Melee. gem Genet. 138, 243--255 (1975) © by Springer-Verlag 1975

Pyrimidine Biosynthesis in Aspergillus nidulans Isolation and P r e l i m i n a r y Characterisation of A u x o t r o p h i c Mutants L. M. Palmer and D. J. Cove Department of Genetics, University of Cambridge, England Received March 17, 1975

Summary. 113 pyrimidine auxotrophs, unable to synthesise UMP have been selected in Aspergillus nidulans. These mutants can be classified by complementation into eight groups, and genetic analysis has shown that five loci are involved. One complex locus consists of the mutually complementing pyrA, pyrB and pyrC groups, as weI1 as the cis-dominant pyrN group, members of which do not complement with members of the A, B or C groups, pyrA mutants have been shown to lack CPSase-ur, pyrB and pyrC mutants have been shown to lack ACTase, and pyrN to lack both these enzymes. This locus appears to code for products which form an enzyme aggregate. The four simple loci, as well as the complex loci have been located genetically, and distinguished from one another on the basis of accumulation of pyrimidine precursors in vivo. The synthesis of ACTase has been shown to be subject to end-product repression. This paper describes studies on the biosynthesis of UMP, and its control, in Aspergillus nidulans. The p a t h w a y of pyrimidine synthesis has been studied extensively in a variety of organisms, and work in this area has been reviewed (O'Donavon and Neuhard, 1970). I n Neurospora crassa (Suyama, Munkres and Woodwood, 1959; Davis, 1967; Caroline, 1969), Saccharomyces cerevisiae (Lacroute, 1968; Denis-Duphil and Lacroute, 1971), and Coprinus lagopus (Hirsch and Gans, 1968) the first two enzymes of the p a t h w a y are coded b y a complex locus, the products of which form an enzyme aggregate which combines CPSase-ur and ACTase activities, and which has been shown to be subject to inhibition b y U T P in S. cerevisiae (Lue and Kaplan, 1969, 1970) and in N. crassa (Williams and Davis, 1970). More recent studies (Lue and Kaplan, 1971) have shown t h a t the aggregate can be dissociated into subunits which possess either CPSase-ur or ACTase activity, suggesting t h a t the aggregate is the product of more than one cistron. The genetic m a p in all three fungi shows non-overlapping regions coding for either CPSase-ur or ACTase (Williams and Davis, 1970; Denis-Duphil and Laeroute, 1971 ; Gans and Masson, 1969). I n all these cases, point m u t a n t s lacking both enzyme activities have been mapped exclusively in the CPSase-ur region; Radford (1970) has identified chain termination mutants among such mutants in N. crassa. Evidence from these studies suggests t h a t the locus codes a polycistronie message, and t h a t the CPSase-ur cistron is translated before the ACTase cistron.

Abbreviations used: ACTase: aspartate earbamoyltransferase (E.C. 2.1.3.2); CA: Nearbamoyl-L-aspartate; CP: carbamoyl-phosphate; CPSase-arg: arginine specific carbamoylphosphate synthase (E.C. 2.7.2.5); CPSase-ur: uridine specific carbamoyl-phosphate synthase (E.C. 2.7.2.5); DItO: dihydroorotic acid; OA: erotic acid; OCTase: ornithine carbamoyltransferase (E.C. 2.1.3.3); OMP: orotidine-5+-phosphate; UMP: uridine monophosphate; UTP: uridine triphosphate. 17

1VIolec. gen. Genet. 138

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L.M. Palmer and D. J. Cove

A l t h o u g h t h e genetic a n d overall e n z y m a t i c characteristics of this c o m p l e x a p p e a r t o be b r o a d l y similar in those fungi s t u d i e d so far, t h e m e t a b o l i c fate of CP varies. I n all species s t u d i e d t h e r e is also a CPSase-arg, which is s u b j e c t t o i n h i b i t i o n a n d / o r repression b y arginine. I n 1V. crassa, t h e t w o pools of CP are f u n c t i o n a l l y s e p a r a t e d (Davis, 1965), whilst in S. cerevisiae pool s e p a r a t i o n is o n l y p a r t i a l (Lacroute, P i e r a r d , Grenson a n d W i a m e , 1965), t h e r e being sufficient i n t e r c h a n g e b e t w e e n pools for m u t a n t s lacking either C P S a s e - u r or C P S a s e - a r g to a p p e a r p r o t o t r o p h i c in t h e absence of t h e e n d - p r o d u c t of t h e i n t a c t p a t h w a y . This p a p e r describes t h e isolation a n d p r e l i m i n a r y c h a r a c t e r i s a t i o n of pyrim i d i n e a u x o t r o p h s of A . nidulans.

Materials and Methods a) A. nldulans Strains and Genetic Techniques. The strains used in these studies carried genetic markers which, with the exception of pyr mutations, whose origins are described below, are those in general use iClutterbnck and Cove, 1974). Genetic techniques were modified after Pontecorvo, Roper, Hemons, Macdonald and Burton (1953), and McCully and Forbes (1965). Heterokaryons and diploids were made using standard methods (Roper, 1952). Complementation in heterokaryons of pyrimidine auxotrophs was tested by first establishing a balanced heterokaryon on minimal medium containing uracil, using strains carrying additional complementary vitamin auxotrophies, and transferring the pre-established heterokaryon to minimal medium lacking uridine, and comparing growth with a control heterokaryon containing similar vitamin auxotrophs but prototrophie with respect to uridine. Pyrimidine mutants were allocated to linkage groups by formation of heterozygous diploids with master strains marked in each linkage group, followed by haploidisation using p-fluorophenylalanine (McCully and Forbes, 1965). Location within a linkage group was achieved using standard meiotic mapping techniques. b) Media and Supplements. Media and supplements used were those described by Pontecorvo et al. (1953) as modified by Cove (1966). Unless stated otherwise 5 mM ammonium tartrate served as nitrogen source. Pyrimidine sources were added to give a final concentration of 5 mM, with the exception of uracil (10 raM). Arginine was added as the free base to give a final concentration of 2.5 m£Vf. c) Chemicals. Analytical grade chemicals were used wherever possible. Pyrimidine bases, nucleosides, nucleotides and precursors were obtained from Sigma Chemical Co. Ltd, Kingstonupon-Thames, Surrey. N-methyl N' nitro N nitrosognanidine was obtained from Kodak Chemical Co. Ltd. d) Selection o/Mutants Auxotrophic ]or Pyrimidine. Pyrimidine anxotrophs were selected by replica plating from media containing uridine to media lacking uridine (see results section for further details of media), using the method of Mackintosh and Pritchard (1963). Master plates were obtained by spreading conidiospores which had been treated with N-methyl N' nitro hT nitrosoguanidine to increase mutation rate, according to the method of Adelberg, Mandel and Chan (1965). Routinely between 80 and 99% of conidiospores were killed by this treatment. Mutants were reisolated from single conidiospores and stored on slopes of complete medium in screw cap vials. Pyrimidine auxotrophs were designated pyr, and allocated an isolation number which was retained irrespective of the complementation group to which they were later assigned. e) Culture and Storage o/Mycelium. Mycelium for analysis of accumulated intermediates and of enzyme content was grown, harvested and stored according to the method of Cove (1966) except that mycelium was stored at -- 70°. For enzyme determinations mycelium was stored for no longer than two weeks, during which time no decrease in extractable ACTase or OCTase activities was observed. Myeelial dry weights were determined by drying samples in a hot air oven at 90° until a constant weight was obtained. /) Preparation o/Cell Free Mycetial Extracts ]or Enzyme Determinations. Cell free extracts were prepared by grinding approximately 1 g of frozen mycelium, with an equal weight of acid washed sand, and 10 ml of extraction buffer (for ACTase: 50 talk NaOH-glycine buffer

Pyrimidine Auxotrophs of

Aspergillus

245

p i t 10.3; for OCTase: 200 mY[ Tris-ttC1 buffer p H 8.5) in a prechilled mortar for 2 rain by hand. The resulting slurry was centrifuged at 40000 × g for 30 rain and the supernatant was used for activity determinations. g) Assay o] Pyrimidine Precursors. The method of Caroline (1969) was used, modified as follows. Myeelium was grown as described in e) above, in minimal medium, with 10 mM NaNOa as nitrogen source, supplemented with 25 ~zM uridine, at 25 ° for 30 hrs. The wet weight of mycelium equivalent to 100 mg dry weight was ground by hand, with a pestle and mortar with 2 ml of 6% HCIOd, and eentrigufed at 500× g. The precipitate was reextracted twice more with 2 ml of distilled water and the total extracts combined. The combined extracts were passed through a column of Amberlite I R 20(R) ion exchange resin, 8 × 8 ram; which was regenerated between different extracts with 1 N HC1. The column was washed with 4 ml distilled water, and this wash was combined with the unabsorbed extract, and neutralised with 1 N K 0 t t . This sample was divided equally into two portions, one for the assay of CA and DHO, the other for the assay of OA and OMP. For the determination of CA and DHO, sodium formate-formic acid buffer p i t 3.2 was added to one half of the sample to give a final concentration of 100 m ~ , and the sample was then applied to a Dowex 1 (formate) column, dimensions 12 × 95 mm pre-equilibrated with the same buffer. The column was eluted with a ~aC1 gradient from 100 mM to 500 mM in a total volume of 200 ml 100 m ~ formate-formic acid buffer pH 3.2, followed by 30 ml of 1 M sodium formate-formic acid buffer, pH 3.2 without NaC1. 9 ml fractions were collected, and 500 ~l samples from each fraction were analysed for CA and DHO content. CA was determined directly by the colourimetric method of Gerhart and Pardee (1962). DHO was converted to US by the addition of 500 ~l of 1 N :NaOH and heating at 28 ° for 20 min. DHO does not interfere in the assay of CA. The column was calibrated by eluting pure samples of CA and DttO. OA and OMP were determined by eluting the second half of the sample from a similar Dowex 1 (formate) column with a p i t gradient of 300 mM sodium formate-formic acid buffer from pH 3.2 to p H 1.8 in a total volume of 120 ml. 5 ml fractions were collected, and their absorbence at 260, 280 and 290 nm determined. This system was found to give a good resolution of artificial mixtures of bases and nueleotides, and the column was calibrated with standard samples of OA and OMP. The identity of the compounds in each fraction was further checked by determining the ratios of absorbence at 260 and 280 nm, and at 260 and 290 nm at known p H and comparing with pure samples of OA and OM_P at the same pit. h) Enzyme Assay. Assay for ACTase and OCTase were modified from established methods (Davis, 1961 ; Gerhart and Pardee, 1962). Both assays depend on the determination of ureido compounds by the test developed by Gerhart and Pardee (1962). The assay mixture for ACTase was as follows: CP (di Li salt) 1 micromole; Na-L-aspartate 40 micromoles; glycine N a O t t Buffer p i t 9.3 150 micromoles; cellfree extract 100 91, in a total volume of 1 ml. The mixture was incubated at 37 °, usually for 20 min. The assay mixture for OCTase was as follows: CP (di Li salt) 5 micromoles; L-ornithine monohydrochloride 125 mieromoles; Tris-HC1 buffer pI-I 8.5 100 micromoles; cell-flee estract 100 91 in a total volume of 1 ml. The mixture was incubated at 37 °, usually for 15 min. For both assays the reaction was stopped by addition of sulphuric acid-butane dione monoxime-diphenylamine p-sulphonate reagent, which is the initial stage in the estimation of CA or eitrulline (Gerhart and Pardee, 1962). Turbidity was corrected for by reference to an assay mixture incubated for 0 min. The assay procedure was standardised by reference to CA and citrulline solutions in the same buffer and assayed at the same time. Soluble protein was determined using the modified Folin reaction of Lowry, Rosebrough, Farr and Randall (1951). Enzyme units are expressed as nanomoles of product (CA for ACTase, citrulline for OCTase) formed per mg of protein per rain. Results

I. Isolation o/ Pyrimidine Auxotrophs Because there might have been an interaction between arginine and pyrim i d i n e b i o s y n t h e s i s , a n u m b e r of d i f f e r e n t m e d i a w e r e u s e d i n t h e r e p l i c a p l a t i n g technique which yielded pyrimidine auxotrophs. Experiments were carried out 17.

246

L. M. Palmer and D. J. Cove Table 1. Results of mutation

Expt. Strain No. mutagenesed

1 2 3

biA1 biA1 pabaA1/wA1uaY9

4

yA1 pyroA4 mauA2 gaIC7

% survival mutagenic treatment 0.6 2.8 1.7 22.0

Replicated From To minimal minimal medium medium plus plus Uridine Uridine Uridine -~ arginine Uridine

Number of colonies screened

Nothing Nothing Nothing

30000 24000 20000

Arginine

40000

replicating from minimal medium plus uridine to minimal medium, from minimal medium plus uridine plus arginine to minimal medium, and from minimal medium plus uridine to minimal medium plus arginine. Table 1 gives the results of these experiments. Pyrimidine auxotrophs were recovered from each type of experiment, but no strain which required both uridine and arginine, or uridine only in the presence of arginine was detected. 113 pyrimidine auxotrophs were isolated from total of 1.1 × 105 colonies examined.

H. Growth Response el Pyrimidine Auxotrophs The response of the auxotrophs obtained to precursors of UMP was tested on solid media incorporating the precursors. No mutant responded to CA, 93 responded to both DHO and OA, 14 to only OA, and 6 to neither DHO nor OA. Orotidine failed to support growth of any mutant responding to OA. Neither DHO nor OA restored full growth of any mutant strain even at elevated concentrations of base, perhaps suggesting that uptake of these compounds is limiting.

III. Complementation el Pyrimidine Auxotrophs All pyrimidine auxotrophs were first put into a heterokaryon with a strain prototrophic with respect to pyrimidine, to check for heterokaryon incompatibility and for the dominance of the pyrimidine auxotrophy. With the exception of pyr53, no pyrimidine auxotroph failed to form heterokaryons, pyr53 consistently produced small stunted heterokaryons, and was therefore outcrossed. Of eight pyrimidine requiring progeny recovered from this cross, five gave rise to similar stunted heterokaryons, but three yielded normal heterokaryons, suggesting that pyr53 carries a mutation preventing heterokaryon formation, which is unlinked to that leading to pyrimidine auxotrophy. An outcrossed strain of pyr53, not carrying this mutation was used for all subsequent tests. All the pyr mutants were shown to be recessive in heterokaryons. For complementation tests the 113 pyr auxotrophs were divided into three groups on the basis of their growth responses, and complementation relationships within each group were investigated. a) Mutants Responding to Both DHO and OA. 15 of this class of strain were chosen at random, and tested for complementation in all pairwise combinations. The results indicated that there were five complementation groups, members of four (pyrA, B, C, D) of which showed full mutual complementation, while members of

Pyrimidine Auxotrophs of Aspergiltu~

247

experiments to isolated pyr mutants Number of mutants classified as

Number or mutants obtained requiring Uridine only

Arginine Only

Uridine + arginine

pyrA Uridine only in the presence of arginine

pyrB

pyrC pyrN

26 12

0 42

0 0

0

O~ 0~ 0

12 7

5 2

2 1

3 1

69

0

0

0

41

2

1

11

6

0

the fifth (pyrN) failed to complement with members of the pyrA, B and C groups, but complemented fully with members of pyrD. One member of each complementation group (pyrA12, pyrB14, pyrC2, pyrD23, pyrN21) was then selected and the remaining 78 strains of this class were tested for complementation with these five strains. No further complementation groups were found. Of the 93 mutants responding to D t t O and OA, 60 were of the pyrA type, 9 pyrB, 4 pyrC, 5 pyrD and 15 pyrN. Among the pyrA, B, C and D types were both temperature sensitive and leaky strains; there were none of such strains among the pyrN group members. Complementation tests among pyrA, B, C and N mutants were extended to a sample comprising of 17 of the pyrA mutants, together with all the pyrB, C and N mutants. No eomplementation was observed between any pyrA, B or C mutants tested and any pyrN mutant. No complementation was observed within the pyrA, B and C groups, with the exception t h a t weak complementation was observed between pyrA18 and A39, and between pyrB15 and B32. All members of the A, B, or C groups complemented fully with all members of the other two groups, except t h a t weak complementation was observed between pyrA27 and B37 and between pyrB30 and C42. b) Mutants Responding Only to OA. The 14 strains of this class (designated pyrE) were tested for complementation. Whilst the majority of pairs of strains failed to complement, complementation was detected between certain pairs. The complementation relationships observed resolve to give a circular map (see Fig. 1). c) Mutants Responding to neither DHO nor OA. Complementation tests between the six strains of this class, revealed t h a t two strains designated pyr G89 and G114 failed to complement with one another but complemented with the remaining four strains of this class. Among these four strains, designated pyrFll, F56, F73 and F83, complementation was only observed between pyrFll and F73.

IV. Genetic Analysis o/pyr Mutants Preliminary recombination tests failed to detect recombination between representative pyrA, ]3, C and N strains, but showed t h a t all recombined freely with pyrD strains. Each member of these five eomplementation groups was then crossed to the particular strain representative of its group (pyrA12, ]314, C2, D23 and N21). No prototrophie recombinant was detected in any ease. The number of progeny aseospores screened was between 10 b and 104. No prototrophie reeombinants were detected in similar samples of progeny from crosses between

248

L.M. Palmer and D. J. Cove

8,19,28,40,67,85

•

18

Fig. 1. Complementation relationships of pyrE mutants. Numbers refer to allele numbers. The eomplementation relationships are represented by the usual convention whereby eomplementation occurs only between non-overlapping sets

members of the pyrE group, between members of the pyrF group, and between the two pyrG mutants. Five loci therefore appear to be involved; one complex, involving four complementation groups. The linkage group involved was determined b y haploidisation of a diploid with a multiply marked strain, pyrA12 and pyrD23 were located in linkage group V I I I , and pyrE8 and pyrG89 were located in linkage group I. A preliminary cross had detected linkage between pyrE8 and pyrFll and so the latter strain was not haploidised. Meiotic mapping was then used to locate the pyr loci in their respective linkage groups. N o linkage was detected between pyrA12 and the following linkage group V I I I markers: sD50, ornB7,/wA1, c n x B l l , uaY9, riboB2, nirB2, p a / E l l , pa/B7, chaA1, abaA1 and uX3. These markers span the entire linkage group. The complex locus, of which pyrA12 is an allele, cannot therefore at present be located. I n crosses with u a Y 9 , / p D 4 3 and palcC4 strains, pyrD23 was located as follows:

Map distance in centrimorgans

uaY

]pD

pyrD

I I

I I

I I

8.1

4.3

pal~C 9.8

I I

Crosses of pyrE8 and pyrF11 strains with anAl, adG14 and luA2 strains, located pyrE and F within linkage group I as follows:

~ap distance in centimorgans

anA

pyrE

adG

pyrF

luA

I I

t t

i I

[ ]

f I

9.3

6.2

9.0

4.5

Pyrimidine Auxotrophs of Aspergillus

249

No linkage was detected between pyrG89 and the following linkage group I markers: pabaA1, anAl, gaID5, adA4, yA1, riboA1, luA2, or niiC628. The pyrF locus cannot therefore at present be located.

V. Accumulation o/Pyrimidine Precursors The complementation and growth tests did not allow the classification of the mutants on a functional basis. Representative strains of each complementation group were therefore examined for the accumulation of pyrimidine precursors. Results are given in Table 2. If it is assumed that the most elaborated precursor accumulated is the substrate of the enzyme missing in the mutant strain, pyrA, B, C and N mutants lack CPSase and/or ACTase, pyrD mutants lack dihydroorotase (E.C. 3.5.2.3), pyre mutants lack orotate reductase (E.G. 1.3.1.14 or 15), pyrF mutants lack orotate phosphoribosyltransferase (E.C. 2.4.2.10) and pyrG mutants lack orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23). Table 2. Accumulation of pyrimidine precursors by pyr mutant strains Strain a

Amount of intermediate accumulated b CA

pyrA12 pyrB14 pyrC3 pyrD23 pyrE8 pyrFtl pyrG89 pyrN21 +

DHO

0 0 0

0 0 0

281 189 185 159

0 61 44 47

0 0

0 0

OA

OM-P

0 0 0

0 0 0

0 0 7.3 1.0

0 0 0 3.3

0 0

0 0

Culture, extraction and assay procedures are given in the methods section. a All strains carried the additional markers pabaA1/wA1 uaY9. b In nanomoles of intermediate per g dry weight of myeelium.

VI. Reversion o] pyrA, B and C Strains Davis (1965) in N. crassa showed that revertants to prototrophy of strains lacking CPSase-ur were of two types. One class were true revertants which had regained CPSase-ur activity, while the other class retained the original mutation which was now suppressed by a second mutation in the gene coding for OCTase. This second mutation caused a decrease in the level of OCTase, and consequently allowed some of the CP from CPSase-arg to become a substrate for ACTase. Because CPSase-arg was inhibited by arginine the second class of revertant still required uridine in the presence of arginine. To see whether a similar situation exists in A. nidulans, and if so, to identify which of the pyrA, B or C genes codes for CPSase-ur, reversion studies were carried out of pyrA4, pyrB14 and pyrC3. Conidiospores, mutagenised with N methyl-N' nitro N nitrosoguanidine, were plated on medium without uridine and revertant strains picked. All four strains gave revertants which had no require-

250

L. N[. Palmer and D. J. Cove Table 3. Ornithine earbamoyl transferase levels in suApyrAd strains Strain

OCTase level

su+pyrA4 suASpyrAd suA8pyrAd suAlOpyrA4 suAldpyrA4 suA2OpyrAd

100 25 62 68 22 48

All strains carried the additional markers pabaA1pyrAd uaY9. Culture, extraction and assay procedures are given in the methods section. YIycelium was cultured at 25° for 28 h in minimal medium with 10 toNI NaNOa is nitrogen source, supplemented with p-aminobenzoic acid and 5 mM uridine. OCTase levels are specific activities expressed as a percentage of the specific activity in an su+pyrA4strain assayed on the same day.

ment for pyrimidines even in the presence of arginine, and which gave only protrophic progeny upon outcrossing to a prototrophic strain. I n the case of pyrA4, however, a class of revertants was obtained, which retained a partial requirement for uridine in the absence of arginine, and which showed a complete requirement for uridine, when arginine was present. When outcrossed to a prototrophic strain this class of revertants gave rise to progeny, a quarter of which were pyrimidine auxotrophs. This class of revertant must therefore have arisen as a result of the suppression of pyrA4 by a mutation in an unlinked gene. Strains carrying a suppressor mutation, designated sul4 pyrA4, were crossed to pyrA1, A12, B14, B15, C3, C50, N21 and N25, and double mutants isolated. Only the pyrA alleles were suppressed. The OCTase level in several su pyrA4 isolates was determined, and compared with levels in pyrA4 strains. The results, presented in Table 3, indicate t h a t these suppressor strains have levels of OCTase lower than nonsuppressor strains. These suppressor mutations therefore probably act b y causing an accumulation of carbamoyl phosphate, synthesised by the CPSase-arg. The sensitivity of suppression to arginine, indicates t h a t CPSase-arg is inhibited b y arginine. The pattern of suppression indicates t h a t only pyrA, and presumably pyrN, mutants lack CPSase-ur.

VII. ACTase Levels in Mutant Strains Table 4 contains data for ACTase levels in wild-type and pyr m u t a n t strains cultured under conditions of uridine limitation. No activity was detected in pyrB, C or N mutants. The enzyme levels in pyrA, D, E, F and G strains are considerably higher than those in the wild-type, which provides evidence, given the uridine limitation, t h a t ACTase is subject to end-product repression. No ACTase actively was detected in extracts of mycelinm of the temperature sensitive strains, pyrB15 and pyrC50, even when cultured and assayed at the permissive temperature of 25 °. Since these strains grow considerably in the absence of uridine at 25 °, we conclude t h a t both produce on ACTase more thermolabile t h a n wild-type, and t h a t both the pyrB and C genes are structural genes for ACTase.

Pyrimidine Auxotrophs of Aspergillus

251

Table 4. Aspartate carbamoyl transferase levels in pyr mutant strains Strain

Growth weight (g blotted weight mycelium/1)

ACTase specific activity

Wild-type

7.6 1.6 1.9 3.1 1.6 6.2 1.8 1.3 1.3 1.8 2.9 2.2 2.5

7.0 18 18 0 0 0 0 27 32 27 36 0 0

pyrA4 pyrA12 pyrB14 pyrB37 pyrC3 pyrC41 pyrD23 pyrE8 pyrF11 pyrG89 pyrN21 pyr:N25

Culture, extraction and assay procedures are given in the methods section. Mycelium was cultured at 25 ° for 28 h in minimal medium with 10 mS~ NaNO a as nitrogen source, and containing 50 mM uridine.

VIII. Reversion o/pyrN Strains Some m u t a n t s of 1V. crassa h a v i n g a p h e n o t y p e similar to A. nidulans pyrN m u t a n t s h a v e been shown to h a v e nonsense m u t a t i o n s in t h e gene specifying C P S a s e - u r (Radford, 1969 a n d 1970). H o w e v e r , whereas in o t h e r fungi t h e r e are o n l y two genes in t h e c o m p l e x locus, in A. nidulans t h e r e are three, pyrA, B a n d C. I f these t h r e e genes were t r a n s c r i b e d as a unit, t h e n as well as nonsense m u t a t i o n s abolishing all t h r e e functions, as specified b y c o m p l e m e n t a t i o n tests, i.e. m u t a n t s w i t h t h e pyrN p h e n o t y p e , it should also be possible to o b t a i n nonsense m u t a t i o n s which abolish o n l y t w o functions. N o m u t a n t s w i t h this p h e n o t y p e h a v e been o b t a i n e d . I t is therefore possible t h a t t h e pyrN p h e n o t y p e m a y h a v e some o t h e r basis. To d e t e r m i n e w h e t h e r deletions were present, conidiospores of pyrN21, 25, 29, a n d 43 s t r a i n s were m u t a g e n i s e d w i t h N - m e t h y l N ' nitro N n i t r o s o g u a n i d i n e a n d p l a t e d on m i n i m a l m e d i u m lacking uridine. W i t h pyrN21, 29 a n d 43, r e v e r t a n t s were o b t a i n e d a t r a t e s b e t w e e n 1.1 a n d 1.8 r e v e r t a n t s p e r l 0 s spores surviving t h e m u t a g e n i c t r e a t m e n t . N o r e v e r t a n t was r e c o v e r e d from pyrN25 strains, a l t h o u g h a t o t a l of 3.7 × 10 s s u r v i v i n g spores were screened. T e n r e v e r t a n t strains f r o m each of pyrN21, 29 a n d 43 were crossed to w i l d - t y p e . F i f t y p r o g e n y from each cross were t e s t e d for p y r i m i d i n e a u x o t r o p h y , b u t none was found. R e v e r s i o n does n o t therefore in these cases a p p e a r to be due to a suppressor m u t a t i o n . N o nonsense suppressors are a v a i l a b l e in Aspergillus nidulans to enable pyrN alleles to be screened further. T h e l a c k of d e t e c t a b l e reversion of pyrN25 can be e x p l o i t e d to allow a funct i o n a l l o c a t i o n of t h e m u t a t i o n involved. I n crosses of pyrN25 to p y r A 1 2 , p y r B 1 4 , pyrC3 a n d pyrN21, p r o t o t r o p h i e r e c o m b i n a n t s were o b t a i n e d a t t h e r a t e s of a b o u t 10 -4 to 10 -~, a n d so pyrN25 c a n n o t be a m a j o r deletion. T h e / u I A 6 m u t a t i o n which leads to resistance to 5-fluorouracil, suppresses pyrA strains in a m a n n e r

252

L.M. Palmer and D. J. Cove Table 5. Aspartate earbamoyl transferase levels in/uIA6 pyrN25 revertant strains

Strain

Wild-type

/uIA6 /uIA6 pyrN25 Revertant 1 Revertant 11 Revertant 12 Revertant 23

ACTase specific activity

Mycelinm cultured in:

Sensitivity to uridine repression. Ratio:

Minimal medium

Minimal medium A-2.5 ~ uridine

Specific activity-minimal medium Specific activity-minimal medium -t- 2.5 mM uridine

7.8 8.1 Not assayed 6.0 5.6 5.9 6.8

2.3 2.3 0 5.4 4.2 2.6 5.7

3.4 3.5 1.1 1.3 2.3 1.2

closely similar to su pyrA4 mutations (Palmer and Cove, unpublished data). A ]ulA6 strain was crossed to a pyrN25 strain, and the double m u t a n t obtained. This is still a pyrimidine auxotroph. Conidiospores from a double m u t a n t strain were mutagenised with N-methyl N' nitro N nitrosoguanidine, and plated onto medium lacking uridine. Rcvertants were obtained at the rate of 0.2X 10-6. A sample of one hundred such revertants was picked, and their growth characteristies examined. All behaved similarly to one another and to su pyrA pyrA double mutants, in showing a pyrimidine requirement only in the presence of arginine. When revertants are crossed to wild-type strains, one quarter of the progeny are like the revertant parent, showing an arginine-sensitive pyrimidine requirement, one quarter are absolute pyrimidine auxotrophs, one quarter are 6-fluorouraeil resistant prototrophs, and one quarter are wild-type. The pyrN revertants are therefore in all respects similar to pyrA-B+C + strains. A cross between a n / u l A 6 pyrN25 revertant strain and a n / u I A 6 strain was made. More than 600 progeny were examined; only progeny of the two parental phenotypes were obtained, and these occurred in approximately equal numbers. Since no pyrN progeny occurred, the mutation altering the pyrN25 phenotype to a TyrA-B+C + phenotype must have occurred at or near to the site of the pyrN25 mutation implying t h a t the pyrN25 mutation has probably occurred in the pyrA region. ACTase levels were examined in four revertants of /ulA6 pyrN25 and the data is presented in Table 5. All had regained ACTase, but all were less sensitive than the wild-type to repression b y uridine.

Discussion The main conclusions which can be drawn from the results presented in this paper are summarised in Table 6. Taken together the growth responses and the determination of intermediates accumulated, allow the function of each gene to be determined with some confidence. The nature of the product(s) of the complex locus, comprised of pyrA, B, C and N, and determining both CPSase-ur and ACTase remains uncertain. Either a single multifunctional peptide is produced,

Pyrimidine Auxotrophs of Aspergillu8

253

Table 6. Summary of principle genetic and biochemical findings, and conclusions Locus

Location

Experimental finding

Conclusion

pyrA

Closely linked to pyrB, Respond to DHO & OA. C & N in linkage group No intermediates VIII. Unlinked to pyrD accumulated or other available markers

pyrB

See pyrA

ACTase structural Respond to DHO & OA. No intermediates accumulated. gene No suppression by partial OCTase block. Lack ACTase. Temperature sensitive mutants produce an ACTase, more labile than wild-type.

pyrC

See pyrA

As pyrB

pyrD

In linkage group VIII, between/pD & palcC

Respond to DHO & OA. Accumulate CA

orotase

pyrE

In linkage group I, between anA & adG

Respond to OA. Accumulate DHO

Lack orotate rednetase.

pyrF

In linkage group I, between adG & luA.

Respond to neither DHO or OA. Lack orotate phosphoribosyl Accumulate OA. transferase

pyrG

In linkage group I. Respond to neither DHO or 0A. Lack orotidineUnlinked to pyrE or F or Accumulate 01~IP 5'-phosphate other available markers' deearboxylase

pyrI~

See pyrA

Lacks CPSase-ur

ACTase structural gene Lack dihydro-

Do not complement with pyrA, Lack both B or C mutants. Respond to CPSase-ur DHO & OA. No intermediates & ACTase. accumulated. No suppression by partial OCTase block. Lack ACTase

or t h r e e discrete p e p t i d e s arc p r o d u c e d which associate to form a heteromer. T h e simple a n d clearcut division of t h e m u t a n t s b y c o m p l e m e n t a t i o n f a v o u r s t h e l a t t e r model, in which case A. nidulans will r e s e m b l e /Y. crassa, C. lagopus a n d S. cerevisiae (see i n t r o d u c t i o n for references) e x c e p t t h a t ACTase in A. nidulans is specified b y two cistrons. T h e n a t u r e of pyrIq m u t a n t s r e m a i n s unresolved. No evidence to i m p l i c a t e chain t e r m i n a t i o n m u t a t i o n s was o b t a i n e d . F i n e s t r u c t u r e genetic m a p p i n g of t h e c o m p l e x locus w o u l d help, h u t in t h e absense of l i n k e d m a r k e r s no such analysis has been a t t e m p t e d . T h e n o n r e v e r t i b l e pyrN25 m u t a t i o n could i n v o l v e a small deletion l e a d i n g to a frameshift. I f this is so, a n d were t h e d e l e t i o n l o c a t e d in t h e pyrA region, t h e r e c o v e r y of pyrB a n d C functions r e p o r t e d here, could arise if a second m u t a t i o n allowed t r a n s l a t i o n to be r e i n i t i a t e d before t h e pyrB a n d C regions. This w o u l d necessarily i m p l y t r a n s l a t i o n from pyrA to pyrB a n d C. T h e r e l a t i v e i n s e n s i t i v i t y of pyrN25 r e v e r t a n t strains to repression of ACTase b y u r i d i n e will be i n v e s t i g a t e d further. T h e r e are now a n u m b e r of e x a m p l e s where

254

L.M. Palmer and D. J. Cove

m u t a t i o n in t h e gene specifying a n e n z y m e can a l t e r t h e r e g u l a t o r y p r o p e r t i e s of t h a t e n z y m e (see Goldberger, 1974; Cove, 1974). The p y r N 2 5 r e v e r t a n t s m a y p r o v i d e evidence for a s i m u l a r m e c h a n i s m involving p y r i m i d i n e biosynthesis. Acknowledgements. We wish to thank the Science Research Council for a research grant which partially supported this work. L. M. Palmer thanks the Science Research Council and Trinity Hall, Cambridge for Research Studentships. We are grateful to Dr. C. Scazzoechio for his stimulating interest in this work.

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Mackintosh, M. E., Pritehard, R. H.: The production and replica plating of micro colonies of Aspergillus nidulans. Genet. Res. 4, 320-322 (1963) MeKully, K. S., Forbes, E.: Use of p-fluorophenylalanine with "master strains" of Aspergillus nidulans for assigning genes to linkage groups. Genet. Res. 6, 352-359 (1965) O'Donovan, G. A., Neuhard, J. : Pyrimidiue metabolism in micro-organisms. Bact. Rev. 34, 278-343 (1970) Ponteeorvo, G., Roper, g.A., Hemmons, L. ~[., MacDonald, K. D., Burton, A. W. J.: The genetics of Aspergillus nidulans. Advane. Genet. 5, 141-238 (1953) Radford, A.: Polarized complementation at the pyrimidine 3 locus of Neurospora. Molee. gen. Genet. 104, 288-294 (1969) Radford, A.: Pyrimidine requiring suppressor mutations of arginine-3 in Nsurospora and their bearing on the structure of the pyrimidine 3 locus. Molec. gen. Genet. 107, 97-106 (1970) Roper, J. A.: Production of heterozygous diploids in filamentous fungi. Experientia (Basel) 8, 14-15 (1952) Suyama, ¥., Munkres, K. D., Woodward, V. W. : Genetic analysis of pyr-3 locus of Neurospora crassa. Genetica 80, 293-311 (1959) Williams, L., Davis, R. H. : Pyrimidine specific carbamyl phosphate synthetase in Neurospora crassa. J. Baet. 108, 335-341 (1970) C o m m u n i c a t e d b y W . Gajewski Dr. L. M. Palmer Department of Energy Thames House South Millbank London, S.W.1 England

Dr. D. J. Cove Department of Genetics, Milton Road, Cambridge CB4 1XH, England