OMP Decarboxylase Research Articles

Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5'-phosphate decarboxylase gene (URA3) and uracil auxotrophic mutants.

A transformation system for Hansenula fabianii J640, a commonly used wastewater treatment yeast, was constructed. As a host cell, a uracil auxotrophic mutant designated as H. fabianii J640 u-1, which was confirmed to have a mutation at the locus of the gene for orotidine-5'-phosphate (OMP) decarboxylase (URA3), was obtained by positive selection using 5-fluoroorotic acid. A plasmid named pHFura3, which includes a 795-bp open-reading frame of the OMP decarboxylase H. fabianii, was obtained by complementation of the Escherichia coli pyrF mutant, pHFura3 could transform H. fabianii J640 u-1 by a non-homologous and frequently multicopy integration into the host genomic DNA.

Low intracellular GTP level induces PKC-β-dependent cell differentiation in human myeloid leukemia cells

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.

Pyrimidine antagonists and antifolates as antimalarial drugs

The malarial parasite, Plasmodium falciparum, synthesises pyrimidines and folate de novo and is unable to synthesise pyrimidines via the alternative salvage pathway from preformed uridine or cytidine (Scheibel and Sherman, 1988). By contrast, purines are not synthesised de novo and must be salvaged from preformed purines such as adenosine which is readily available within the human erythrocyte (Szabados et al., 1996). Drug resistance has become a major problem for the treatment of malaria and many potential antagonists of nucleotide and folate biosynthesis have been tested for selective toxicity against the parasite. Because the parasite only has the de novo pyrimidine pathway and humans have both the de novo and salvage pathways, inhibitors of the de novo pyrimidine pathway could be effective antimalarial drugs. We have synthesised several potent inhibitors of the enzyme dihydroorotase (CA-asp→ DHO, eq 1; Christopherson et al, 1989), 6-L-thiodihydroorotate (TDHO) blocks de novo pyrimidine biosynthesis in parasites, inducing accumulation of CA-asp and depletion of UTP and CTP (Seymour et al., 1994). Atovaquone blocks the respiratory chain of malarial mitochondria at Complex III, leading to inhibition of dihydroorotate dehydrogena&e (DHO→ Oro, eq 1) and consequent accumulation of DHO and CA-asp (Seymour et al. 1994). Pyrazofurin as the nucleoside 5′-mo-nophosphate derivative, inhibits OMP decarboxylase (OMP→ UMP, eq 1) with accumulation of orotidine and Oro in cells (Seymour et al., 1994). 5-Fluoroorotate (FOro) is a potent inhibitor of the growth of P. falciparum and has an ID50 value of 42 nM while 5-fluorouracil (FUra) is far less toxic with an ID50 of 5.2 μM (Queen et al., 1990). FOro is converted to FUTP which may inhibit the malarial carbamyl phosphate synthetase (Seymour et al., 1994).

Perspectives for the chemotherapy of respiratory syncytial virus (RSV) infections

Respiratory syncytial virus (RSV) is the major respiratory pathogen in infants and young children. Ribavirin is the only antiviral agent approved for the treatment of RSV infections, but its efficacy has remained controversial. In the past few years several compounds have been described that in vitro exhibit marked activity against RSV at a 50% effective concentration that is significantly lower, and with a selectivity index that is significantly higher, than that of ribavirin. Among the most potent and selective RSV inhibitors are various polyanionic substances (polysulfates, polysulfonates and polyoxometalates), EICAR (an IMP dehydrogenase inhibitor), pyrazofurin (an OMP decarboxylase inhibitor) and cyclopentenylcytosine (Ce-Cyd, a CTP synthetase inhibitor). These compounds should be further explored for their therapeutic potential in the treatment of RSV infections, following systemic or, preferably, topical administration (i.e. as an aerosol), as topical application may better mimic the potency and selectivity exhibited in vitro by these compounds.

Use of the yellow fever virus vaccine strain 17D for the study of strategies for the treatment of yellow fever virus infections

We have employed the attenuated vaccine strain 17D of yellow fever virus (YFV) to evaluate the inhibitory effect of a selected series of compounds on YFV in Vero cells. Use of the vaccine strain does not require high-level microbiological containment facilities and should allow extensive screening. In addition, YFV may serve as a model for other flaviviruses including hepatitis C virus (HCV), and thus strategies for the treatment of YFV infections may apply to flavivirus infections in general. In the present study, several compounds belonging to different classes of nucleoside analogues and polyanions were evaluated for their inhibitory effect on the replication of YFV. Compounds that are targeted at: (i) IMP dehydrogenase (ribavirin, EICAR, tiazofurin, selenazofurin and mycophenolic acid), (ii) OMP decarboxylase (pyrazofurin and 6-azauridine), (iii) CTP synthetase (carbodine and cyclopentenyl cytosine), (iv) dihydrofolate reductase (methotrexate) and the (v) sulfated polymers (dextran sulfate and PAVAS) proved inhibitory to the replication of YFV. Mycophenolic acid (EC 50: 0.08 μg/ml), EICAR (EC 50: 0.8 μ/ml) and methotrexate (EC 50: 0.07 μg/ml) were the most effective. The finding that EICAR and mycophenolic acid, despite their potent anti-YFV activity, had little or no effect on the replication of the bunyavirus Punta Toro or herpes simplex virus in Vero cells, indicates that their anti-YFV activity is rather specific and does not merely result from cytotoxicity. Inhibitors of S-adenosylhomocysteine hydrolase (SAH hydrolase) and thymidylate synthase were found to be devoid of anti-YFV activity.

Inhibitors of infectious pancreatic necrosis virus (IPNV) replication

In attempts to detect inhibitors of infectious pancreatic necrosis virus (IPNV) replication, we have evaluated, by an IPNV plaque inhibition assay, a group of compounds that have broad spectrum antiviral activity for both single- and double-stranded RNA viruses. The inosine monophosphate dehydrogenase (IMP dehydrogenase) inhibitors 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR), and the orotidine monophosphate decarboxylase (OMP decarboxylase) inhibitor 4-hydroxy-3-β-D-ribofuranosylpyrazole-5-carboxamide (pyrazofurin), were found to inhibit IPNV replication. For EICAR and pyrazofurin the concentrations that inhibited the IPNV plaque formation by 50% (EC 50) were 0.01 μg/ml and 0.5 μg/ml, respectively. The cytotoxic concentrations required to reduce cell viability by 50% (CC 50) were 50 μg/ml and 100 μg/ml, respectively, and the concentrations that reduced [ methyl- 3H] thymidine incorporation by 50% (IC 50) were 0.5–1 and 50 μg/ml. Thus, for both compounds the IPNV-inhibitory concentration was 50–100 times lower than the concentration that affected DNA synthesis in growing cells. EICAR and pyrazofurin seem to be good candidates for further evaluation in an in vivo model of IPNV infection.

A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase.

Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. Alpha-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5'-monophosphate (OMP) in the presence of divalent cations, preferably Mg2+. OMP is thereafter converted to uridine 5'-monophosphate by OMP decarboxylase. We have determined the 2.4 angstrom structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer. We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase.OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis. Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S. typhimurium OPRTase.OMP and E. coli OPRTase are found in the substrate binding regions: the 5'-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73).

A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5′-monophosphate decarboxylase

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5′-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lpl), which grows as an endophyte in perennial ryegrass ( Lolium perenne). Three positive clones, λMC11, λMC12 and λMC14, were isolated. Two of these clones, λMC12 and λMC14, were overlapping clones from the same locus, while λMCll was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from λMC12 and λMC14 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5′ non-coding sequences were the least conserved with 88.5% identity and the 3′ non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lpl demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lpl showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307–1317] that the two pyr4 in Lpl arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5′ non-coding regions of pyr4, from possible ancestors to Lpl supports this hypothesis. Comparisons of these sequences to the 5′ non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lpl.

Sequence of the pyr4 gene encoding orotidine-5′-phosphate decarboxylase from the biocontrol fungus Trichoderma harzianum

The pyr4 gene, encoding orotidine-5′-phosphate decarboxylase (OMP decarboxylase) from the biocontrol fungus Trichoderma harzianum, has been isolated by hybridization, using a polymerase chain reaction (PCR)-derived fragment as a probe. The PCR primers corresponded to conserved regions of OMP decarboxylase-encoding genes from other filamentous fungi. A 2490-bp genomic fragment, which complemented a pyr4-auxotrophic T. reesei mutant, was sequenced. The gene showed high homology to pyr4 genes from other pyrenomycetes.

Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.

The uraA locus and homologous recombination in Mycobacterium bovis BCG

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity. The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines.

The effects of boron containing peptides on L1210 lymphoid leukemia metabolism

The purpose of this study was to establish the efficacy and mode of action of peptide boron derivatives as antineoplastic agents and to evaluate their safety in vivo. Boron-containing phenylalanine and tyrosine methyl esters were found to be potent cytotoxic agents in a number of murine and human cancer cell lines. DNA, RNA and protein syntheses were inhibited by selected agents, e.g. [(trimethylamine boryl)carbonyl]-phenylalanine-acetyl ester (9) andN-acetyl-p-boron-phenyl-alanyl-phenlalanine-methyl ester (10), in L1210 lymphoid leukemia cells. IMP dehydrogenase, OMP decarboxylase, m-RNA, t-RNA, r-RNA polymerase and ribonucleoside reductase activities were inhibited. d(CTP) levels were reduced. DNA strand scission occurred after 24 hr incubation. Acute toxicity studies in mice demonstrated that the key derivative was safe at therapeutic levels with no effects on histology of major organs, hematopoietic parameters and clinical values.

Molecular approaches for the treatment of hemorrhagic fever virus infections

Viruses causing hemorrhagic fevers in man belong to the following virus groups: togavirus (Chikungunya), flavivirus (dengue, yellow fever, Kyasanur Forest disease, Omsk hemorrhagic fever), arenavirus (Argentinian hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever), filovirus (Ebola, Marburg), phlebovirus (Rift Valley fever), nairovirus (Crimian-Congo hemorrhagic fever) and hantavirus (hemorrhagic fever with renal syndrome, nephropathic epidemia). Hemorrhagic fever virus infections can be approached by different therapeutic strategies: (i) vaccination; (ii) administration of high-titered antibodies; and (iii) treatment with antiviral drugs. Depending on the molecular target of their interaction, antiviral agents could be classified as follows: IMP dehydrogenase inhibitors (i.e., ribavirin and its derivatives); OMP decarboxylase inhibitors (i.e., pyrazofurin); CTP synthetase inhibitors (i.e., cyclopentylcytosine and cyclopentenylcytosine); SAH hydrolase inhibitors (i.e., neplanocin A); polyanionic substances (i.e., sulfated polymers); interferon and immunomodulators.

Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria

Orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains using polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC-deficient E. coli, the strains were able to grow on minimal media without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4-9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.

The orotidine-5'-monophosphate decarboxylase gene of Myxococcus xanthus. Comparison to the OMP decarboxylase gene family.

The nucleotide sequence of the Myxococcus xanthus orotidine-5'-monophosphate decarboxylase (OMP DCase) gene was determined. The derived protein sequence is not closely related to other prokaryotic OMP DCase sequences; nor is it closely related to any eukaryotic OMP DCase sequences. Progressive multiple alignment of the M. xanthus OMP DCase protein sequence with 19 other OMP DCase sequences revealed four conserved regions present in all 20 sequences. Ten entirely conserved residues were found in these four regions and one region contains a tight cluster of 5 conserved residues, certain of which may be catalytically active residues. A second open reading frame was found upstream of uraA and oriented in the same direction as uraA. A stretch of 21 consecutive pyrimidine (C or T) residues were found in the intercistronic region between the potential ribosome-binding site of uraA and the UGA stop codon of the upstream open reading frame. RNA directly upstream of the pyrimidine run, including the UGA stop codon of the upstream open reading frame, could be folded into a stable hairpin structure resembling Rho-independent terminators of Escherichia coli. Expression of the uraA gene may be regulated by an intercistronic transcription termination mechanism.

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.

Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP synthetase

Cyclopentenylcytosine (Ce-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [herpes (cytomegalo), pox (vaccinia)], (+)RNA viruses [picorna (polio, Coxsackie, rhino), toga (Sindbis, Semliki forest), corona], (−)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), arena (Junin, Tacaribe), rhabdo (vesicular stomatitis)] and (±)RNA viruses (reo). Ce-Cyd is a more potent antiviral agent than its saturated counterpart, cyclopentylcytosine (carbodine, C-Cyd). Ce-Cyd also has potent cytocidal activity against a number of tumor cell lines. The putative target enzyme for both the antiviral and antitumor action of Ce-Cyd is assumed to be the CTP synthetase that converts UTP to CTP. In keeping with this hypothesis was the finding that the antiviral and cytocidal effects of Ce-Cyd are readily reversed by Cyd and, to a lesser extent, Urd, but not by other nucleosides such as dThd or dCyd. In contrast, pyrazofurin and 6-azauridine, two nucleoside analogues that are assumed to interfere with OMP decarboxylase, another enzyme involved in the biosynthesis of pyrimidine ribonucleotides, potentiate the cytocidal activity of Ce-Cyd. Ce-Cyd should be further pursued, as such and in combination with OMP decarboxylase inhibitors, for its therapeutic potential in the treatment of both viral and neoplastic diseases.

Tranformation of Neurospora pyr-4 with defective donor genes

Using the Vollmer/Orbach transformation protocol, transformation frequencies of a pyr-4 (OMP decarboxylase) strain of Neurospora crassa of circa 10(3)/µg are routinely achievable. At these levels of transformation, it is feasible to screen out ectopic integrations and look specifically for homologous integration events. Homologous integrants were sought by transforming a pyr-4 recipient with interrupted or incomplete copies of the cloned pyr-4 gene derived from the pyr+ clone in plasmid pFB6, selecting by complementation of the pyrimidine auxotrophy in the recipient

Molecular cloning of human UMP synthase.

The last two steps of de novo synthesis of UMP are catalyzed by the sequential actions of orotate phosphoribosyltransferase (orotate PRTase; pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (OMP decarboxylase; orotidine 5′-phosphate carboxy-lyase; EC 4.1.1.23)