Defective splicing of thyroglobulin gene transcripts in the congenital goitre of the Afrikander cattle.

Ricketts, M.; Pohl, Viviane; Martynoff, Guy de; Boyd, Charles D.; Bester, A J; Jaarsveld, P. P. Van; Vassart, Gilbert

doi:10.1002/j.1460-2075.1985.tb03690.x

Cited by 41 publications

(22 citation statements)

References 20 publications

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“…The failure to synthesize appreciable amounts ofTG in the case ofhereditary goiters ofthe goat and cow have been investigated at the molecular level (4,8 (1987) immunologically detectable in vitro translation products of this mRNA were obtained. In the bovine goiter, both normaland reduced-sized TG mRNAs were found in reduced amounts; these were translated in vitro into Mr 75,000 and Mr 250,000 polypeptides (8). The two mRNAs are the result of two kinds of abnormal splicing, the first leading to a frameshift and the second to the precise excision of the large ninth exon.…”

Section: Resultsmentioning

confidence: 99%

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The congenital goiter mutation is linked to the thyroglobulin gene in the mouse.

Taylor¹,

Rowe²

1987

Proc. Natl. Acad. Sci. U.S.A.

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Rat thyroglobulin (TG) cDNA clones were used to identify DNA restriction fragment variants among inbred mouse strains. One of these variants was shown to be closely linked to the recessive mutation congenital goiter (cog), which had previously been mapped to mouse chromosome 15. These results indicate that the structural gene for thyroglobulin is on chromosome 15 and suggest that a mutation at the site of the TG gene is the basis of the cog defect. No differences were observed between cog/cog and +/+ DNA in Southern blots using TG cDNA probes corresponding to 88% of the coding sequences, suggesting that the cog mutation is not due to a large deletion of this portion of the gene. Neither was there any obvious qualitative or quantitative difference between mutant and normal TG mRNA as judged by blot hybridization of electrophoretically fractionated thyroid RNAs. The thyroglobulin gene locus (Tgn) was mapped near the glutamic-pyruvic transaminase isoenzyme locus Gpt-1. The Tgn locus is syntenic with the c-myc protooncogene locus (Myc) in the mouse as in the rat and man.The thyroid hormones thyroxine (T4) and triiodothyronine (T3) are synthesized in a complex process from the Mr 660,000 thyroglobulin (TG) protein precursor (1). TG is a large glycoprotein consisting of two identical subunits whose primary polypeptide chain is of Mr 300,000. Two pairs of primary hormonogenic tyrosine residues have been identified near the amino and carboxyl termini ofthe polypeptide (2, 3). These tyrosine residues are iodinated and coupled by ether bonds. Thyroid hormones are released when TG is endocytosed by the thyrofollicular cells from the follicular lumen, where it is stored, and degraded in the lyzosomes by proteolytic digestion. Parts of the TG gene have been cloned in several species including the cow, goat, rat, and man (3-7). The rat TG gene consists of42 exons, spanning more than 170 kilobases (kb), and is transcribed into an -8500-nucleotide mRNA (5). The human TG gene is >300 kb long (6). Hereditary hypothyroidism due to disorders of TG biosynthesis has been described in both man and several species of domestic animals (1,4,8). Recently, a mouse mutation, congenital goiter (cog), was described that in homozygous condition causes primary hypothyroidism with goiter (9). We have used rat TG cDNA clones to identify and map DNA restriction fragment variants associated with the mouse TG gene. We show that the cog mutation is closely linked to these TG restriction fragment variants and is therefore probably a mutation in the mouse TG gene.

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Section: Resultsmentioning

confidence: 99%

“…The human TG gene is >300 kb long (6). Hereditary hypothyroidism due to disorders of TG biosynthesis has been described in both man and several species of domestic animals (1,4,8). Recently, a mouse mutation, congenital goiter (cog), was described that in homozygous condition causes primary hypothyroidism with goiter (9).…”

mentioning

confidence: 99%

The congenital goiter mutation is linked to the thyroglobulin gene in the mouse.

Taylor¹,

Rowe²

1987

Proc. Natl. Acad. Sci. U.S.A.

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“…1) was subcloned in both orientations in M13mpl9 and sequenced (16). The corresponding Ras I/HinclI fragment from a normal animal of European origin was subcloned and sequenced in exactly the same manner, except that the starting material was a cosmid clone harboring the first 16 exons of the Tg gene (9).…”

Section: Methodsmentioning

confidence: 99%

“…The hereditary goitre of the Afrikander cattle is inherited as an autosomal recessive disease (7). The goitre ofhomozygotes contains no normal Tg (8) and contains both a shorter and a normal-sized Tg mRNA (9); exon 9 is absent in the shorter Tg mRNA, suggesting that a splicing error is responsible for the disease (9). To identify precisely the mutation, we have cloned and sequenced the cDNA of the affected region of the smaller mRNA and the exon 9-intron 9 region of the genomic DNA from both a normal individual and an individual with goitre.…”

mentioning

confidence: 99%

A nonsense mutation causes hereditary goitre in the Afrikander cattle and unmasks alternative splicing of thyroglobulin transcripts.

Ricketts¹,

Simons²,

Parma³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

100

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The hereditary goitre of Afrikander cattle is an autosomal recessive disease characterized in homozygotes by the production of abnormal thyroglobulin (Tg) and the coexistence in the thyroid of normal-sized 8.4-kilobase (kb) Tg mRNA with a misspliced 7.3-kb message having lost exon 9. We have cloned and sequenced the cDNA segment corresponding to the abnormal exon 8-exon 10 junction and the relevant genomic DNA region. The mutation responsible for the disease is a cytosine to thymine transition creating a stop codon at position 697 in exon 9. The original reading frame is maintained in the 7.3-kb mRNA, which, as it lacks the mutated exon, is translatable into a potentially functional protein. This puzzling phenotype in which a mutated exon is apparently removed selectively from transcripts by alternative splicing leads us to suggest that the 7.3-kb transcript could be present in normal animals. Using a sensitive oligonucleotide hybridization assay, we have demonstrated that a 7.3-kb mRNA lacking exon 9 does exist in normal thyroids as a minor mRNA species. As it is fully translatable, the 7.3-kb mRNA is expected to be more stable than the normal-sized 8.4-kb message. This probably accounts for the higher proportion of 7.3-kb transcript found in the goitre.Thyroglobulin (Tg), a 660-kDa homodimer, is the biosynthetic precursor of the thyroid hormones (1). The Tg gene is -250,000 nucleotide pairs long, ofwhich 8431 nucleotides are represented in the mRNA (2). Congenital goitres with defective Tg production have been described in humans (3) and in animal models (4, 5). They are usually associated with hypothyroidism and may lead, in humans, to the development of the "cretin" phenotype (6). The hereditary goitre of the Afrikander cattle is inherited as an autosomal recessive disease (7). The goitre ofhomozygotes contains no normal Tg (8) and contains both a shorter and a normal-sized Tg mRNA (9); exon 9 is absent in the shorter Tg mRNA, suggesting that a splicing error is responsible for the disease (9). To identify precisely the mutation, we have cloned and sequenced the cDNA of the affected region of the smaller mRNA and the exon 9-intron 9 region of the genomic DNA from both a normal individual and an individual with goitre. We show that a cytosine to thymine transition in exon 9, changing codon 697 from CGA (arginine) to TGA (stop) is responsible for the disease. The nonsense mutation in exon 9 is apparently associated with and partially cured by removal of the defective exon from a portion ofTg transcripts. An explanation for this curious phenotype was obtained by the identification of a minor mRNA species lacking exon 9 in the thyroids of normal animals. Different stability of the shorter (fully translatable) and larger (containing an early stop codon) messages probably accounts for their relative amounts in goitre tissue.

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“…Various observations support this hypothesis. Thyroid hormone formation within truncated NH 2 -terminal Tg fragments, derived from the abortive translation of normal-sized mRNAs, was probably responsible for the correction of hypothyroidism, by iodide supplementation, in a strain of Dutch goats with congenital goiter (39 -41), and for euthyroidism in Afrikander cattle (42)(43)(44). Efficient T 4 formation was demonstrated in isolated fragment 1-171 of human Tg (22,34).…”

Section: Efficiency Of Tyr-1375 As a T 4 -And T 3 -Forming Site-the Dmentioning

confidence: 99%

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

Gentile

Ferranti²,

Mamone³

et al. 1997

Journal of Biological Chemistry

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A fragment of bovine thyroglobulin encompassing residues 1218 -1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T 3 ), and tetraiodothyronine (thyroxine, T 4 ) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T 4 of thyroglobulin (0.30 mol of T 4 /mol of 660-kDa thyroglobulin), and 8% of total T 3 (0.08 mol of T 3 /mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.Thyroglobulin (Tg), 1 a homodimeric glycoprotein with a molecular mass of 660 kDa, is the site of the biosynthesis of 3,5,3Ј-triiodothyronine (T 3 ) and 3,5,3Ј,5Ј-tetraiodothyronine (thyroxine, T 4 ) (reviewed in Ref. 1). T 3 and T 4 are synthesized via the iodination and coupling of a small subset of tyrosyl residues within the polypeptide chains of Tg. The coupling reaction takes place by the transfer of an iodophenyl group from a donor 3-monoiodotyrosine or 3,5-diiodotyrosine to an acceptor 3,5-diiodotyrosine. This causes the formation of T 3 or T 4 , respectively, at the acceptor site and dehydroalanine at the donor site (2, 3). Both reactions are catalyzed by thyroid peroxidase. Different tyrosyl residues have different reactivities toward iodine, so that iodination proceeds in a sequential order, which is controlled by the native structure of Tg (4, 5). Early iodinated tyrosyl residues are preferentially involved in iodothyronine synthesis (6); the coupling of iodotyrosines, in turn, has stringent steric requirements (7). In fact, out of 72 tyrosyl residues per bovine Tg monomer, only 15 are iodinated and a maximum of 6 -8 of them undergo coupling to form T 3 and T 4 (8, 9). So far, four major hormonogenic tyrosines have been identified, by the isolation and sequencing of hormone-rich peptides from Tgs of various animal species and comparison of their sequences with the cDNA-deduced sequences of bovine (10) and human Tg (11). Ty...

show abstract

Defective splicing of thyroglobulin gene transcripts in the congenital goitre of the Afrikander cattle.

Cited by 41 publications

References 20 publications

The congenital goiter mutation is linked to the thyroglobulin gene in the mouse.

The congenital goiter mutation is linked to the thyroglobulin gene in the mouse.

A nonsense mutation causes hereditary goitre in the Afrikander cattle and unmasks alternative splicing of thyroglobulin transcripts.

Identification of Hormonogenic Tyrosines in Fragment 1218-1591 of Bovine Thyroglobulin by Mass Spectrometry

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