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J. Biol. Chem., Vol. 263, Issue 30, 15335-15341, Oct, 1988
L Ercolani, B Florence, M Denaro and M Alexander
Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.2.12) (GAPDH) mRNA levels,
protein, and enzymatic activity increase in 3T3-F442A adipocytes after
exposure to physiological concentrations of insulin (Alexander, M., Curtis,
G., Avruch, J., and Goodman, H. (1985) J. Biol. Chem. 260, 11978-11985). In
order to understand the mechanism of this regulation, we have isolated and
sequenced 5.4 kilobase pairs of a 12- kilobase pair human genomic clone
encoding a functional GAPDH gene. The gene consists of 9 exons and 8
introns with eukaryotic signals necessary for the transcription and
translation of GAPDH mRNA. The exon sequence confirms previously published
cDNA sequences for human GAPDH in muscle, liver, and erythrocytes. The
organization of the human and the unique chicken GAPDH genes is strikingly
similar. Although chicken exons VIII-XI have been fused into human exon 8,
introns which separate exons encoding the NAD binding, catalytic, and
helical domains of the GAPDH protein have been retained. Stable
transfection of rodent cells with the intact human GAPDH gene resulted in
the expression of a correctly initiated human GAPDH mRNA and an
enzymatically active human GAPDH polypeptide. Thus, the gene contains a
functional promoter and intact coding sequences. Although many processed
GAPDH pseudogenes and GAPDH-like sequences are present in the human genome,
Southern blot analysis of human genomic DNA using a probe derived from the
3'- untranslated region of the GAPDH gene detected only two genes, a 10-
copy processed pseudogene and a single copy of the isolated gene. In
contrast, a probe derived from an intron segment of the isolated gene
detected only a single copy of the GAPDH gene. Collectively, these findings
strongly suggest that the human genome encodes a single functional GAPDH
gene.
Isolation and complete sequence of a functional human glyceraldehyde-3- phosphate dehydrogenase gene
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
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