of GC boxes and CACCC elements in the 5′-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-β gene shows a structure distinct from the porcine FSH-β and common α genes, which are counterparts of the LH-β gene, reflecting differential control of their synthesis during gametogenesis.

ABSTRACT

To elucidate the structure and control of expression of the porcine FSH-β subunit gene, two genomic clones were isolated and the entire gene structure was determined to the extent of 10 kb, consisting of 6 kb of the 5′-flanking region and 4 kb of the transcriptional unit. The porcine FSH-β gene consisted of three exons the same as the human and bovine genes, but the positions of both splicing sites of porcine intron-1 were unique. It is known that the synthesis of FSH is regulated by gonadal steroids, gonadotrophin-releasing hormone (GnRH) and inhibin. However, the consensus steroid-responsive element was unexpectedly absent in the 5′-flanking region of 6 kb. On the other hand, the potential binding sites for activator protein-1 (AP1) and AP2, which might be stimulated by the GnRH—protein kinase C cascade, were present at seven and five positions respectively. An imperfect cyclic AMP-responsive element was also present. Southern blot analyses, using the cDNA and genomic fragments as probes, gave smear patterns suggesting the presence of repetitive sequences in the porcine FSH-β gene. A survey of homology with the repetitive sequences revealed that short interspersed repeated sequences (SINES)-type non-viral retroposons were present with about 250 bp length repeats twice in the 5′-flanking region and once each in intron-1 and the 3′-flanking region. Other SINES-like sequences were also found in intron-1, exon-2 and exon-3. In comparison with the 5′-flanking sequences of the porcine α and LH-β genes, there were no significantly conserved regions, implying a lack of common modulation of the three subunit genes.

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.

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.

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.

Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in response to fasting also suggest that the regulation of nucleus/cytoplasm translocation actually functions in vivo.

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.

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.