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The human L-threonine 3-dehydrogenase gene is an expressed pseudogene.

Edgar AJ - BMC Genet. (2002)

Bottom Line: These truncated proteins are the result of 3 mutations within the gene.There is a SNP, A to G, present in the genomic DNA sequence of some individuals which results in the loss of the acceptor splice site preceding exon 4.The acceptor splice site preceding exon 6 was lost in all 23 individuals genotyped and there is an in-frame stop codon in exon 6 (CGA to TGA) resulting in arginine-214 being replaced by a stop codon.

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Affiliation: Tissue Engineering & Regenerative Medicine Centre, Division of Investigative Science, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Chelsea & Westminster Hospital, London, United Kingdom. alasdair.edgar@ic.ac.uk

ABSTRACT

Background: L-threonine is an indispensable amino acid. One of the major L-threonine degradation pathways is the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine. L-threonine dehydrogenase (EC 1.1.1.103) is the first enzyme in the pathway and catalyses the reaction: L-threonine + NAD+ = 2-amino-3-ketobutyrate + NADH. The murine and porcine L-threonine dehydrogenase genes (TDH) have been identified previously, but the human gene has not been identified.

Results: The human TDH gene is located at 8p23-22 and has 8 exons spanning 10 kb that would have been expected to encode a 369 residue ORF. However, 2 cDNA TDH transcripts encode truncated proteins of 157 and 230 residues. These truncated proteins are the result of 3 mutations within the gene. There is a SNP, A to G, present in the genomic DNA sequence of some individuals which results in the loss of the acceptor splice site preceding exon 4. The acceptor splice site preceding exon 6 was lost in all 23 individuals genotyped and there is an in-frame stop codon in exon 6 (CGA to TGA) resulting in arginine-214 being replaced by a stop codon. These truncated proteins would be non-functional since they have lost part of the NAD+ binding motif and the COOH terminal domain that is thought to be involved in binding L-threonine. TDH mRNA was present in all tissues examined.

Conclusions: The human L-threonine 3-dehydrogenase gene is an expressed pseudogene having lost the splice acceptor site preceding exon 6 and codon arginine-214 (CGA) is mutated to a stop codon (TGA).

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The human TDH pseudogene is expressed. (A) Clones from 2 individuals were sequenced and mapped to the TDH gene. Clone 1 skipped exon 6, and the resulting frame-shift generated a premature stop codon in exon 7 (TGA). Clone 2 skipped exon 4 and utilised a cryptic splice site in exon 6, and the resulting frame-shift generated a premature stop codon in exon 6. (B) The translation of clone 1 (exon 6 skipped) encoded a truncated 230 residue ORF. The skipped exon 4 in clone 2 does not alter the reading frame. However, the use of a cryptic splice site in exon 6, results in a premature stop codon in exon 6. Stop codons are indicated by *.
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Figure 8: The human TDH pseudogene is expressed. (A) Clones from 2 individuals were sequenced and mapped to the TDH gene. Clone 1 skipped exon 6, and the resulting frame-shift generated a premature stop codon in exon 7 (TGA). Clone 2 skipped exon 4 and utilised a cryptic splice site in exon 6, and the resulting frame-shift generated a premature stop codon in exon 6. (B) The translation of clone 1 (exon 6 skipped) encoded a truncated 230 residue ORF. The skipped exon 4 in clone 2 does not alter the reading frame. However, the use of a cryptic splice site in exon 6, results in a premature stop codon in exon 6. Stop codons are indicated by *.

Mentions: The human TDH gene is expressed. PCR amplification from cDNA libraries using primers designed to amplify the TDH ORF was carried out and amplicons of approximately 1000 bp were obtained from cDNAs from 2 individuals, cloned and sequenced. The 1019 bp sequence of clone 1 (AY101186) skipped exon 6, resulting in a premature stop codon in exon 7, (Fig. 8A) and would encode a 230 residue ORF (Fig. 8B). The 935 bp sequence of clone 2 (AY101187) skipped exon 4 and utilised a cryptic splice site in exon 6, 24 bp downstream of the expected site (Fig 5B), resulting in a premature stop codon in exon 6 (Fig. 8A) and would encode a 157 residue ORF (Fig. 8B). The human TDH gene is therefore a pseudogene since all individuals examined contain at least 2 mutations that on translation would generate truncated proteins that would be non-functional since they would be unable to make appropriate contacts with the substrates, L-threonine and NAD+.


The human L-threonine 3-dehydrogenase gene is an expressed pseudogene.

Edgar AJ - BMC Genet. (2002)

The human TDH pseudogene is expressed. (A) Clones from 2 individuals were sequenced and mapped to the TDH gene. Clone 1 skipped exon 6, and the resulting frame-shift generated a premature stop codon in exon 7 (TGA). Clone 2 skipped exon 4 and utilised a cryptic splice site in exon 6, and the resulting frame-shift generated a premature stop codon in exon 6. (B) The translation of clone 1 (exon 6 skipped) encoded a truncated 230 residue ORF. The skipped exon 4 in clone 2 does not alter the reading frame. However, the use of a cryptic splice site in exon 6, results in a premature stop codon in exon 6. Stop codons are indicated by *.
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Figure 8: The human TDH pseudogene is expressed. (A) Clones from 2 individuals were sequenced and mapped to the TDH gene. Clone 1 skipped exon 6, and the resulting frame-shift generated a premature stop codon in exon 7 (TGA). Clone 2 skipped exon 4 and utilised a cryptic splice site in exon 6, and the resulting frame-shift generated a premature stop codon in exon 6. (B) The translation of clone 1 (exon 6 skipped) encoded a truncated 230 residue ORF. The skipped exon 4 in clone 2 does not alter the reading frame. However, the use of a cryptic splice site in exon 6, results in a premature stop codon in exon 6. Stop codons are indicated by *.
Mentions: The human TDH gene is expressed. PCR amplification from cDNA libraries using primers designed to amplify the TDH ORF was carried out and amplicons of approximately 1000 bp were obtained from cDNAs from 2 individuals, cloned and sequenced. The 1019 bp sequence of clone 1 (AY101186) skipped exon 6, resulting in a premature stop codon in exon 7, (Fig. 8A) and would encode a 230 residue ORF (Fig. 8B). The 935 bp sequence of clone 2 (AY101187) skipped exon 4 and utilised a cryptic splice site in exon 6, 24 bp downstream of the expected site (Fig 5B), resulting in a premature stop codon in exon 6 (Fig. 8A) and would encode a 157 residue ORF (Fig. 8B). The human TDH gene is therefore a pseudogene since all individuals examined contain at least 2 mutations that on translation would generate truncated proteins that would be non-functional since they would be unable to make appropriate contacts with the substrates, L-threonine and NAD+.

Bottom Line: These truncated proteins are the result of 3 mutations within the gene.There is a SNP, A to G, present in the genomic DNA sequence of some individuals which results in the loss of the acceptor splice site preceding exon 4.The acceptor splice site preceding exon 6 was lost in all 23 individuals genotyped and there is an in-frame stop codon in exon 6 (CGA to TGA) resulting in arginine-214 being replaced by a stop codon.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Engineering & Regenerative Medicine Centre, Division of Investigative Science, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Chelsea & Westminster Hospital, London, United Kingdom. alasdair.edgar@ic.ac.uk

ABSTRACT

Background: L-threonine is an indispensable amino acid. One of the major L-threonine degradation pathways is the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine. L-threonine dehydrogenase (EC 1.1.1.103) is the first enzyme in the pathway and catalyses the reaction: L-threonine + NAD+ = 2-amino-3-ketobutyrate + NADH. The murine and porcine L-threonine dehydrogenase genes (TDH) have been identified previously, but the human gene has not been identified.

Results: The human TDH gene is located at 8p23-22 and has 8 exons spanning 10 kb that would have been expected to encode a 369 residue ORF. However, 2 cDNA TDH transcripts encode truncated proteins of 157 and 230 residues. These truncated proteins are the result of 3 mutations within the gene. There is a SNP, A to G, present in the genomic DNA sequence of some individuals which results in the loss of the acceptor splice site preceding exon 4. The acceptor splice site preceding exon 6 was lost in all 23 individuals genotyped and there is an in-frame stop codon in exon 6 (CGA to TGA) resulting in arginine-214 being replaced by a stop codon. These truncated proteins would be non-functional since they have lost part of the NAD+ binding motif and the COOH terminal domain that is thought to be involved in binding L-threonine. TDH mRNA was present in all tissues examined.

Conclusions: The human L-threonine 3-dehydrogenase gene is an expressed pseudogene having lost the splice acceptor site preceding exon 6 and codon arginine-214 (CGA) is mutated to a stop codon (TGA).

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