Premature translation termination of the Pre-E1α subunit of the branched chain α-ketoacid dehydrogenase as a cause of maple syrup urine disease in polled hereford calves

Bei Zhang, Peter J. Healy, Yu Zhao, David Crabb, Robert Harris

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain α-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain α-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E la mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1α subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1α subunit revealed a single base substitution at codon -6 (GAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1α subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1β protein stability on the co-expression of the E1α.

Original languageEnglish
Pages (from-to)2425-2427
Number of pages3
JournalJournal of Biological Chemistry
Volume265
Issue number5
StatePublished - 1990

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Maple Syrup Urine Disease
Messenger RNA
Polymerase chain reaction
Fibroblasts
Complementary DNA
Animal Disease Models
Polymerase Chain Reaction
Mutation
Terminator Codon
Protein Stability
DNA-Directed RNA Polymerases
Protein Sorting Signals
Codon
Oligonucleotides
Northern Blotting
Reverse Transcription
Transcription
Western Blotting
Alleles

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Premature translation termination of the Pre-E1α subunit of the branched chain α-ketoacid dehydrogenase as a cause of maple syrup urine disease in polled hereford calves",
abstract = "Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain α-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain α-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E la mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1α subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1α subunit revealed a single base substitution at codon -6 (GAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1α subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1β protein stability on the co-expression of the E1α.",
author = "Bei Zhang and Healy, {Peter J.} and Yu Zhao and David Crabb and Robert Harris",
year = "1990",
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AU - Zhang, Bei

AU - Healy, Peter J.

AU - Zhao, Yu

AU - Crabb, David

AU - Harris, Robert

PY - 1990

Y1 - 1990

N2 - Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain α-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain α-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E la mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1α subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1α subunit revealed a single base substitution at codon -6 (GAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1α subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1β protein stability on the co-expression of the E1α.

AB - Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain α-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain α-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E la mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1α subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1α subunit revealed a single base substitution at codon -6 (GAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1α subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1β protein stability on the co-expression of the E1α.

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