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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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Expression of the TDH pseudogene in human tissues and cell types. (A) Expression of the TDH pseudogene in human tissues. The lanes are: 100 bp marker, m; heart, H; brain, B; placenta, Pl; lung, Lu; liver, L; skeletal muscle, M; kidney, K; pancreas, Pa; spleen, S; thymus; Th; prostate, Pr; testis, Te; ovary, O; small intestine, I; negative control, (-). (B) Expression of the TDH pseudogene in human cells. RT-PCR for TDH (top panel) and β-actin (bottom panel). The cell types examined were: foetal osteoblast, fO; adult osteoblasts, aO; pulmonary foetal fibroblast, HFL-1, fF; adult fibroblasts, aF; placental microvascular endothelial, mE; umbilical vein endothelial, uE; alveolar epithelial adenocarcinoma, A549, EA; bronchial epithelial adenocarcinoma, H322, EH; bronchial smooth muscle, SM; colorectal adenocarcinoma, CaCo2, CE; Epstein-Barr-transformed lymphocyte Ly; erythroleukaemia, K562, EK; erythroleukaemia TF1, ET; glioma, U87, G8; glioma, U373, G3; and negative control (-).
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Figure 9: Expression of the TDH pseudogene in human tissues and cell types. (A) Expression of the TDH pseudogene in human tissues. The lanes are: 100 bp marker, m; heart, H; brain, B; placenta, Pl; lung, Lu; liver, L; skeletal muscle, M; kidney, K; pancreas, Pa; spleen, S; thymus; Th; prostate, Pr; testis, Te; ovary, O; small intestine, I; negative control, (-). (B) Expression of the TDH pseudogene in human cells. RT-PCR for TDH (top panel) and β-actin (bottom panel). The cell types examined were: foetal osteoblast, fO; adult osteoblasts, aO; pulmonary foetal fibroblast, HFL-1, fF; adult fibroblasts, aF; placental microvascular endothelial, mE; umbilical vein endothelial, uE; alveolar epithelial adenocarcinoma, A549, EA; bronchial epithelial adenocarcinoma, H322, EH; bronchial smooth muscle, SM; colorectal adenocarcinoma, CaCo2, CE; Epstein-Barr-transformed lymphocyte Ly; erythroleukaemia, K562, EK; erythroleukaemia TF1, ET; glioma, U87, G8; glioma, U373, G3; and negative control (-).

Mentions: Reverse-transcriptase PCR was used to determine the presence of the TDH pseudogene mRNA in different tissues and cell types. TDH expression was found in all tissues examined (Fig. 9A). TDH mRNA was present in most cell types examined, but was below the level of detection in endothelial cells, glioma cell lines and some leukaemia cell lines (Fig. 9B). Together, this suggests that the TDH promoter is still active and is regulated in different cell types.


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

Edgar AJ - BMC Genet. (2002)

Expression of the TDH pseudogene in human tissues and cell types. (A) Expression of the TDH pseudogene in human tissues. The lanes are: 100 bp marker, m; heart, H; brain, B; placenta, Pl; lung, Lu; liver, L; skeletal muscle, M; kidney, K; pancreas, Pa; spleen, S; thymus; Th; prostate, Pr; testis, Te; ovary, O; small intestine, I; negative control, (-). (B) Expression of the TDH pseudogene in human cells. RT-PCR for TDH (top panel) and β-actin (bottom panel). The cell types examined were: foetal osteoblast, fO; adult osteoblasts, aO; pulmonary foetal fibroblast, HFL-1, fF; adult fibroblasts, aF; placental microvascular endothelial, mE; umbilical vein endothelial, uE; alveolar epithelial adenocarcinoma, A549, EA; bronchial epithelial adenocarcinoma, H322, EH; bronchial smooth muscle, SM; colorectal adenocarcinoma, CaCo2, CE; Epstein-Barr-transformed lymphocyte Ly; erythroleukaemia, K562, EK; erythroleukaemia TF1, ET; glioma, U87, G8; glioma, U373, G3; and negative control (-).
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Figure 9: Expression of the TDH pseudogene in human tissues and cell types. (A) Expression of the TDH pseudogene in human tissues. The lanes are: 100 bp marker, m; heart, H; brain, B; placenta, Pl; lung, Lu; liver, L; skeletal muscle, M; kidney, K; pancreas, Pa; spleen, S; thymus; Th; prostate, Pr; testis, Te; ovary, O; small intestine, I; negative control, (-). (B) Expression of the TDH pseudogene in human cells. RT-PCR for TDH (top panel) and β-actin (bottom panel). The cell types examined were: foetal osteoblast, fO; adult osteoblasts, aO; pulmonary foetal fibroblast, HFL-1, fF; adult fibroblasts, aF; placental microvascular endothelial, mE; umbilical vein endothelial, uE; alveolar epithelial adenocarcinoma, A549, EA; bronchial epithelial adenocarcinoma, H322, EH; bronchial smooth muscle, SM; colorectal adenocarcinoma, CaCo2, CE; Epstein-Barr-transformed lymphocyte Ly; erythroleukaemia, K562, EK; erythroleukaemia TF1, ET; glioma, U87, G8; glioma, U373, G3; and negative control (-).
Mentions: Reverse-transcriptase PCR was used to determine the presence of the TDH pseudogene mRNA in different tissues and cell types. TDH expression was found in all tissues examined (Fig. 9A). TDH mRNA was present in most cell types examined, but was below the level of detection in endothelial cells, glioma cell lines and some leukaemia cell lines (Fig. 9B). Together, this suggests that the TDH promoter is still active and is regulated in different cell types.

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
Related in: MedlinePlus