Liver fructose-1,6-bisphosphatase cDNA: trans-complementation of fission yeast and characterization of two human transcripts

Abstract

The SV40 early promoter is active both in mammalian cells and in the fission yeast Schizosaccharomyces pombe, and is used to drive full-length cDNA in polyvalent pcD-libraries. Two such liver libraries, of human and rat origin, were used to trans-complement a S. pombe mutant deficient in fructose-1,6-bisphosphatase (Fru-1,6-Pase) activity, a key gluconeogenic enzyme restricted to liver, kidney and intestine in mammals. A rat liver Fru-1,6-Pase cDNA was readily cloned and sequenced. Complementary PCR experiments revealed full-length Fru-1,6-Pase cDNA also present in the human liver library, however at a low abundance. Two human liver transcripts were thus characterized. Contrary to expectation, they were not differentially spliced products. They both encoded the same protein and were generated by a polyadenylation choice mechanism. The longest transcript comprised two polyadenylation signals and a consensus GT-rich element for the 3′ processing of the upstream site. Rapid amplification of cDNA ends- polymerase chain reaction (RACE-PCR) analysis of 3′ ends from hepatic, renal and intestinal mRNA disclosed that both Fru-1,6-Pase transcripts are expressed in the three main gluconeogenic cell types and are subject to insulin differential modulation. On the other hand, overcoming liver cell heterogeneity problems, sequence analysis of 16 independent clones of 3′ end-cDNA demonstrated that, in addition to a monocytic type corresponding to a previously described λgt11 clone, human liver does not contain a hepatic type Fru-1,6-Pase comprising a liver-specific carboxyl-terminal extension like its rat counterpart. This liver-specific extension is involved in enzyme up-regulation and appears to give a conclusive advantage to the rat hepatic enzyme over the human one when trans-complementing mutant yeast. These data are discussed in terms of (1) tissue-specific gene expression, (2) post-transcriptional gene regulation involving alternative polyadenylation sites, and (3) expression cloning of trans-acting factors that control “gluconeogenic” genes either with S. pombe or hepatoma cells.