Signal transduction pathways involved in the expression of the uridine diphosphoglucose pyrophosphorylase gene of Dictyostelium discoideum

The uridine diphosphoglucose pyrophosphorylase (UDPGP1) gene of Dictyostelium discoideum is an excellent marker to study the pathways that control the expression of genes during development. We have previously shown that the UDPGP1 gene is regulated by exogenous cAMP acting on cell-surface cAMP receptors. Various steps in the signal transduction pathway between receptor stimulation and the induction of the gene can now be studied. Induction does not require the synthesis of intracellular cAMP, but does require new protein synthesis. By deletion and transformation with altered genes, two cis-acting sequences that are required for UDPGP1 expression have been identified. A GC-rich palindromic sequence located between -410 and -374 is essential for induction of the gene by extracellular cAMP, but not for its basal expression. A sequence element located between -374 and -337 is required for any basal expression of this gene. When the polarity of the palindromic sequence was reversed such that it resembled the H2K enhancer element, the gene could still be induced by exogenous cAMP. Two DNA binding activities were detected in gel mobility shift assays using a fragment containing both of the regulatory sequence elements of UDPGP1 gene. Transformation with a vector that resulted in the synthesis of anti-sense UDPGP1 RNA led to almost total elimination of the enzyme antigen and no detectable enzyme activity. However, these transformants developed normally, indicating that either UDPGP is not required for development or residual synthesis of UDPGP may be sufficient for normal development.     

Structure and sequence of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum.

Cell-cell contact and exogenous cAMP regulate the expression of uridine diphosphoglucose pyrophosphorylase (UDPGP) of Dictyostelium discoideum (B. Haribabu, A. Rajkovic and R. P. Dottin, 1986, Dev. Biol., Vol. 113, 436-442). cAMP appears to regulate gene expression in Dictyostelium by transmembrane signal transduction (B. Haribabu and R. Dottin, 1986, Mol. Cell. Biol. 6, 2402-2408). To further characterize the mechanism of action of cAMP on the expression of this gene and the nature of the defects in UDPGP mutants that abort development, we sequenced the cDNA and the genomic DNA, including intervening and flanking sequences. The deduced amino acid sequence predicts a polypeptide of 57,893 d. molecular weight. Three short (100-200 nucleotides) A+T rich introns occur within the coding sequences but only one of them contains a sequence TAACTAAC, similar to the yeast lariat acceptor site. The 5' flanking sequences are also A+T rich and contain an oligo A tract (-14 to -24), a TATA box (-25 to -32), and a short G+C rich region (-63 to -101) which may be a control region. From -196 to -209 is a sequence AAAGTAGTATTCAA which matches in 11 of its 14 nucleotides, a sequence found upstream from the hormonally regulated P-enolypyruvate carboxykinase gene of rat.     

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.