Sequence analysis of the DdPYR5-6gene coding for UMP synthase in Dictyostelium discoideum and comparison with orotate phosphoribosyl transferases and OMP decarboxylases

Abstract

A Dictyostelium discoideum DNA fragment that complements the ura3 and the ura5 mutants of Saccharomyces cerevisiae has been sequenced. It contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. This gene, named DdPYR5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (OPRTase) and the orotidine-5'-phosphate decarboxylase (OMPdecase) domains described for UMP synthase in mammals. The existence of separate domains for the two activities was suspected because deletion of the N-terminal coding segment of the gene eliminated the ura5 but not the ura3 complementing activity. We have now confirmed that the two parts of the open reading frame share homology with known OPRTase and OMPdecase sequences. Several blocks of sequence are conserved among OPRTase from bacteria, fungi and slime mold and one of them corresponds to the consensus sequence for phosphoribosylbinding sites. The OMPdecase domain shows extensive similarity with the yeast and Neurospora crassa enzymes, suggesting that they have evolved from an ancestral gene which was fused to the OPRTase gene in D. discoideum. It is less related to the bacterial enzyme but all these sequences present conserved blocks of homology which could identify the active site. The codon usage is strongly biased in a manner similar to that found for other D. discoideum genes. The flanking DNA contains homopolymers of A and T and alternating sequences that are characteristic of the gene organization in D. discoideum.

Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

“…The cloning strategy used to acquire the gene for gp130 and details regarding its genomic sequence are described separately (Supplementary Data). The UMP synthase gene cassette had been released by digestion with ClaI from a gene replacement vector provided by Dr. M. Fechheimer (University of Georgia-Athens; Rivero et al, 1996) and was originally derived from plasmid pJB1 (Jacquet et al, 1988). The disrupted gp130 sequence was cut out of the pCR2.1 vector with sequential digestions using NotI and BamHI, and used to transform DH1 cells by electroporation (Knecht and Pang, 1995).…”

“…DH1 cells, auxotrophic for uracil (Caterina et al, 1994), were transformed with a construct where the UMP synthase gene cassette (Jacquet et al, 1988) was inserted into the genomic sequence of gp130 ( Figure 3A). Transformed cells were selected by their ability to grow in FM without uracil.…”

Orotate Phosphoribosyltransferase fromCorynebacterium ammoniagenesLacking a Conserved Lysine

“…Thus, both wild-type and pyrE-deficient strains can be positively selected (37). Because of these key roles in nucleotide metabolism and the ubiquitous distribution, the pyrE genes from many organisms have been cloned and sequenced (7,9,13,14,(22)(23)(24)33), and more than 13 homologous enzymes that carry out the OMP formation reaction have been identified in bacteria, fungi, insects, and mammals (26,29).…”

Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction

“…In some lower eukaryotes such as Dictystelium discoicleiim and in higher eukaryotes like mammals (Traut and Jones, 1911), Drosophilci (Rawls, 1978) and plants (Waltheret al, 1984;Santoso, 1995;Maier et al, 1995), this single enzyme has both orotate phosphoribosyl transferase (OPRTase) and OMP decarboxylase (ODCase). UMP synthase genes or cDNAs have been isolated from some eukaryotes including D. discoideum (Boy-Marcott and Jacquet, 1982;Jacquet et al, 1988), Arabidopsis thaliana (Nasr et al, 1994) and N. tabaciim (Maier et al, 1995). The non-toxic antimetabolite 5-fIuoroorotic acid (5F0A) can be utilized by UMP synthase to form toxic 5-fluoro-UMP (FUMP).…”

“…However, in higher eukaryotes, these two enzymatic steps have become fused into a single bifunctional protein that catalyzes the final two steps of this pathway. This bifunctional protein, termed UMP synthase, is characteristic of higher eukaryotes, including Dictyosteliiim discoideum (Jacquet et al, 1988), insects (Eisenberg et al, 1990), vertebrates (Suttle et al, 1988 and plants (Maier et al, 1995;Nasr et al, 1994).…”