The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the single-phosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pdsA gene and four fgd genes affect the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.     

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes.     

A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing

Genes with sequence similarity to the yeast tRNA(His) guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNA(His) maturation to 5'-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3'-5' nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G(-1) addition enzyme likely to be responsible for tRNA(His) maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5'-tRNA editing, based on their ability to efficiently repair the 5' ends of mitochondrial tRNA editing substrates. Although 5'-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5'-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3'-5' nucleotide addition reactions for diverse biological functions in the same organism.     

Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum

Developmental variations in the expression of two genes of the de novo pyrimidine biosynthetic pathway have been examined in Dictyostelium discoideum. One gene, DdPYR4, encodes the dihydroorotate dehydrogenase (EC 1.3.3.1); the other, DdPYR5-6, encodes the UMP synthase which in D. discoideum is a bifunctional enzyme harboring both the orotate phosphoribosyl transferase activity (EC 2.4.2.10) and the OMP decarboxylase activity (EC 4.1.1.23). The relative amount of mRNA for both genes has been estimated by hybridization with the previously cloned DNAs and compared with the amount of actin mRNA. The level of both mRNAs is dramatically reduced after 4 h of development and remains at a low level later in development. In contrast to these variations, the specific activity of the enzymes encoded by these genes during development is similar to that measured during exponential growth. These results lead us to propose that DdPYR4 and DdPYR5-6 genes encode for relatively stable proteins and that their synthesis is reduced to maintain a constant level of enzymes in non-growing cells. This mode of regulation could apply to a large number of housekeeping genes.     

Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa

The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway. The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined. Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced. Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast. Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks. Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified.     

AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae.

We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.     

apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

Carbamoyl phosphate synthetase (CPSase) in the PYR1-3 multigene of Dictyostelium discoideum.

We have isolated and sequenced the genomic DNA from the slime-mould Dictyostelium discoideum multi-gene (PYR1-3) encoding the carbamoyl phosphate synthetase II domain (CPSase, EC.6.3.5.5). We describe sequencing by oligo-walking directly on PCR product in the solid-phase, avoiding subcloning procedures. The 2.4 kb fragment completes the sequence of the PYR1-3 gene, has no introns, and has the same structure as the rudimentary gene of Drosophila melanogaster. Comparison with the carbamoyl phosphate synthetases (CPSase I and CPSase II) of other species supports the hypothesis that this gene has arisen by tandem duplication from a smaller common ancestral gene in the progenote.     

Characterization and comparative structural features of the gene for human interstitial retinol-binding protein

We have cloned the gene for human interstitial retinol-binding protein (IRBP) and compared its nucleotide sequence with that of the corresponding cloned cDNA. The human IRBP gene is approximately 9.5 kilobase pairs (kbp) in length and consists of four exons separated by three introns. The introns are 1.6-1.9 kbp long. The gene is transcribed by photoreceptor and retinoblastoma cells into an approximately 4.3-kilobase mRNA that is translated and processed into a glycosylated protein of 135,000 Da. The amino acid sequence of human IRBP can be divided into four contiguous homology domains with 33-38% identity, suggesting a series of gene duplication events. In the gene, the boundaries of these domains are not defined by exon-intron junctions, as might have been expected. The first three homology domains and part of the fourth are all encoded by the first large exon, which is 3,180 base pairs long. The remainder of the fourth domain is encoded in the last three exons, which are 191, 143, and approximately 740 base pairs long, respectively. This unusual structure is shared with the bovine IRBP gene. A large (1.7 kbp) fragment appears to have been lost from the 3'-noncoding region of the last human exon. We conclude that the human and bovine genes have similar evolutionary histories.     

Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G-rich sequence element lying between them with the characteristics of a basal promoter element

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G-rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP-inducibility. A synthetic oligonucleotide, containing two copies of the G-rich element from the CP1 gene, will reconstitute cAMP-inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.     

An alternative non-tyrosine protein kinase product of the c-src gene in chicken skeletal muscle.

While the c-src locus is expressed as a 4.0-kilobase (kb) mRNA coding for pp60c-src in various chicken tissues, including embryonic muscle, it is expressed as a novel 3.0-kb mRNA in adult skeletal muscle. We have analyzed the primary structure of this alternatively transcribed and spliced c-src mRNA. The sequence revealed three open reading frames, with the previously defined c-src exons 1 through 5 or 6 comprising the third, on the 3' untranslated region of this 3-kb mRNA. The exons coding for the tyrosine kinase domain of pp60c-src were excluded. On the 5' side, 2 kb of sequence upstream from the previously defined exon 1 of the c-src gene was included in this mRNA. The start site for the 3-kb mRNA probably lies downstream of that for the 4-kb mRNA. The first reading frame of the 3.0-kb mRNA, called sur (for src upstream region), encoded a 24-kilodalton (kDa) protein product rich in cysteine and proline residues. In vitro analysis indicated that the 24-kDa sur protein was membrane associated. Antibodies to sur protein detected in vivo a 24-kDa muscle-specific protein which was developmentally regulated and corresponded to the switch from the 4-kb to the 3-kb c-src mRNA. A striking kinetic pattern of appearance of sur protein and disappearance of pp60c-src suggests that the expression of these two proteins is inversely related.     

Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.