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At least 19 recordsLinked to original sources
PubMed1985

Molecular cloning of the human thyrotropin-beta subunit gene.

Genomic DNA fragments that carried a gene for human thyrotropin-beta (hTSH beta) subunit were isolated. Nucleotide sequence analysis of the gene showed that the hTSH beta subunit precursor consists of 138 amino acid residues. There is an N-terminal sequence of 20 amino acids as a signal peptide, followed by 112 amino acids, whose sequence is in agreement with that known for the secretory form of hTSH beta subunit. This is followed by an additional stretch of 6 hydrophobic amino acids, which may be eliminated post-translationally. The coding region is separated by an intron of about 460 bp. Genomic Southern blot hybridization analysis suggested that the hTSH beta gene is a unique single copy gene.

Amino Acid Sequence↗
PubMed1985

Evidence for a single rat thyrotropin-beta-subunit gene: thyroidectomy increases its mRNA.

We have isolated and characterized cDNAs representing the rat thyrotropin-beta-subunit. The cDNAs were prepared from poly(A)+ RNA obtained from rat pituitary glands and encode the precursor of the rat thyrotropin-beta-subunit which contains a leader or signal peptide of 20 amino acids, and an apoprotein of 118 amino acids. Blot hybridization analysis of total rat liver DNA digested with several restriction enzymes indicates the likelihood of a single gene encoding the rat thyrotropin-beta-subunit. In addition, analyses of pituitary RNA from normal and thyroidectomized rats indicate that the mRNA encoding the rat thyrotropin-beta-subunit is approximately 700 bases in length and its level increases 8--10-fold after thyroid gland ablation.

Amino Acid Sequence↗
PubMed1992

Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.

In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-beta subunit glycosylation recognition sequence, Asn-Thr-Thr (codons 23-25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Effects of irradiation and semistarvation on rat thyrotropin beta subunit messenger ribonucleic acid, pituitary thyrotropin content, and thyroid hormone levels.

The effect of radiation-induced anorexia on serum thyrotropin (TSH), pituitary TSH-beta mRNA, pituitary TSH content, serum thyroxine (T4), and serum 3,5,3'-triiodothyronine (T3) was investigated using feed-matched controls. Rats received 10 Gy gamma whole-body irradiation and were examined 1-3 days postirradiation. Feed-matched and untreated controls were also studied. The average food intake of the irradiated and feed-matched groups was approximately 18% of the untreated controls. Over the three day period both the irradiated and feed-matched groups lost a significant amount of body weight. The serum T4 levels of both the irradiated and feed-matched groups were not significantly different from each other, but were significantly depressed when compared to the untreated control group. The serum TSH and T3 were, however, significantly greater in the irradiated than the feed-matched groups at day 3 posttreatment. To determine if the difference in the serum TSH level between the two groups was due to a pretranslational alteration in TSH production, we measured the TSH-beta mRNA using an RNA blot hybridization assay. We found that the TSH-beta mRNA level was the same in the irradiated and feed-matched groups, suggesting that the mechanism responsible for the radiation-induced increase in the serum TSH level is posttranscriptional. Pituitary TSH content in the irradiated rats was significantly less than in pair-fed controls, suggesting that irradiation may permit enhanced secretion of stored hormone.

Animals↗
PubMed2003

Cloning of the gene for the thyrotropin beta subunit in the Japanese crested ibis, Nipponia nippon.

We isolated a putative gene for the thyrotropin beta subunit (TSHbeta) from two types of genomic libraries of the Japanese crested ibis, Nipponia nippon. Exon-intron structure was deduced by comparing the determined sequence with those of TSH beta cDNA of other birds. The deduced amino acid sequence shows extensive similarities to those of the other birds, which assures our assumption that the acquired nucleotide sequence represents the TSHbeta gene. The assembled genomic fragment is 4192 bp in size and consists of 1937 bp of putative 5' flanking region followed by exon-intron structure with three exons and two introns, similar to those observed in rat, human and goldfish counterparts. Locations of introns are also similar to those in mammals and goldfish. Comparison of the 5' flanking region of the ibis TSHbeta gene with those of mammals reveals that several regulatory sequences, such as negative thyroid hormone responsive element (nTRE), Pit-1 responsive element, and AP-1 responsive element, which were characterized in mammalian TSHbeta genes, are also found in the promoter region. This is the first report on the exon-intron structure and 5' flanking region of the TSHbeta gene in an avian species.

Animals↗
PubMed1999

Isolation and characterization of the goldfish thyrotropin beta subunit gene including the 5'-flanking region.

The complete gene encoding the beta subunit of thyrotropin (thyroid-stimulating hormone, TSH) was isolated from a goldfish genomic library. The goldfish TSHbeta subunit gene, which is approximately 2.0 kilobase pairs (kb) in length, consisted of three exons and two introns. The first intron was much longer (0.89 kb) than the second intron (0.3 kb) as are TSHbeta genes in mammalian species. On the basis of the location of the first intron, the goldfish TSHbeta gene belongs to the mammalian TSHbeta/FSHbeta gene group, which is distinct from the LHbeta group. Inspection of the 5'-flanking and exon 1 regions of the goldfish TSHbeta gene (1.2 kb) revealed the presence of several putative cis-acting elements, including the negative triiodothyronine (T(3))-responsive element (nTRE), Pit-1 element, and GATA-2 element. Comparison of the goldfish sequence with mammalian TSHbeta promoter sequences showed an identical region, nTRE, in the first exon-intron junction region. An in vitro study using dispersed goldfish pituitary cells showed that T(3) treatment (20 ng/ml) suppressed the TSHbeta mRNA level in the cells. These data indicate that (1) the basic structure of TSHbeta genes is highly conserved in vertebrates and that (2) T(3) acts directly on the pituitary and inhibits TSHbeta gene expression in goldfish, probably via the nTRE in the TSHbeta gene.

Amino Acid Sequence↗
PubMed1986

Mapping thyrotropin beta subunit gene in man and mouse.

Thyrotropin (TSH) is composed of two subunits: alpha and beta. Previously, we have mapped the TSH alpha gene to human chromosome 6 and mouse chromosome 4. In this study we have located the human TSH beta gene on chromosome 1 and the mouse TSH beta gene to chromosome 3. These data suggest that the TSH beta gene lies in a conserved linkage group with the genes for amylase 1 and 2, nerve growth factor, and the protooncogene Nras.

Animals↗
PubMed1993

[Cloning and sequence of thyrotropin beta subunit of a teleost fish: the eel (Anguilla anguilla L.)].

We obtained the sequence of eel thyrotropin beta-subunit cDNA. About 1,100 bp long, it encodes a 147-amino acid protein including a 20-residue signal peptide. We analyse homologies between angTSH beta and the other known beta-subunits taking in account the ability of mammal gonadotropins (GTH) to stimulate teleost thyroid. A peptide corresponding to mammalian CAGYC (implicated in subunit association) is original and different from its eel beta GTH2 counterpart.

Amino Acid Sequence↗
PubMed1992

Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.

cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.

Animals↗
PubMed1992

The proto-oncogenes c-fos and c-jun modulate thyroid hormone inhibition of human thyrotropin beta subunit gene expression in opposite directions.

The sequence from -1 to +6 bp in the hTSH beta gene contains overlapping putative thyroid hormone and AP-1 response elements. We demonstrate interaction between the AP-1 constituents c-fos and c-jun and thyroid hormone receptor in this region by transient transfection experiments using a -125 to +37 bp hTSH beta fragment. T3 inhibition was completely abolished by c-jun, but increased threefold by c-fos. A single transversion mutation at +2 bp restored T3 inhibition in the presence of c-jun and markedly reduced binding of purified c-jun by gel mobility shift assay. Thus, c-fos and c-jun influence T3 inhibition of hTSH beta expression in opposite directions acting through a response element shared with thyroid hormone receptor. Control of the relative cellular levels of these two proto-oncogenes may play a major role in modulating thyroid hormone inhibitory responses.

Base Sequence↗
PubMed1989

Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene.

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Thyroid hormone regulates expression of the thyrotropin beta-subunit gene from both transcription start sites in the mouse and rat.

Thyroid hormones suppress transcription of the gene for the beta-subunit of thyrotropin (TSH beta). Since the TSH beta gene in both the mouse and the rat contains two start sites of transcription in exon 1, we have investigated whether expression of the gene from each start site is differentially regulated by thyroid hormones in each species. RNase protection analysis was used to assay the levels of mRNA specifically transcribed from the upstream (TSS 1) and downstream (TSS 2) transcription start sites in the mouse and rat pituitary. In euthyroid and hypothyroid pituitaries there was an approximately 5-fold and 2-fold greater abundance of mRNA derived from TSS 2 than TSS 1, respectively. Hypothyroidism induced an 18- and a 9-fold increase in TSH beta gene expression from TSS 1 and TSS 2, respectively. Treatment of hypothyroid animals for 1 day with triiodothyronine (T3) reduced expression from both start sites by about 50%; after 4 days of T3 treatment, TSH beta mRNAs derived from both start sites were below detectable levels. These results were confirmed in the rat by primer extension analysis. Expression from TSS 1 in the mouse was also shown to be dependent on thyroid status using the polymerase chain reaction (PCR) technique. In contrast to previous results from primer extension studies, PCR analysis demonstrated that alternative splicing of the TSH beta RNA primary transcript can occur when transcription is initiated at the upstream start site. We conclude that, in both the mouse and the rat pituitary, expression of the TSH beta gene from both transcription start sites is regulated by thyroid hormones.

Animals↗
PubMed1993

AP-1 antagonizes thyroid hormone receptor action on the thyrotropin beta-subunit gene.

Thyrotropin-releasing hormone (TRH) stimulates and thyroid hormone (T3) inhibits transcription of the thyrotropin beta-subunit gene (TSH-beta). The first exon contains DNA sequences necessary for both responses and binds both AP-1 and thyroid hormone receptor (T3R). T3 did not inhibit TSH-beta gene expression in a T3R-deficient cell line. Transfection of a T3R expression vector, however, resulted in a 70% inhibition of expression by T3, which was abolished by cotransfection of c-jun and c-fos expression vectors. Mutations surrounding the transcription initiation site and DNA binding studies demonstrate both a functional and structural interaction between c-jun and T3R. Thus, TRH, acting through AP-1, may alter the set point and magnitude of thyroid hormone negative feedback of the TSH-beta gene through an interaction between AP-1 and T3R. Other regulatory pathways acting through AP-1 may alter thyroid hormone action in man.

Animals↗
PubMed1987

Isolation and characterization of the rat thyrotropin beta-subunit gene. Differential regulation of two transcriptional start sites by thyroid hormone.

The gene encoding the beta-subunit of rat thyrotropin (TSH beta) has been isolated and characterized. Blot hybridization of restriction enzyme digests of rat genomic DNA suggests that the gene is present in a single copy. The transcriptional unit is 4.9 kilobases in size representing 3 exons interrupted by 2 introns of 3.9 and 0.4 kilobases. Its nucleotide sequence reveals that the locations of the exon/intron junctions are one nucleotide upstream from the translational start and between codons +34 and +35. The location of the second intron is apparently strictly conserved among the glycoprotein hormone beta-subunit genes being four codons downstream from a region encoding a consensus sequence: Cys-Ala-Gly-Tyr. Using S1 nuclease mapping and oligonucleotide-primed reverse transcription of normal and thyroidectomized rat pituitary mRNA, two transcriptional start sites were identified in the rat TSH beta gene that are 28 and 71 nucleotides upstream from the translational start site. The level of TSH beta mRNA containing the downstream site is altered by thyroidal status whereas the other mRNA utilizing the upstream cap site appears to be constitutively expressed. Characteristic promoter elements are present in the 5'-flanking region including TATAAA or Goldberg-Hogness consensus regions which are present 29 and 26 bases upstream from the respective starts of transcription. Also, several CAAT boxlike sequences are located between 95 and 300 bases upstream from the start of translation. Isolation and characterization of the gene encoding the TSH beta gene will facilitate the study of the molecular mechanisms by which hormones regulate TSH beta gene expression.

Animals↗
PubMed1987

Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor.

Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.

Animals↗
PubMed1988

The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

Animals↗