JOURNAL OF BACTERIOLOGY, Dec. 1969, p. 1371-1377Copyright 0 1969 American Society for Microbiology

Vol. 100, No. 3Printed in U.S.A.

Pyrimidine Synthesis in Neurospora crassa:

Gene-Enzyme RelationshipsDINA F. CAROLINE'

Departmentt of Botaniy, University of Michigan, Ann Arbor, Michigani 48104

Received for publication 15 May 1969

A new series of pyrimidine-requiring mutants of Neurospora has been isolated andall enzymes involved in pyrimidine biosynthesis are represented by at least one

mutant. Among these mutants is included a single isolate for a new locus, pyr-6.This mutant is deficient in dihydroorotase (DHOase) and represents the only enzy-

matic step in orotate synthesis for which no mutant previously had been found.This mutant, which mapped genetically on the right arm of linkage group V, isunlinked to any of the other pyrimidine mutants. The DHOase-deficient mutant isalso characterized by an unexpected growth behavior. The pyr-l locus has beenspecifically associated with a lack of dihydroorotate dehydrogenase (DHOdehase).Mutants isolated in this series for other pyrimidine loci have been related to previ-ously isolated mutants by allelism, recombination, and accumulation studies.

Pyrimidine biosynthesis was studied exten-sively and was shown to follow the same sequencein all organisms and tissue systems analyzed,ranging from bacteria to mammals (8, 9, 26, 27)(Fig. 1). The first step is the formation of car-bamyl phosphate (CAP), which is also a pre-cursor for arginine biosynthesis (10). In Neuro-spora, there are two pathway-specific CAP poolswhich are independently synthesized in normalcells (3). This channeling of CAP occurs to alesser extent in yeast and does not appear to existin Escherichia coli (4). The pyrimidine-specific,glutamine-dependent carbamyl phosphate syn-thetase (G-CPSpyr) was characterized in Neuro-spora (Williams and Davis, 1968. Abstr. Genet.Soc. Amer.). An arginine-specific, glutamine-dependent CPS was also characterized (4). Thesequence of uridylate (UMP) formation in Neu-rospora is shown in Fig. 1.Although many pyrimidine-requiring mutants

were isolated in Neurospora, there were severalgaps in the gene-enzyme relationships in thispathway. In the present study, mutants for all thesteps of UMP synthesis were isolated, mutationswere correlated with enzymatic deficiencies fordihydroorotase (DHOase) and dihydroorotatedehydrogenase (DHOdehase), and previouslydetermined relationships were confirmed. Theisolation of a complete set of mutations in a singlegenetic background provides favorable materialfor studying other problems. Regulation of the

1 Present address: Genetics Foundation, University of Texas,Austin. Tex. 78712.

pyrimidine enzymes is one such study and is pre-sented in the accompanying paper (1).

MATERIALS AND METHODSStrains. Wild-type Neurospora crassa strain 74A

was used as the normal standard strain for all enzymeassays and for the selection of uridine-requiring mu-tants. The mutants were then reisolated by crossingto wild-type strain 73a. Various pyrimidine mutantswhich are available from the Fungal Genetics StockCenter, Dartmouth College, were used. These includepyr-3d (45502), pyr-l (H263), pyr-2 (37709), andpyr-4 (36601). "Alcoy," a multiple-translocationstrain with visible markers (17), was used for thepreliminary genetic analysis of pyr-6.

Genetic techniques. Crosses were made on Difcocorn meal agar, supplemented, if necessary, with0.2% sucrose and 500 ,g of uridine per ml. All growthmedia utilized Vogel's minimal salts (23), and solidmedia contained 1.5% agar (Difco). This was supple-mented with 1% sorbose, 0.05% glucose, and 0.05%fructose for spore plating; 0.8% sorbose and 0.4%sucrose for spot-testing strains on supplements;and 1.5% sucrose for maintenance of stock cultures.Uridine (100 jAg/mI) was used as a supplement whennecessary.

Mutagenesis and mutant selection. N-methyl-N'-nitro-N-nitrosoguanidine (0.1 mg/ml, Aldrich Chem-ical Co.) in 0.1 M sodium citrate-citric acid buffer(pH 5.0) was combined with an equal volume ofconcentrated wild-type (strain 74A) conidia andincubated for 60 min at room temperature. Theconidia were then washed, suspended in 100 ml ofminimal medium, and put on a reciprocal shaker. Wild-type conidia were eliminated by the filtration-concen-tration method of Woodward et al. (25). The conidia

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CAROLINE

Fpr-3MNX

glutamine G-CP5p, CtII

Co ATCes4 CHI pyr-6Co3 P0-CAPACose I HOe CHt DHOdegse

(jHNHCNH IV b I IH

CH2

wreldosuccinote dihydrooroteteCHNHg (US) (OHO)coo-

Aspertete

J. BACrERIOL.

0

IX04 ',coo-

H

IO)

,yr- 2 zC pyr-4 C

OMPPose HN H OMP decase HNIi 'H

PRPP PPi \-"N "COO COt 0' ,NpoDPo1HuIOsPICt|0WOH

orotidylote eridylite(OMP) lUMP)

FIG. 1. Pyrimidine biosynthesis in Neurospora with enzymes and metabolic positions ofgenes shown.

remaining after 2 to 3 days were then plated on petridishes containing 0.8% sorbose, 0.4% sucrose and100,g of uridine per ml. Individual colonies were

isolated in tubes (10 by 75 mm) supplemented withuridine, and the isolates were later tested for uridinedependence.

Complementation. Uridine-requiring mutants wereinitially assigned to complementation groups bypairwise heterokaryon tests in which the ability ofmutants to form uridine-independent heterokaryonswas tested. Conidia of two mutant strains wereinoculated into tubes (10 by 75 mm) containing 2 mlof liquid minimal medium and incubated at 30 C.Complementation was recorded from 2 to 6 daysafter inoculation.

Growth, extraction of mycelia and protein deter-mination. Mycelia were grown from 5-day-old conidiafor 17 to 24 hr in 700 ml of Vogel's medium. Therate of growth was observed by measuring the opticaldensity of the culture with a no. 54 filter in a Klett-Summerson colorimeter, or the dry weights weredetermined by drying a sample of the culture withreagent-grade acetone. For enzyme determinations,acetone-dried pads were extracted with ground glasshomogenizers or were first ground to a powder in aSorvall Omnimixer in cold acetone, followed bydrying on a Buchner funnel. Protein was measuredby the biuret method (13). Specific activities ofenzymes are given as micromoles of product per mil-ligram of protein per hour.

Assay ofATCase (carbamoylphosphate: L-aspartatecarbamoyl-transferase E.C. 2.1.3.2). Acetone padsor powders were extracted (5 ml/100 mg of mycelium)in 0.02 M sodium citrate buffer, pH 6.0, which con-tained 5% Triton X-100 (Rohm and Haas Co.).The homogenate was centrifuged at 25,000 X g

for 20 min, and the supernatant extract was assayedby the method of Davis (3). Ureidosuccinate (US)formed was determined by the method of Koritzand Cohen (11) or by the method of Gerhart andPardee (7). The use of citrate, which does not influencethe ATCase reaction, made it possible to assay DHO-

ase and ATCase in the same extract. Triton X-100also has no effect on the ATCase activity.

Assay of DHOase (L-4,5-dihydro-orotate amido-hydrolase, E.C. 3.5.2.3). The extract was preparedas described for ATCase. Reaction mixtures con-tained 7 ,umoles of dihydroorotate (DHO), 100 jmolesof tris(hydroxymethyl)aminomethane (Tris)-hydro-chloride (pH 8.0) extract, and water to a final volumeof 1 ml. Assay tubes were incubated at 37 C for 15min; the reaction was stopped with 1 ml of 12%HClO4, and precipitated protein was removed bycentrifugation. Time-zero control tubes were pre-pared in which acid was added prior to substrate.The US formed was measured by the method ofGerhart and Pardee (7).

Neurospora DHOase, like that of other organisms,is extremely unstable (12). In this study, consistentactivities were obtained by extracting acetone padswith citrate buffer at pH 6.0. Triton X-100 led tosome loss of activity but was included in the extractionto permit measurement of ATCase in the same ex-

tract. For maximal activity, the enzyme has to beassayed in crude, freshly prepared extracts.

Within the limits used here, enzyme activity waslinear with time and enzyme concentration. Boiledextract gave no activity. The optimal pH is 9.0.DHO was used at a concentration of 7 mm in theassay; this concentration saturates the enzyme, whilecausing minimal interference with the color reaction.

Assay of DHOdehase (L4,5-dihydro-orotate:oxygen oxidoreductase, E.C. 1.3.3.1). Acetone pads(100 mg) were extracted with a ground glass homog-enizer in 5 ml of 0.02 M potassium phosphate buffer(pH 7.0). The homogenate was centrifuged for 20 minat 25,000 X g. The supematant fluid was discarded,and the pellet was reextracted in the same amount ofbuffer containing 5% Triton X-100. This extract wasthen centrifuged for 20 min at 25,000 X g, andthe resulting supernatant fluid was assayed for DHO-dehase. The reaction mixture, modified from Taylorand Taylor (21), contains 5 umoles of DHO, 300jumoles of Tris-hydrochloride (pH 8.0), 3 jumoles of

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PYRIMIDINE SYNTHESIS IN N. CRASSA

potassium ferricyanide, and 1.5 mg of protein in atotal volume of 3 ml. Tubes were incubated at 25 Cfor 30 min, and the reaction was stopped with 0.5ml of 30% HClO4. The tubes were heated at 60 Cfor 5 min to induce full precipitation of protein andwere then centrifuged. Time-zero controls were usedin which HClO4 was added prior to substrate. Thereduction of ferricyanide, representing oxidation ofDHO to orotate (OA), was measured using the no.42 filter (maximum transmission 420 nm). A changeof 73 Klett units represents reduction of 1 /Amole offerricyanide in the assay.The assay was approximately linear with time and

enzyme concentration. The optimal pH was foundto be about 8.5. The first supernatant fluid obtainedafter extracting the mycelium in buffer has no DHO-dehase activity and must be discarded because of aninterfering, substrate-independent reaction. Eighty-seven percent of the total activity measured in thepellet after the first centrifugation is released into thesecond supernatant fluid by the Triton X-100 treat-ment. A small amount of the remaining activity maybe removed by a second extraction.

Isolation of accumulated intermediates. For measure-rment of accumulated intermediates, mutant myceliawere grown in shake flasks with a growth-limitingamount of uridine (20 gg/ml). Measurement andidentification of US and DHO were carried out byextracting wet mycelial pads (equivalent to 100 mg,dry weight) of a culture in stationary phase in 2 mlof 6% HClO4. After low-speed centrifugation, theresidues were washed twice with 2 ml of water, andthe combined extracts were passed thiough a shortDowex-50 column (4 by 1.1 cm) and washed twicewith 2 ml of water (6). The HClO4 was neutralizedwith KOH. The extract, after removal of KC1O4, wasapplied to a Dowex-1 formate column (7 by 1.1 cm,200 to 400 mesh, 8% crosslinkage) and eluted with0.055 M Na+ formate buffer adjusted to pH 3.2 withformic acid. Samples (10 ml) were collected on a frac-tion collector. US was measured in a 0.5 ml portionof each sample by the method of Gerhart and Pardee(7). DHO was identified and measured by convertingit to US by the addition of 0.05 ml of 9 M NaOH toeach 0.5-ml sample. DHO itself does not react in thecolorimetric test for US.

RESULTS

Complementation analysis. Seventy-seven py-rimidine-requiring mutants were selected from2,500 conidia isolated after mutagenic treatmentand the filtration-concentration procedure. Ten ofthe mutants were tested in all possible combina-tions for their ability to form uridine-independentheterokaryons in a two-axis complementationgrid. From these, mutually complementing strainswere selected as tester strains to group the remain-ing mutants. Isolates representing all distinguish-able complementation groups were further ex-amined by crosses and enzyme analysis todetermine each specific deficiency. The comple-mentation groups found represented all previ-

ously known groups, as well as one new group(Table 1).The new locus found in this study, pyr-6, com-

plements all the other pyrimidine-deficient muta-tions, and, as shown below, is deficient inDHOase. Conversion of US to DHO was theonly enzymatic step in the synthesis of UMP forwhich there was previously no known mutant.Pyr-6 (DFC-37) was first isolated in a doublemutant, carrying an unlinked morphologicalmarker. The latter was removed by recombina-tion in an outcross to wild type. All reisolatescarrying pyr-6 are somewhat leaky in minimalagar medium.

Allelism and recombination. Representativeisolates were crossed to known pyrimidine mu-tants to test for allelism. Analysis of crosses wasmade both by plating large numbers of sporesdirectly on minimal medium to test for the pres-ence of prototrophs and by isolating individualspores on uridine-supplemented medium and thenspot-testing them on minimal medium. The datashow no recombination between DFC-3 andpyr-3d (45502), between DFC-33 and pyr-l(H263), between DFC-9 and pyr-2 (37709), andbetween DFC-90 and pyr-4 (36601) (Table 2). Inmany of these crosses, even when uridine wasadded to the crossing medium, germination waspoor. A low frequency of prototrophs in intra-allelic crosses could represent true recombinants,or possible wild-type contaminants, since nooutside markers were used.The recombination between the linked pyrimi-

dine mutants pyr-J, pyr-3, pyr-2, and strainsisolated here is consistent with previously estab-lished linkage relationships. DFC-9 (pyr-2) whencrossed to pyr-la yielded 14.8% prototrophs andis consistent with reported map distances betweenpyr-l and pyr-2. DFC-33, crossed to DFC-3, gave3.3%, and DFC-33 crossed to DFC-8 gave 4.5%recombination. These figures are within the rangeof recombination values reported between pyr-Jand pyr-3 [Table 3 (6)].

Pyr-6 (DFC-37) is not linked to any of the otherloci of the pathway (Table 3). The original py-rimidine-requiring morphological mutant wascrossed to wild-type 73a, and a morphologicallywild-type, pyrimidine-requiring isolate was chosenfor study. After preliminary genetic analyses, athree-point cross was done by mating pyr-6 to astrain carrying the group VR markers pab-2(H193) and inos (36401). Analysis of the crossshows that pyr-6 is located 8.7 units from pab-2and 24.1 units from inos (Table 4). The coefficientof coincidence is 0.77. The symbol "pyr-S" is notused for the DHOase-deficient mutants since itwas first used to designate the isolate KS-12, a

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1374 CAROLINE J. BACTERIOL.

TABLE 1. Complementation analysis and identification of enzymatic deficienciesin uridine-requiring mutants

Enzyme assaysaCmpe-tto No. of Isolates tested for - ____-_________ Presumed Rfrnegrnatoup mutants enzyme activity DHO- funtional locusgroup ~~~~~~~~ATCaseDHOase dehase OMPdecase

Wild type 0 74A 2.8 4.5 + 45 No enzy-matic de-ficiency

I 13 DFC-1 11.9 3.1 + pyr-3M 19DFC-8 3.1 4.5 +

I-II 55 DFC-3 0.0 4.4 + pyr-3MN 3, 5

II 1 DFC-7 0.0 3.1 + pyr-3N 2

III 1 DFC-37 3.9 0.0 + pyr-6 This study

IV 3 DFC-33 2.8 4.6 0 pyr-l WoodwardDFC-46 5.9 8.1 0 et al., Proc.DFC-66 3.1 6.6 0 X, Int.

Cong.Genet.,1959; thisstudy

V 3 DFC-9 3.1 4.1 + 40 pyr-2 R. H. Davis,DFC-32 unpublished

data; 16

VI 1 DFC-90 4.1 11.1 + 0.05 pyr-4 14, 16, 18

a Some of these assays were made under conditions conducive to identifying deficiencies, i.e., onlimiting uridine or late in growth; therefore, some of the values reflect regulatory elevation of enzymelevels. Specific activities for ATCase, DHOase, and OMPdecase are given as micromoles per milligramof protein per hour. DHOdehase is measured qualitatively.

TABLE 2. Allelism ofpyrimidine mutants isolated inthis study with previously identified markers

SporesPer cent ualivy Random

Cross of germ- ated ponres platedCross - i~~~solatdSOination from on msnimal'uridinea

45502 A (pyr-3N) X 50 0/100 1/11,000DFC-3 a

H263 a (pyr-1) X 50 0/180 1/6,000DFC-33 A

37709 a (pyr-2) X 30 0/300 0/13,000DFC-9 A

36601 A (pyr-4) X 10 0/159 0/10,000DFC-90 a

a Values expressed a

total number of spores.s ratio of prototrophs to

strain later shown to be a double mutant contain-ing both pyr-J and pyr-3 markers (6).

Identification of enzymatic deficiencies. In thisstudy, four of the six enzymes leading to the syn-

thesis of uridylic acid have been specifically corre-lated with separate, single mutants. Indirect evi-dence that pyr-3a lacks G-CPS is abundant (3, 4,6). Recently Williams and Davis (Abstr. Genet.Soc. Amer., 1968) have developed an assay forthis enzyme and have related all types of pyr-3

TABLE 3. Recombination between variouspyrimidine markers

Per cent Prototropbs per M~ap~cCross of germ- total no. of spores isnce

ination snferred

DFC-37 A X 98 24/100 (24%) unlinkedDFC-8 a

DFC-37 A X 30 26/104 (25%) unlinked37709 a (pyr-2)

DFC-9 A x 80 14/100 (14%) 28H263 a (pyr-1)

DFC-33 A X 90 8/242 (3.3%) 6.6DFC-3 a

DFC-33 A X 90 10/245 (4.0%) 8.0DFC-8 a

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PYRIMIDINE SYNTHESIS IN N. CRASSA

TABLE 4. Mappinga ofpyr-6 (DFC3to the linkage group VR marke?

and pab-2 (H193)

Class of progeny Zygote geno

Parental types inos pab-2+ + j

Single crossovers, inos + pjregion I + pab-2

Single crossovers, + +region II inos pab-2 1

Double crossovers + pab-2 Iinos +

Total progeny

a Map:Linkage group VR

0- I_-centromere inos

15.4

mutants to the expected enzymaOrotidylate pyrophosphorylasewhich is missing in pyr-2 mutant,lished data), was not assayed. Fo2enzyme levels in the various mutwere grown on unlimiting uridine

Pyr-3. Among the pyr-3 mulATCase activity, both DFC-7 alshown to lack activity, whereas Imal ATCase. Since DFC-8 failswith TIFPr' hi-it Anae r-fmnlamaini

I7R2A) by mating dry weight), but then continues growth to a maxi-rs inos (37401) mum dry weight characteristic of wild type (Fig.

2). When grown on the reciprocal shaker or inNo. of round-bottom flasks with forced air at 25 C Qnitype isolates minimal medium, the mutant exhibits no growth

after 4 days. Extracts made from DFC-37 which+ 80 had been grown on limiting uridine, but whichpyr-6 70 achieved wild-type growth (Fig. 2), were all

tested for DHOase activity. At no point in theyr-6 12 growth cycle could any DHOase be detected.+ 16 When grown on unlimiting uridine, the mutant

+ 8 had wild-type levels of the other pyrimidinepyr-6 7 enzymes.

Pyr-1. It has previously been shown that mu-pyr-6 1 tants carrying pyr-J are blocked between US and+ 1 orotate, but the specific step was not clear (26).

Using the assay developed for DHOdehase, both195 DFC-33 and the standard pyr-J strain, H263,

were found to lack this enzyme. One-hundredthof the wild-type activity would have been detected.

8.7 An experiment in which mutant and wild-typeextracts were mixed shows that neither deficiencyrb-2 pyr-6 can be attributed to the presence of a free inhibi-

tor. ATCase and DHOase activities are in thewild-type range in pyr-1 mutants grown on un-tticphentyps. limiting uridine.

1 (OMPPase), Pyr-4. The pyr-4 isolate 36601 has been showns (Davis, unpub- to accumulate orotate (18) and orotidine (14) andr comparison of is deficient in orotidylic decarboxylase (OMPde-ants, the strains case) (18). DFC-90, isolated in this study, which

is allelic to 36601 and DFC-9, a pyr-2 allele, weretants tested for assayed for OMPdecase. The assay was kindlynd DFC-3 were performed by Robert Krooth by using his methodDFC-8 has nor- for assaying the enzyme in human tissue (9).to complement DFC-90 was shown to have a negligible amount

WILII LJ UL-JUJ IUCJ OR1UIpClllWiLLWLrLL - /, it

can be deduced that DFC-8 lacks pyrimidine-specific, glutamine-dependent carbamyl phos-phate synthetase (G-CPSpyr); DFC-7 lacksATCase; and the noncomplementing DFC-3lacks both of the pyr-3 enzymes (24). Activity ofother pyrimidine enzymes is normal.

Pyr-6. DFC-37 is the first (and only) recognizedmutant deficient in DHOase activity in an assay inwhich a specific activity of 0.1 umole per mg ofprotein per hr (about 2% of wild-type activity)would have been detected. A mixture of equalamounts of protein from DFC-37 and wild-type74A extracts was assayed and found to have aspecific activity equal to one-half that of wild-type extract alone. This shows that there is nofree inhibitor in the DFC-37 extract which couldmask wild-type DHOase activity.DFC-37 has an unusual growth pattern. When

grown on limiting uridine (20 ,ug/ml) in a low-form culture flask on a reciprocal shaker, adistinctly biphasic growth curve is obtained. Themutant shows a lag in growth at the point thatmost mutants cease growing (about 0.9 mg/ml,

3.C

2E ioaCPEO-.C.2 o.5

03

10 15 20 25 30

HoursFIG. 2. Growth curve of strain DFC-37 (pyr-6).

Mycelia were grown on 20 pg of uridine per ml (0)and 100 ;.g of uridine per ml (@) in reciprocal shakerflasks.

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CAROLINE

of OMPdecase, whereas DFC-9 has the sameamount as wild-type (Table 1).

Utilization of intermediates. All of the pyrimi-dine mutants studied grew well when supple-mented with uridine but not when supplementedwith uracil or cytosine. None of the mutants grewon US, even those blocked prior to US in thepathway. This observation is consistent withprevious data (2). With the exceptions of DFC-9and DFC-90, all strains could grow on orotate, al-though growth was quite erratic. DFC-9 andDFC-90 are blocked after the production oforotate, and thus they would not be expected torespond to this compound.Accumulation of intermediates. Supplementary

information consistent with the enzymatic data isthat DFC-37 (DHOase-deficient) accumulatesonly US, and that DFC-33 (DHOdehase-defi-cient) accumulated both US and DHO. DFC-9(pyr-2) also accumulates both US and DHO.Neither wild-type 74A nor DFC-3, a pyr-3MNmutant, accumulated US or DHO, as expected.A double mutant made by crossing DFC-8 (apyr-3M mutant) to DFC-33 (pyr-1) also failed toaccumulate US (Table 5). This shows that, despitethe presence of ATCase, the pyr-3 mutation pre-vents the accumulation of US in a pyr-J mutant.This supports the view that such pyr-3M mutantslack an earlier function, probably the synthesis ofpyrimidine-specific CAP. This confirms a similarconclusion based on the pyr-3- pyr-J doublemutant, KS-12 (6).

DISCUSSIONIn this study DHOase and DHOdehase have

been confirmed to be biosynthetic enzymes inNeurospora. Assays for these enzymes have beendeveloped, and a uridine-requiring mutant foreach enzyme has been found. Pyr-1, representedby DFC-33 as well as the previous isolate H263,lacks DHOdehase, and pyr-6, a new genetic locusrepresented by DFC-37, lacks DHOase. Each

TABLE 5. Accumulation of US and DHO inpyrimidine mutants and wild type

Strain Dz -DIbryStrain ~weight" at Ub DHOb

harvest

74A 1.34 0 0DFC-33 A (pyr-1) 0.87 110 24DFC-37 A (pyr-6) 0.86 75 0DFC-9 A (pyr-2) 1.04 159 23DFC-3 A (pyr-3MN) 0.66 0 0DFC-8, DFC-33 a (pyr- 0.85 0 03M, pyr-1)

aValues expressed as milligrams per milliliter.bValues expressed as micromoles per gram of

mycelium.

mutant contains a single enzymatic deficiencyand segregates in crosses with wild type as onegene. These mutants have also been shown toaccumulate only the intermediates prior to theblock in the pathway which they affect. A diagramof the pyrimidine pathway with the mutant locusfor each enzyme is shown in Fig. 1. One analyticalcriterion could not be successfully used with thispathway, namely growth tests of mutants on in-termediates of the pathway. "Early" mutants(e.g. pyr-3) were unable to grow on US or DHO.The inability of these intermediates to support thegrowth of mutants may be tied to the functionalorganization of the enzymes or to the impermea-bility of the cells to the substrates.A mutant lacking DHOase has been isolated

here for the first time and several possibilitiesmight aid in explaining the difficulty in detectinga mutant lacking DHOase. The pyr-6 mutant,DFC-37, was isolated here as a double mutantcombined with an unlinked morphological markerwhich causes very slow growth. After the morpho-logical marker was eliminated by out-crossing, itwas observed that the DHOase-deficient strain isleaky. However, DHOase was not detected in thisstrain when it was growing in the absence of sup-plement. If future pyr-6 mutants are similarlyleaky, it may explain their rarity in past selectionexperiments and indicate that one was isolatedhere only by virtue of its association with a de-pauperate morphological mutation. It is possiblethat, in the absence of DHOase, US can be con-verted to DHO by dihydropyrimidinase (E.C.3.5.3.3), an enzyme of the pyrimidine degrada-tive pathway which converts dihydrouracil to,B-ureidopropionate. Delayed induction of somealternate enzyme would also explain the biphasicgrowth curve of DFC-37 observed on limitinguridine. Another explanation for the leakiness ofthe strain could be the presence of an extremelyunstable form of DHOase which cannot be de-tected by the assay method used. However, thescarcity of mutants for this locus and the biphasicgrowth curve of the mutant favor the hypothesisthat a secondary enzyme is induced.

In Neurospora only a single, particulateDHOdehase has been detected. This contrastswith several microorganisms in which the biosyn-thetic enzyme is particulate but in which there isalso a soluble, degradative enzyme (15, 22). Sincethe pyr-J mutation in Neurospora causing a de-ficiency in DHOdehase results in a complete py-rimidine requirement, it is likely that only a singleDHOdehase is present in Neurospora. In the pokymutant of Neurospora, a DHOdehase activity hasbeen localized in the mitochondrial fraction (R. T.Eakin, personal communication).The pyr-3 locus controls the synthesis of the

first two enzymes specific to pyrimidine biosyn-

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PYRIMIDINE SYNTHESIS IN N. CRASSA

thesis, and the mutants could be identified withsome confidence by complementation patternalone. One of the complementation groups (pyr-3M) consists of mutants which have ATCase butcannot form US. These mutants are deficient inG-CPSpyr (Williams and Davis Abstr. Genet.Soc. Amer., 1968). The second group, pyr-3N,consists of mutants (here a single isolate) whichlack ATCase but complement the pyr-3M mu-tants (2). The noncomplementers (pyr-3MN)lack both activities and fail to complement withany other pyr-3 mutant. Strong additional evi-dence that pyr-3M mutants lack G-CPSpyr isprovided in the present study by the failure of thedouble mutant, pyr-3M pyr-J (DFC-8, DFC-33)to accumulate US.Pyr-3 mutants are by far the most numerous

type of pyrimidine-requiring mutant isolated inthis and previous work, and this locus has beenthe most extensively studied. As in the previousstudy (5, 24), only a single mutant lacking onlyATCase was obtained. This mutant (DFC-7)complements with the pyr-3 isolates which haveATCase (e.g., DFC-1 and DFC-8). All of theother isolates which lack ATCase fail to comple-ment with any of the other pyr-3 isolates. Only 13of the pyr-3 isolates in this study belong to thenoncomplementing pyr-3 class, whereas the greatmajority of isolates (55) have ATCase but lackG-CPSpyr (pyr-3M). This contrasts with the olderseries of mutants in which 23 of 30 mutants be-long to the noncomplementing class (5).The disparity in the numbers of mutants among

the pyrimidine loci could be due to differences inmutability, gene size, or to the selection methodused. It is also possible that some enzymes con-tain dispensable segments, and mutants for thatenzyme would be detected only if a mutationaffected a small, critical region of the enzyme.

ACKNOWLEDGMENTS

I wish to thank Rowland H. Davis for his direction and ad-vice. I also am grateful to Robert S. Krooth for his helpful dis-cussion and for performing the OMPdecase assays.

This investigation was supported by Public Health Servicetraining grant 2-TOI-GM-00071 from the National Institute ofGeneral Medical Sciences, National Institutes of Health, and waspresented as partial fulfillment of the requirement for the Ph.D.degree at the University of Michigan.

LITERATURE CITED1. Caroline, D. F., and R. H. Davis. 1969. Pyrimidine synthesis

in Neurospora crassa: regulation of enzyme activities. J.Bacteriol. 100:1378-1384.

2. Davis, R. H. 1960. An enzymatic difference among pyr-3mutants of Neurospora. Proc. Nat. Acad. Sci. U.S.A.46:677-682.

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