LIVER FRUCTOSE-1,6-BISPHOSPHATASE CDNA - TRANSCOMPLEMENTATION OF FISSION YEAST AND CHARACTERIZATION OF 2 HUMAN TRANSCRIPTS

The SV40 early promoter is active both in mammalian cells and in the f ission yeast Schizosaccharomyces pombe, and is used to drive full-leng th cDNA in polyvalent pcD-libraries. Two such liver libraries, of huma n and rat origin, were used to trans-complement a S. pombe mutant defi cient in fructose-1,6-bisphosphatase (Fru-1,6-Pase) activity, a key gl uconeogenic enzyme restricted to liver, kidney and intestine in mammal s. A rat liver Fru-1,6-Pase cDNA was readily cloned and sequenced. Com plementary PCR experiments revealed full-length Fru-1,6-Pase cDNA also present in the human liver library, however at a low abundance. Two h uman liver transcripts were thus characterized. Contrary to expectatio n, they were not differentially spliced products. They both encoded th e same protein and were generated by a polyadenylation choice mechanis m. The longest transcript comprised two polyadenylation signals and a consensus CT-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 glu coneogenic cell types and are subject to insulin differential modulati on. On the other hand, overcoming liver cell heterogeneity problems, s equence analysis of 16 independent clones of 3' end-cDNA demonstrated that, in addition to a monocytic type corresponding to a previously de scribed lambda gt11 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 th e rat hepatic enzyme over the human one when trans-complementing mutan t yeast. These data are discussed in terms of (1) tissue-specific gene expression, (2) post-transcriptional gene regulation involving altern ative polyadenylation sites, and (3) expression cloning of trans-actin g factors that control ''gluconeogenic'' genes either with S. pombe or hepatoma cells.