Expression of the rat L-type pyruvate kinase gene from its dual erythroid- and liver-specific promoter in transgenic mice.
The gene for the L-type pyruvate kinase possesses two promoters which are located 500 base pairs apart. The L promoter is specific to liver and regulated by hormones and diet; the L' promoter is specific to erythroid cells. We produced two series of transgenic mice carrying either the entire rat L-pyruvate kinase gene or a minigene devoid of exons two to nine, with 2.7 kilobases of flanking sequences 5' to the cap site of the L' promoter and 1.4 kilobases 3' to the downstream polyadenylation site. In both series the patterns of expression from the two promoters were similar to those of the endogenous rat gene. The rat L promoter was expressed strongly in liver and weakly in kidney and gut of adult transgenic mice. Moreover, it was regulated like the endogenous rat L-pyruvate kinase gene upon hormonal and nutritional adaptation: the level of L-pyruvate kinase mRNA was decreased dramatically by 24 h of starvation, while refeeding a carbohydrate-rich diet strongly stimulated expression of the transgenes. This stimulation was prevented by glucagon. Use of alternative polyadenylation sites in the last exon of the rat L-type pyruvate kinase gene was similar for both types of transgenes and similar to that in rat and not control mice, suggesting that the transgenes contain the sequences that control the choice of polyadenylation site. Transcription of the minigene was higher than that of the entire transgene, probably due to the high copy number of the minigene. At the protein level, rat L subunits encoded by the entire transgene were more abundant than mouse subunits in the liver of adult transgenic mice. In contrast, expression of the rat L' promoter in fetal liver was only 5% of that in fetal rat liver, and we were unable to detect rat L' subunits of pyruvate kinase enzyme in the red blood cells from transgenic mice. Our results suggest that the integrated DNA contains all elements necessary for tissue specificity (L' and L) as well as hormonal and nutritional control (L) of expression of the rat transgene. Nevertheless, a L'-specific activating element may be missing.
Tremp
G L
GL
Unité de Recherches en Génétique et Pathologie Moléculaires, Institut National de la Santé et de la Recherche Médicale Unité, Paris, France.
Boquet
D
D
Ripoche
M A
MA
Cognet
M
M
Lone
Y C
YC
Jami
J
J
Kahn
A
A
Daegelen
D
D
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004040:Dietary Carbohydrates / Q000494:pharmacology; D005091:Exons; D005260:Female; D005333:Fetus; D015870:Gene Expression / Q000187:drug effects; D005796:Genes / Q000187:drug effects; D005934:Glucagon / Q000494:pharmacology; D007527:Isoenzymes / Q000097:blood / Q000235:genetics*; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009928:Organ Specificity; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000097:blood / Q000235:genetics*; D051381:Rats; D012016:Reference Values; D015183:Restriction Mapping; D014158:Transcription, Genetic
D004040:Dietary Carbohydrates; D007527:Isoenzymes; D015345:Oligonucleotide Probes; D005934:Glucagon; D011770:Pyruvate Kinase