Isolation of a functional, insulin regulatable glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6360815

Alexander-Bridges, M; Ramaika, C; Lomanto, M; Florence, B; Ercolani, L

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme which is regulated by insulin in 3T3 adipocytes and liver. They have isolated a 10 Kb BAM fragment which contains the entire GAPDH coding sequence and 1 Kb of the 5' flanking region. This clone has been mapped and sequenced to show the presence of a TATAA box; ATG, TAA, ATAA sites; and introns. When transiently expressed in L cells, the gene encodes a full length mRNA and functional protein. Thus, they have isolated a functional gene and not a pseudogene. When the gene is expressed in insulin-sensitive cells, the human GAPDH mRNA level was increased 3-fold in H35 hepatoma cells similar to the fold effect seen for endogenous rat mRNA in the same experiment. A 600 bp fragment of the GAPDH gene 5' flanking sequence was subcloned into a vector containing the chloramphenicol acetyl transferase (CAT) gene and cotransfected with the neomycin-resistant gene in H35 hepatoma cells to select stable lines. RNA isolated from control- and insulin-treated transfected cells show an appropriately sized S1 nuclease-protected fragment indicating that both the control species and insulin-stimulated species are primed off the GAPDH protomer. These studies indicate that insulin regulates GAPDH gene expression through an interaction with specific DNA sequences in rat hepatoma cells.

• Research Organization: Massachusetts General Hospital, Boston
• OSTI ID: 6360815
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
BIOLOGICAL FUNCTIONS
DNA SEQUENCING
ENZYMES
FUNCTIONS
GENE REGULATION
GENES
GENETIC MAPPING
HORMONES
INSULIN
MAMMALS
MAN
MAPPING
MESSENGER-RNA
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PEPTIDE HORMONES
PRIMATES
RATS
RNA
RODENTS
STRUCTURAL CHEMICAL ANALYSIS
TUMOR CELLS
VERTEBRATES