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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.

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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Amplicons from individuals with different genotypes for the exon 4 splice acceptor site SNP were digested with the restriction enzyme AciI (upper panel) and DNA sequenced (lower panel). Undigested samples, UD and digested samples, D. Arrows indicate the polymorphic nucleotide. In an individual homozygous for the splice acceptor site (AG, middle panel) no AciI digest site is present. In an individual homozygous for loss of the splice acceptor site (GG, right panel) the 158 bp amplicon is cut into fragments of 104 and 54 bp. In an individual heterozygous for the AG splice acceptor site (RG, left panel) both alleles are present and the amplicon is only partially digested.
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Figure 6: Amplicons from individuals with different genotypes for the exon 4 splice acceptor site SNP were digested with the restriction enzyme AciI (upper panel) and DNA sequenced (lower panel). Undigested samples, UD and digested samples, D. Arrows indicate the polymorphic nucleotide. In an individual homozygous for the splice acceptor site (AG, middle panel) no AciI digest site is present. In an individual homozygous for loss of the splice acceptor site (GG, right panel) the 158 bp amplicon is cut into fragments of 104 and 54 bp. In an individual heterozygous for the AG splice acceptor site (RG, left panel) both alleles are present and the amplicon is only partially digested.

Mentions: To determine whether these mutations in the 2 human genomic sequences were sequencing errors or just present in those individuals utilised for determining the human genome, the region encompassing the mutations was amplified by PCR from a number of individuals, sequenced and digested with restriction enzymes. Amplicons from the exon 4 splice acceptor site were digested with the restriction enzyme AciI. If the acceptor splice site was not present (G^CGG rather than GCAG, acceptor splice site underlined) the 158 bp amplicon was digested to produce restriction fragments of 104 and 54 bp. The restriction enzyme digest pattern and DNA sequence of 3 individuals are illustrated (Fig. 6) in which one individual has the splice site intact, another has lost the splice site and the third has the heterozygous condition. Digestion is not as complete as would be expected in the heterozygous condition, presumably due to heteroduplex formation during the PCR. This SNP was found in 20 out of 40 chromosomes examined.


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

Edgar AJ - BMC Genet. (2002)

Amplicons from individuals with different genotypes for the exon 4 splice acceptor site SNP were digested with the restriction enzyme AciI (upper panel) and DNA sequenced (lower panel). Undigested samples, UD and digested samples, D. Arrows indicate the polymorphic nucleotide. In an individual homozygous for the splice acceptor site (AG, middle panel) no AciI digest site is present. In an individual homozygous for loss of the splice acceptor site (GG, right panel) the 158 bp amplicon is cut into fragments of 104 and 54 bp. In an individual heterozygous for the AG splice acceptor site (RG, left panel) both alleles are present and the amplicon is only partially digested.
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Related In: Results  -  Collection

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Figure 6: Amplicons from individuals with different genotypes for the exon 4 splice acceptor site SNP were digested with the restriction enzyme AciI (upper panel) and DNA sequenced (lower panel). Undigested samples, UD and digested samples, D. Arrows indicate the polymorphic nucleotide. In an individual homozygous for the splice acceptor site (AG, middle panel) no AciI digest site is present. In an individual homozygous for loss of the splice acceptor site (GG, right panel) the 158 bp amplicon is cut into fragments of 104 and 54 bp. In an individual heterozygous for the AG splice acceptor site (RG, left panel) both alleles are present and the amplicon is only partially digested.
Mentions: To determine whether these mutations in the 2 human genomic sequences were sequencing errors or just present in those individuals utilised for determining the human genome, the region encompassing the mutations was amplified by PCR from a number of individuals, sequenced and digested with restriction enzymes. Amplicons from the exon 4 splice acceptor site were digested with the restriction enzyme AciI. If the acceptor splice site was not present (G^CGG rather than GCAG, acceptor splice site underlined) the 158 bp amplicon was digested to produce restriction fragments of 104 and 54 bp. The restriction enzyme digest pattern and DNA sequence of 3 individuals are illustrated (Fig. 6) in which one individual has the splice site intact, another has lost the splice site and the third has the heterozygous condition. Digestion is not as complete as would be expected in the heterozygous condition, presumably due to heteroduplex formation during the PCR. This SNP was found in 20 out of 40 chromosomes examined.

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).

Show MeSH