Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Nitrogen metabolite repression of fluoropyrimidine resistance and pyrimidine uptake in Neurospora crassa

Summary Wild type Neurospora crassa was shown to be more resistant to 5-fluoro-uracil, 5-fluoro-uridine and 5-fluoro-2-deoxyuridine in the presence of ammonia than in its absence. This differential resistance may in part be accounted for by the observation that both uracil and uridine uptake in germinating conidia is under nitrogen metabolite regulation. The uptake of uracil and uridine is increased on poor nitrogen sources in wild type, is unaffected by nitrogen source in a nit-2 mutant strain while a gln-1 mutant strain showed intermediate behaviour.Wild type also showed increased resistance to all three fluoropyrimidines with increased temperature and both uracil and uridine uptake in the wild type increased with temperature.Supported by S.R.C. grant GR/A/64655F.P. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

A protonmotive force as the source of energy for galactoside transport in energy depletedEscherichia Coli

Summary Uracil transport inSaccharomyces cerevisiae is mediated by a specific permease which does not recognize other pyrimidines such as uridine, cytosine, thymine, 2-hydroxypyrimidine or 5-amino-uracil; hypoxanthine and 6-amino-uracil slightly inhibit the uptake of uracil in a strain lacking cytosine permease activity. Wild type cells concentrate extracellular uracil before its transformation into UMP and subsequent incorporation into nucleic acids. A strain lacking UMP pyrophosphorylase and uridine ribohydrolase (strainfur 1–8 rh, in which the endogenous production as well as the utilization of uracil are lacking) is able to concentrate¹⁴C-2 uracil from the medium. At the same time no other¹⁴C-2 labelled compound could be detected in this strain, thus suggesting that the uptake of uracil in yeast occurs by active transport which is not coupled to the UMP pyrophosphorylase. The optimal pH of uracil uptake in standard growth conditions was 4.3. It was deduced from experiments performed on strainfur 1–8 rh with³H-5 and¹⁴C-2 uracil that the intracellular pool of uracil is recycled once the steady-state has been reached. First order kinetics with similar rate constants were observed for uracil efflux in strainfur 1–8 rh (k min^–1=0.75±0.08) as well as in the strain lacking uracil permease,fur 1–8 rh fur 4–6 (k min^–1=0.60±0.08). The intracellular pool of¹⁴C-2 uracil can be chased in strainfur 1–8 rh by addition of³H uracil without inducing a large initial acceleration of the exit rate (the rate constant remained at 0.60). 2-4-dinitrophenol inhibits the uptake of uracil but also reduces the efflux of uracil in strainfur 1–8 rh fur 4–6. These data and the comparison with cytosine transport in the same organism support the hypothesis that, whereas uracil uptake is a permease mediated active transport, the efflux of uracil does not involve the uracil uptake permease. A coefficient of permeability of 7.4×10^–7 cm sec^–1 was calculated for uracil.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Elevated uridine nucleotide pools in fluorouracil/fluorouridine resistant mutants ofNocardia lactamdurans

Summary Selection of spontaneous mutants ofNocardia lactamdurans MA2908 for resistance to 5-fluorouracil results in the simultaneous development of resistance to 5-fluorouridine. The resulting mutants fall into four distinct classes based on the amount of uracil accumulating in fermentation broths. An additional characteristic of these mutants is a reduction in the ability to incorporate exogenous uracil into nucleic acids even though transport and conversion to the nucleotide level appears normal. Finally, production of efrotomycin is increased in these mutants in both chemically defined and complex fermentation media to levels equivalent to those of MA4820, the first productivity mutant isolated in a conventional strain improvement program. Resistance development and uracil excretion are adequately explained by an elevation of the intracellular uridine nucleotide pool, in particular UMP. The role of the uridine necleotides in the efrotomycin fermentation is unknown.     

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogenCandida albicans

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeastCandida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg⁺⁺ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. Ping pong mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (K _m=70.5 and 186 µM for uracil and PRPP, respectively) as compared with those ofE. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.Abbreviations UPRTase Uracil phosphoribosyltranferase - PRTases phosphoribosyltransferases - PRPP 5-phosphoribosyl-1-pyrophosphate - 5-FC 5-fluorocytosine - 5-FU 5-fluorouracil - PEI polyethyleneimine - DTT dithiothreitol - DMSO dimethyl sulphoxide - PMSF phenylmethylsulphonyl fluoride - UMP uridine mono-phosphate     

UMP synthesis in the kinetoplastida

All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular form of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmi, whereas orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

The Dhod locus of Drosophila: mutations and interrelationships with other loci controlling de novo pyrimidine biosynthesis

Summary Mutations at the Dhod locus have been isolated following ethylmethanesulfonate mutagenesis. These mutants express those phenotypes common to other mutations of the de novo pyrimidine pathway: specific wing and leg defects and female sterility. Dihydroorotate dehydrogenase activity is severely reduced in all Dhod mutants, whereas levels of the other pathway enzymes are largely unaffected. The twelve Dhod mutations described here comprise a single complementation group. All of these mutations are nonlethal and the collection includes apparent amorphic as well as hypomorphic alleles. These results are discussed relative to the properties of the complex loci that encode the other steps of de novo pyrimidine biosynthesis.Abbreviations DHOase dihydroorotase (EC 3.5.2.3.) - DHOdehase dihydroorotate dehydrogenase (EC 1.3.3.1.) - EMS ethylmethanesulfonate - ODCase orotidylate decarboxylase (EC 4.1.1.23) - OPRTase orotate phosphoribosyltransferase (EC 2.4.2.10) - UMP uridine 5-monophosphate     

Errata

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterized the transport mechanism of uracil and uridine. Mutant U^?, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR^?, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U^?UR^?isolated from a culture of mutant U^?, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR^?R^? was isolated from a culture of mutant UR^?, and is resistant to high concentration of showdomycin. This mutant is defective for transport of intact uridine and in addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U^? and UR^?, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these process.     

Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine.

We have previously described a mutant of Toxoplasma gondii that was 100-fold more resistant to 5-fluorodeoxyuridine, as measured by growth in human fibroblast cultures. Various pyrimidine salvage enzymes were measured in the wild type and the mutant parasites to determine the biochemical basis for resistance to fluorodeoxyuridine. Both the resistant mutant and the wild type parasite had little or no uridine kinase, an enzyme readily detectable in the human fibroblast host cells. Uridine and deoxyuridine phosphorylases were found in both parasites while human fibroblasts had much less of these enzymes. The critical difference between the mutant and the wild type parasites proved to be a 100-fold lower concentration of uracil phosphoribosyltransferase in the fluorodeoxyuridine-resistant mutant. A back mutant of the resistant strain, selected for its ability to use uracil, simultaneously regained uracil phosphoribosyltransferase and sensitivity to fluorodeoxyuridine. This enzymic evidence together with previously published data show that in wild type T. gondii, deoxyuridine is incorporated into nucleic acids through a phosphorolysis to produce uracil which is then converted to uridylic acid by uracil phosphoribosyltransferase.     

Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization

Summary Strains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.Abbreviations and Symbols FU 5-fluorouracil - FUR 5-fluorouridine - FUdR 5-fluoro-2'deoxyuridine - FCR 5-fluorocytidine - FCdR 5-fluorodeoxycytidine - THUR 3, 4, 5, 6-tetrahydrouridine - UMP uridine monophosphate - CMP cytidine monophosphate - dUMP deoxyuridine monophosphate. Genes coding for: cytidine deaminase - edd uridine phosphorylase - udp thymidine phosphorylase - tpp purmnucleoside phosphorylase - pup uridine kinase (=cytidine kinase) - udk UMP-pyrophosphorylase - upp. CytR regulatory gene for cdd, udp, dra, tpp, drm and pup Enzymes EC 2.4.2.1 Purine nucleoside phosphorylase or purine nucleoside: orthophosphate (deoxy)-ribosyltransferase - EC 2.4.2.4 thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - EC 2.4.2.3 uridine phosphorylase or uridine: orthophosphate ribosyltransferase - EC 3.5.4.5 cytidine deaminase or (deoxy)cytidine aminohydrolase - EC 4.1.2.4 deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - EC 2.4.2.9 UMP-pyrophosphorylase or UMP: pyrophosphate phosphoribosyltransferase - EC 2.7.1.48 uridine kinase or ATP: uridine 5-phosphotransferase     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

The derepression of enzymes of de novo pyrimidine biosynthesis pathway in Brevibacterium ammoniagenes producing uridine-5-monophosphate and uracil

A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyltransferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.     

Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants.

Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.