Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid

Summary In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase^– mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura ^– phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.     

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles.     

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M.?circinelloides OPRTase with those of E. coli and S.?typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid     

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl₂ for maximum activity but was inhibited by MnCl₂; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K_m values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 μm and 11 μm; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5′-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5′-phosphate and 5-fluorouridine 5′-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.     

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells

The de novo biosynthesis of pyrimidine nucleotides is completed by two sequential enzyme activities that convert orotate plus 5-phosphoribosyl-1-pyrophosphate to orotidine-5′-monophosphate (OMP) and PP_i and then decarboxylate OMP to produce 5′-uridylic acid. In mammalian cells the two enzyme activities, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, form a normally inseparable enzyme complex. It was previously reported that this complex is able to channel the intermediate product, OMP (Traut, T. W., and Jones, M. E., 1977, J. Biol. Chem.252, 8374–8381). The studies reported here indicate that one advantage of this channeling of OMP is to spare OMP from being degraded to orotidine by a potentially competitive nucleotidase activity. Yeast cells have two separate enzymes instead of an enzyme complex, and lack the ability to channel OMP. The OMP formed in yeast cells is not degraded because these cells lack significant nucleotidase activity. These results suggest that the capability for channeling OMP may have been important in evolving the enzyme complex found in mammalian cells.     

Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

6-azauracil-resistant variants of cultured plant cells lack uracil phosphoribosyltransferase activity

6-Azauracil-resistant variants of Haplopappus gracilis (Nutt.) Gray and Datura innoxia Mill. lack activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme that catalyzes the conversion of uracil and 6-azauracil to uridine-5′-monophosphate and 6-azauridine-5′-monophosphate, respectively. Resistant cells are competent to take up uracil from their growth medium but do not convert it into a form that can be used for macromolecular synthesis. In extracts from resistant cells, orotate monophosphate decarboxylase, a target enzyme of 6-azauridine monophosphate, is fully sensitive to the phosphorylated analog. These results strongly suggest that uracil phosphoribosyltransferase is the major pathway of pyrimidine salvage in cells of these species and that loss of this enzyme activity confers on the variants resistance to 6-azauracil.     

Development of Pyrimidine-metabolizing Enzymes in Cotyledons of Germinating Peas

Mechanisms controlling conversion of orotic acid-6-¹⁴C to uridine-5′-phosphate in cotyledons of germinating Alaska peas (Pisum sativum L.) were investigated. The content of 5-phosphoribosyl-1-pyrophosphate was very low in dry seeds, increased to a maximum after about 12 hours of imbibition, and then rapidly declined. Orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase activities more than doubled during the first 24 hours of germination and then also decreased. These results do not account for the continuous increases of orotate anabolism in such cotyledons as we observed previously. The initial increases in activities of these two enzymes were unaffected by cycloheximide, while the subsequent decreases were less rapid in the presence of this inhibitor. Activities of cotyledonary cytidine deaminase and uridine hydrolase also increased during imbibition, but the activity of only the latter showed a decrease after imbibition was completed. Cycloheximide inhibited the initial rapid increase in uridine hydrolase activity but had little effect on its subsequent decline. Cycloheximide had only slight inhibitory effects on the development of cytidine deaminase activity during the first 62 hours. The evidence suggests that uridine hydrolase might be synthesized de novo during the first few days of germination, but that the other three enzymes might not be.     

Increased levels of UMP synthase protein and mRNA in pyrazofurin-resistant rat hepatoma cells

Rat hepatoma cells that have undergone stepwise selection in increasing concentrations of pyrazofurin have coordinately increased levels of both orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23) activity. These two activities catalyze the conversion of orotic acid to UMP in de novo pyrimidine biosynthesis. Cells selected in 50 microM pyrazofurin have over 40 times the wild type level for both activities. A single polypeptide of approximately 55,000 daltons is increased in the resistant cells in amounts corresponding to the increase in the two activities. Resistant cell lines that are grown for extended periods in the absence of pyrazofurin are unstable, losing their elevated levels of both enzyme activities and the increased specific protein. Antibody prepared against a purified protein containing both enzyme activities binds specifically to this increased protein. These results corroborate other evidence indicating the two enzyme activities are contained within a single polypeptide called UMP synthase. Poly(A+) mRNA isolated from wild type and resistant lines was analyzed by in vitro translation for production of UMP synthase protein. Immunoprecipitation of the translation products shows the resistant cells have a 17-fold increase in translatable mRNA activity coding for UMP synthase. The synthase accounts for 0.24% of the total in vitro translation products synthesized with poly(A+) mRNA from the pyrazofurin-resistant cells as opposed to 0.014% with wild type mRNA. A cloned UMP synthase cDNA sequence hybridizes strongly to a 1.8-kilobase mRNA in the resistant cells. This mRNA is only barely detectable in equivalent preparations from wild type cells. Quantitation of the mRNA by dot hybridization techniques indicates a 16-fold increase in UMP synthase mRNA in the resistant cells. Although this increase in mRNA for UMP synthase could explain most of the increased protein, it is not sufficient to totally account for the 40-fold increase in UMP synthase.     

Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase

The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.     

Potential for ambiguity in the interpretation of immunochemical studies on multienzymic proteins. The case of uridylate synthase

Rabbit antibodies directed against homogeneous uridylate synthase multienzyme from mouse Ehrlich ascites carcinoma precipitate both the orotidine-5'-monophosphate decarboxylase (EC 4.1.1.23) and orotate phosphoribosyltransferase (EC 2.4.2.10) activities of mouse and human erythrocyte uridylate synthase. When the partially purified human enzyme is used as antigen the two activities coprecipitate with the same apparent titer; however, when the mouse carcinoma protein was studied under the same conditions the decarboxylase activity immunoprecipitated with significantly higher avidity than did the transferase activity. Since the mouse multienzyme has been shown to be a single polypeptide that contains both activities (McClard, R.W., Black, M.J., Livingstone, L.R. and Jones, M.E. (1980) Biochemistry 19, 4699-4706), these results were, at face value, surprising. However, when the mouse orotate phosphoribosyltransferase activity (which is largely lost upon dilution into the immunoassay medium) was stabilized with 5-phosphoribosyl 1-pyrophosphate, both enzyme activities displayed the same apparent antibody titer. The immunochemical studies indicate that the antibodies, as a population, preferentially bind to a form or forms of the enzyme which contain(s) denatured transferase domains. A calculation based on a simple model yields a value of approximately 100 for the relative selectivity of the antibody for the denatured form of uridylate synthase. These results illustrate an ambiguity that is inherent in the interpretation of immunochemical studies on such multienzymic proteins; that is, it is possible to conclude incorrectly that two enzyme activities are not functionally associated if one of the catalytic domains is particularly unstable and thereby displays greater immunoreactivity for the specific antiserum.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles. Received: 29 March 1999 / Accepted: 25 May 1999     

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

Summary 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.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998