Molec. gen. Genet. 160, 165--173 (1975)~ © by Springer-Verlag 1975

Pyrimidine Biosynthesis in Aspergillus nidulans I s o l a t i o n a n d C h a r a c t e r i s a t i o n of M u t a n t s R e s i s t a n t to F l u o r o p y r i m i d i n e s L. M. Palmer, C. Scazzocchio, a n d D. J. Cove Department of Genetics, University of Cambridge, England Received June 16, 1975

Summary. Mutants resistant to 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine have been selected in Aspergillus nidulans. Growth tests combined with genetic analysis showed that mutations conferring resistance to fluoropyrimidines could occur in at least seven genes. Three of these, ]uIE,/ulF and jurA were concerned with either the uptake of pyrimidines or their conversion to uridine monophosphate. The other four genes did not affect these functions. Mutations in fuIA probably confer resistance by lowering ornithine transcarbamoylase, thereby making the norraMly arginine-speeific earbamoyl phosphate pool available for increased uracil synthesis. Mutations i n / u l D may make the arginine-specific carbamoyl phosphate synthetase insensitive to inhibition or repression by arginine, and so lead to increased carbamoyl phosphate pool sizes, and increased uracil synthesis. Both/ulA and/uID mutants suppress pyrA mutants which lack the uracil-specific carbamoyl phosphate syntheruse. Mutations in ]ulB and/ulC do not suppress pyrA, and so may act more directly to increase uracil synthesis. The synthesis of aspartate carbamoyl transferase in ]ulB7 strains is not repressed by uracil. ]ulC mutants are closely linked to the pyrA, B, C, N region which codes for the first two enzymes of pyrimidine biosynthesis, and may result in these enzymes being less sensitive to inhibition by uracil. W e h a v e r e c e n t l y described (PMmer a n d Cove, 1975) studies on p y r i m i d i n e biosynthesis i n Aspergillus nidulans, i n which auxotrophic m u t a n t s were isolated a n d characterised. This paper described c o m p l e m e n t a r y studies, u n d e r t a k e n to elucidate t h e m e c h a n i s m of t h e control of p y r i m i d i n e biosynthesis, i n which mut a n t s r e s i s t a n t to fluoropyrimidines have been selected a n d studied. Whereas a m a j o r i t y of such m u t a n t s are altered i n the u p t a k e , or i n t h e p a t h w a y s of salvage of exogenous pyrimidines, some h a v e alterations i n their r e g u l a t i o n of p y r i m i d i n e biosynthesis. Materials and ~Iethods

a) A. nidulans Strains and Genetic Techniques. The strains used in these studies carry genetic markers which are those in general use (Clutterbnck and Cove, 1974), with the exception of pyr mutations, whose origins and properties are described in Palmer and Cove, 1975, and /ul and ]ur mutations which are described below. Genetic techniques are those in general use (Pontecorvo, Roper, Hemmons, Macdonald and Burton, 1953; MeCully and Forbes, 1965). b) Media and Supplements. Media and supplements used were those described by Pontecorvo et al. (1953) as modified by Cove (1966). 5 mM ammonium tartrate served as nitrogen source in minimal medium. Pyrimidine sources were added to give a final concentration o~ 5 raM, with the exception of uracil (10 mM). Arginine was added as the free base to give a final concentration of 2.5 mM. c) Chemicals. 5FU, 5FUR and 5FdUR were grits from Hoffman La Roche, 5FU was also obtained from Fluka AG, 5 fluoroorotic acid from the Nutritional Biochemicals Corporation, Cytosine arabinoside and all other pyrimidine bases, nucleotides and precursors from the Sigma Chemical Co. Ltd.

Abbreviations used: 5FU: 5-fluorouracil; 5FUR: 5-fluorouridine; 5FdUR: 5-fluorodeoxyuridine.

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d) Selection o/ Mutants Resistant to Fluoropyrimidines. Spontaneous mutants resistant to 5FU and 5FUR were obtained as vigorous sectors arising from an inoculum of conidiospores of biA1 onto the appropriate solid medium, after incubation at 37 ° for 3-5 days. Only one resistant sector was picked from each conidial inoeulum. Resistant mutants were obtained on minimal medium containing either (a) 500 ~M 5FU or (b) 100 ~M 5FUR or (c) both 500 y2¢I5FU and 100 ~M 5FUR. Mutants resistant to 5FdUR, which inhibits conidiation but not growth of wild-type strains, were obtained by spreading spores of biA1 which had been treated with N-methyl N'-nitro N-nitrosoguanidine to increase the mutation rate (Adelberg, Mandel and Chan, 1965), onto minimal medium containing 800 rag/1 sodium deoxycholate, which leads to the formation of compact colonies (Mackintosh and Pritchard, 1963), and 500 ~M 5FdUR and picking conidiating colonies which developed after 3-4 days incubation at 37 °. Routinely between 80% and 99 % of conidiospores were killed by the mutagenic treatment. e) Techniques/or the Determination o/Aspartate Carbamoyltransfera~e (E.C. 2.1.3.2.). The techniques for the culture, harvest and storage of mycelinm, the preparation of cell free extracts, the assay of enzyme activity and the estimation of protein are all those given in Palmer and Cove, 1975. Results

I. E//ects o/ Pyrimidins Analogues on Wild-type Strains T h e g r o w t h of w i l d - t y p e strains a t 37 ° was c o m p l e t e l y i n h i b i t e d b y t h e addit i o n of 1 m M 5 F U or 5 F U R t o m i n i m a l m e d i u m . A t lower c o n c e n t r a t i o n s g r o w t h was n o t c o m p l e t e l y p r e v e n t e d , b u t c o n i d i a t i o n was inhibited. A t 1 mM, 5 F d U R d i d n o t i n h i b i t growth, b u t p r e v e n t e d conidiation. Microscopic e x a m i n a t i o n of m y c e l i u m growing in t h e presence of 1 m M 5 F d U R showed t h a t eonidiation was a r r e s t e d a t t h e p r i m a r y conidiophore stage. 5 fluoroorotic acid was n o t t o x i c a t 1 raM, a n d m a y n o t t h e r e f o r e be t a k e n u p or m e t a b o l i s e d t o 5 F U R . Cytosine a r a b i n o s i d e was also f o u n d n o t t o be t o x i c a t 1 mM. Cytosine a r a b i n o s i d e has b e e n r e p o r t e d b o t h to i n h i b i t D N A synthesis ( F u r t h a n d Cohen, 1968) a n d t o i n h i b i t a s p a r t a t e c a r b a m o y l t r a n s f e r a s e ( O ' D o n o v a n a n d N e u h a r d , 1970). I n b o t h cases, t o x i c i t y is d e p e n d e n t on conversion t o c y t i d i n e arabinoside. T h e l a c k of t o x i c i t y in A. nidulans m a y therefore be t h e result of l a c k of u p t a k e or a failure t o c o n v e r t cytosine a r a b i n o s i d e t o its active t o x i c form. To d e t e r m i n e t h e i n t e r a c t i o n s b e t w e e n p y r i m i d i n e s a n d r e l a t e d m e t a b o l i t e s in u p t a k e a n d salvage p a t h w a y s , t h e effects of various bases a n d nucleosides on t h e t o x i c i t y of 5 F U , 5 F U R a n d 5 F d U R were t e s t e d (see T a b l e 1).

II. The Selection and Phenotype o/Mutants Resistant to 5-/luoropyrimidines M u t a n t strains r e s i s t a n t to 5 F U , 5 F U R a n d 5 F d U R were i s o l a t e d b y t h e met h o d s described in t h e MateriMs a n d M e t h o d s section. Once isolated, t h e s t r a i n s were t e s t e d for t h e i r cross resistance to t h e o t h e r analogous. T a b l e 2, which summarises m a n y of t h e p r o p e r t i e s of t h e s e strains t o be described below, give details of t h e selection p r o c e d u r e a n d resistance p h e n o t y p e of t h e strains obtained. F o u r d i s t i n c t p h e n o t y p e s were shown: rulE m u t a n t s are r e s i s t a n t t o t h e effects of 5 F U a n d 5 F U R on b o t h g r o w t h a n d conidiation, b u t r e m a i n sensitive t o t h e effect of 5 F d U R on conidiation; ]ulA, B, C a n d D m u t a n t s a r e r e s i s t a n t , b u t less so t h a n / u l E m u t a n t s , t o t h e effects of 5 F U a n d 5 F U R on growth, b u t r e m a i n sensitive t o t h e effects of 5 F U , 5 F U R a n d 5 F d U R on c o n i d i a t i o n ; / u l F m u t a n t s

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167

Table 1. Interactions between fluoropyrimidines and pyrimidines Fluoropyrimidine

5 fluorouraeil 5 fluorouridine 5 fluorodeoxyuridine

Pyrimidine uracil

uridine

deoxyuridine cytosine eytidine

+ ---

+ + +

+ + +

----

-+ +

+ = reversal of toxicity, -- = no reversal of toxicity. Bases and nueleosides added at 5 raM, except for uracil (10 mM), 5FU and 5FdUR added at 500 tLM, 5~UR at 100 ~M. Conidia of biA1 were inoculated and growth was compared to that on minimal medium after 48 hrs at 37 ° C. 5FU and 5FUR toxicity scored as an effect on growth, 5FdUR toxicity as an effect on conidiation.

are r e s i s t a n t to t h e effects of 5 F U on g r o w t h a n d conidiation, b u t r e m a i n sensitive t o t h e effects of 5 F U R on g r o w t h a n d couidiation, a n d of 5 F d U I ~ on conidiation; a n d / u r A m u t a n t s are r e s i s t a n t o n l y t o t h e effects of 5 F d U R on couidiation. G r o w t h t e s t s were also carried o u t at 25 °./uIClO a n d /ulC15 were no longer r e s i s t a n t t o 5 F U a n d 5 F U R a t this t e m p e r a t u r e , / u l D 1 6 was u n a b l e t o grow on m i n i m a l medi u m a t 25 °, b u t g r o w t h was r e s t o r e d b y t h e a d d i t i o n of arginine t o t h e m e d i u m .

III. GeneticAnalysis o/~'luoropyrimidine Resistant Mutants T h e following m u t a n t strains were crossed t o a s u i t a b l e f l u o r o p y r i m i d i n e sensitive s t r a i n : /uIA6, /uIBT, /uIC8, /uID16, /uIEl01, /uIEl03, /u/F12, /ulF19, /urA2 a n d / u r A d . I n each case a t least 200 p r o g e n y were e x a m i n e d , a n d t h e seg r e g a t i o n d i d n o t d e v i a t e significantly f r o m 1 : 1 r a t i o for t h e r e s i s t a n t a n d sensitive p h e n o t y p e s . I n all cases w h e r e t h e p h e n o t y p e was complex, no r e c o m b i n a t i o n b e t w e e n t h e v a r i o u s c o m p o n e n t s of t h e p h e n o t y p e was observed, including t h e cold-sensitive arginine a u x o t r o p h y o f / u l D 1 6 . I t is t h e r e f o r e l i k e l y t h a t t h e p h e n o t y p e s in all cases r e s u l t f r o m a single m u t a t i o n .

IV. I n vivo Tests/or Pyrimidine Uptake and Salvage Activity in Fluoropyrimidine Resistant Mutants To t e s t w h e t h e r a n y f l u o r o p y r i m i d i u e r e s i s t a n t m u t a n t h a d become r e s i s t a n t because of a loss in u p t a k e or salvage a c t i v i t y , a n u m b e r of f l u o r o p y r i m i d i n e r e s i s t a n t m u t a n t s were crossed t o a pyrA34 s t r a i n (pyrA m u t a t i o n s l e a d to t h e absence of t h e u r i d i n e specific c a r b a m o y l p h o s p h a t e s y n t h e t a s e , E.C. 2.7.2.5., P a l m e r a n d Cove, 1975) a n d t h e d o u b l y m u t a n t strains were e x a m i n e d for t h e i r a b i l i t y to be r e p a i r e d b y exogenous p y r i m i d i n c s . The use of pyrA34, which is t e m p e r a t u r e sensitive for its p y r i m i d i n e r e q u i r e m e n t e n a b l e d t h e r e c o v e r y of t h e double m u t a n t a t t h e permissive t e m p e r a t u r e (25 °) even if t h e f l u o r o p y r i m i dine resistance m u t a t i o n p r e v e n t e d r e p a i r of t h e p y r i m i d i n e a u x o t r o p h y . The results of such crosses are s u m m a r i z e d in T a b l e 2. I t will be seen t h a t / u l E mut a t i o n s p r e v e n t r e p a i r of pyrA34 b y uracil or uridine, /ulF m u t a t i o n s p r e v e n t r e p a i r b y uracil only, a n d furA m u t a t i o n s p r e v e n t r e p a i r b y u r i d i n e only. N o

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168

Table 2. Summary of principle g e n e t i c Locus

Selection procedure

Resistance phenotype

Allele no.

Inhibition of growth by

Inhibition of conidiation by

5FU

5FUR 5FU

5FUR 5FOUR

Resistance to

]ulA

6

5FU

R

R

S

S

S

/ulB

7

5FU

R

R

S

S

S

/ulC

8, 10, 15

5FU

R

R

S

S

S

]ulD

16

5FU

R

R

S

S

S

]ulE

2, 5, 21, 25, 101-125

5FU 5FU -k 5FUR

R

R

R

R

S

]uIF

12, 19, 23, 24 31-38 1, 2, 4-9 12, 14, 19 22-27

5FU

R

S

R

S

S

5FUR 5FdUR

S

R

S

R

R

furA

R ~ resistant, S ~ sensitive.

recombinants between pyrA34 and ]ulCS, 10 and 15 were recovered (in each case, 350 progeny were scored). ]ulA6 a n d / u / D 1 6 partially suppressed the pyrimidine requirement of pyrA34 at 37°; and did not prevent its full repair b y exogenous uracil and uridine. ]~/B7 neither prevented the repair of pyrA34 b y uracil or uridine, nor suppressed its pyrimidine requirement. Further crosses were carried out to investigate whether ]ulA6 and /u/D16 suppressed other pyr mutations. Only 19yrA mutations were suppressed [pyr alleles tested A4, A12 (lack uridine specific carbamoyl synthetase); B14, B15, B37, C3, C41, C50 (lack aspartate carbamoyl transferase); D6, D23 (lack dihydro-orotase) ; E8 (lacks orotato reductase) ; F l l (lacks orotate phosphoribosyl transferase); G89 (lacks orotidine-5'-phosphate decarboxylase); N21, N25, N43 (lack both uridine specific carb~moyl synbhe~ase and aspartate carbymoyl transferase)--see Palmer and Cove, 1975 f o r details of these m u t a n t alleles].

FIuoropyrimidino Resistant Mutants of Aspergillus

169

and biochemical findings, and conclusions Uptake/Salvage of

Other data

Possible basis of fluoropyrimidine resistance

uracil

uridine

yes

yes

Linkage group III, suppresses pyrA, but only in absence of arginine

Lowered levels of ornithine carbamoyl transferase lead to earbamoyl phosphate accumulation, and cross feeding of uridine biosynthesis

yes

yes

Linkage group I, aspartate carbomoyl transferase insensitive to uridine repression

Uridine monophosphate biosynthesis insensitive to repression by pyrimidines

yes

yes

Linkage group VIII, closely linked pyrA, B, C, N. No aspartate earbamoyl transferase detectable in ]ulC8

Carbamoyl phosphate synthetase/aspartate carbamoyl transferase insensitive to inhibition by pyrimidines

yes

yes

Linkage group VIII, closely linked argC. Requires arginine at 25 ° Suppresses pyrA

Arginine specific carbamoyl phosphate synthetase insensitive to inhibition by arginine. Carbamoyl phosphate cross feeds uridine biosynthesis

no

no

Prevents repair of pyrA by uracil or uridine

Unable to convert uridine to uridine monophosphate

no

yes

Prevents repair of pyrA by uracil

Either unable to take up uracil or to convert it to uridine

yes

no

Prevents repair to pyrA by uridine

Unable to take up uridine or deoxyuridine

V. Genetic Location o/fulA, B, C a n d D As a r e s u l t of t h e crosses to p y r i m i d i n e a u x o t r o p h s , it h a d a l r e a d y been established t h a t ]ulC m a p p e d a t or v e r y close t o t h e pyrA, B, C, N c o m p l e x locus in l i n k a g e g r o u p V I I I . To e s t a b l i s h t h e c h r o m o s o m a l l o c a t i o n of t h e /ulA, B a n d D genes, a d i p l o i d b e t w e e n a f l u o r o p y r i m i d i n e r e s i s t a n t strain, a n d a m u l t i m a r k e d m a s t e r s t r a i n m a d e was, a n d s u b j e c t e d to analysis b y h a p l o i d i s a t i o n (McCully a n d F o r b e s , 1 9 6 5 ) . / u I A is l o c a t e d in l i n k a g e g r o u p I I I , / u l B in l i n a k g e g r o u p I, a n d / u l D in l i n k a g e g r o u p V I I I . No linkage was d e t e c t e d b e t w e e n / u l A 6 a n d argB2, b u t / u / D 1 6 was f o u n d to be closely l i n k e d to argC3. I t will be recalled t h a t t h e / u l D 1 6 m u t a t i o n l e a d s to a r e q u i r e m e n t for arginine a t 25 °. To i n v e s t i g a t e w h e t h e r t h e f l u o r o p y r i m i d i n e resistance a n d t h e arginine a u x o t r o p h y were pleiot r o p i c effects of t h e s a m e m u t a t i o n , / u I D 1 6 was r e v e r t e d on m i n i m a l m e d i u m a t 25 °. Of 50 s p o n t a n e o u s r e v e r t a n t s , 11 showed t h e s a m e level of f l u o r o p y r i m i d i n e resistance as /ulD16, 7 were as sensitive as t h e w i l d - t y p e , a n d t h e r e m a i n d e r

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170

Table 3. Aspartate earbamoyl transferase levels in/ulB7,/uIC8 and wild-type Genotype

I~o uridine

2.5 mM uridine

biA1 biA1/uIB7 biA1 ]ulC8

8.0 8.4

2.5 6.4

0

0

Myeelium was grown shaken, in liquid minimal medium, supplemented appropriately, for 28 h at 25°. Enzyme levels are in nanomoles of carbamoyl aspartate produced per mg soluble protein per rain.

showed varying levels of intermediate resistance. Revertants were also obtained after N-methyl Iq' nitro N nitrosoguanidine mutagenesis. A sample of 50 induced revertants were tested and all showed intermediate levels of resistance.

VI. Phenotype o/fulA, B, C and D Double Mutant Strains To see if/ulA, B, C and D mutations conferred resistance to fluoropyrimidines b y similar mechanisms, double mutants were obtained b y crosses between ]ulA6, /ulB7, /ulC8 and ]ulD16 strains, and their fluoropyrimidine resistance levels scored. All double mutants were more resistant t h a n either component single mutants, with the exception o f / u l B 7 / u l C 8 indicating that the ]ulB and C mutations perhaps confer fluoropyrimidine resistant b y similar mechanisms.

VII. Aspartate Carbamoyltrans/erase Levels in fulB7 and C8 Aspartate carbamoyltransferase levels in myeelium grown in the presence and absence of uridine were determined f o r / u / B 7 , /ulC8 and wild-type strains. From the results summarised in Table 3, it will be seen t h a t whereas enzyme levels in the wild-type are repressed b y uridine, those in ]ulBT, are almost insensitive to repression b y uridine. No activity could be detected in fulC8 myeelinm grown in either the presence or absence or uridine. Discussion

1. The rulE, fulF, furA Genes and the Uptake and Salvage o/Pyrimidines Mutations in t h e / u l E , [ulF and ]urA genes interfere with the repair of pyrimidine auxotrophies by exogenous pyrimidines. These genes are therefore likely to be involved in either the uptake of pyrimidines, or their conversion to uridine monophosphate. Since rulE mutants are resistant to both fluoro~racil and fluorouridine, these compounds must share a common step in the p a t h w a y leading to inhibition. Similarly/urA mutants are resistant to both fluorouridine and fluorodeoxyuridine, and so these compounds too share a common step in the pathway leading to inhibition. A model which is also consistent with the patterns of protection b y pyrimidines against the effects of fluoropyrimidines is given in Fig. 1. Uridine and deoxyuridine and their 5-fluoro derivatives are seen as sharing a common uptake system in the specification of which t h e / u r A gene might be involved. This is consistent with the reversal of both 5FUR and 5FdUR toxicity b y

Fluoropyrimidine Resistant Mutants of Aspergillus Uracil

Urld[ne

/

Deo~ridlua

171

OuLs~de

I (11 missing in £ulF

Uridlne m~n~phcs~haLe

Deom]uri41ue ~nv~ho~pha~e

Fig. 1. Modelto account for the properties of/ulE,/ulF and/urA mutants, and the protection afforded by pyrimidines against fluoropyrimidinetoxicity

both uridine and deoxyuridine. Uracil and 5FU have a separate uptake system which may be lacking in [ulF mutants. On the other h a n d / u l F mutants may be defective in the conversion of uracil to uridine. As uridine reverses the toxicity of 5FU, either uptake or this conversion step must be prevented by uridine. Finally ~alE mutants could be defective in the conversion of uridine to uridine monophosphate. While the simple model accounts for the phenotypes of/uIE, [uIF and/urA mutants, as well as the patterns of protection afforded by pyrirnidines other models are possible. In Saveharomyces cerevisiae uracil and uridine are thought to share the same uptake system (Jund and Laeroute, 1970) in contrast with the model proposed above for A. nidulans.

2. The fulA and D Genes Mutations in t he / ul A and D genes which lead to fluoropyrimidine resistance do not prevent repair of pyrimidine auxotrophies by exogenous pyrimidines. Such mutations do not therefore confer resistance by reducing uptake or salvage. It is likely that /ulA and D mutants are resistant because they show increased synthesis of endogenous pyrimidines. Since both classes of mutation suppress partially pyrA mutations (which abolish the uridine specific carbam,)yl phosphate synthetase, Palmer and Cove, 1975), but suppress no other class of pyrimidine auxotroph, both the /ulA and /ulD mutations probably lead to the normally arginine-specffic carbamoyl phosphate pool being made available for uridine biosynthesis. The effect of mutations isolated directly as suppressors of pyrA4 has been explained in similar terms (Palmer and Cove, 1975). Such su pyrA4 mutations have reduced levels of ornithine carbamoyl transferase, and only suppress pyrA4 in the absence of arginine, confirming that cross feeding of the uridine pathway is occuring from the normally arginine-specific carbamoyl phosphate pool, presumably as ~ result of a partial loss of ornithine transcarbamoylase. The suppression of pyrA by/ulA6 was shown to be similarly abolished by exogenous arginine, indicating that ]ulA6 and su pyrA4 suppress by a similar mechanism. They may even be allelic. The suppression of pyrA4 by/ulD 16 is not however abolished by arginine, and so the likely basis of suppression is the loss of inhibition or repression by arginine of the arghfine-specific carbamoyl phosphate synthetase. The close linkage of/ulD16 to argC, and the pleiotropic cold-sensitive arginine 6

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172

L.M. Palmer et al.

auxotrophy of ]ulD16 suggest that the two mutations m a y occur in the same gene which would most probably specify the arginine-specific carbamoyl phosphate synthetase. The argO3 allele would presumably lack enzyme activity under all conditions, while the ]ulD16 allele would specific an enzyme insensitive to arginine inhibition at 37 °, and lacking activity at 25 °. Mutants of Coprinus, having an arginine-specific carbamoyl phosphate which is insensitive to arginine inhibition have been described by Prevost (1966).

3. The fulB and C genes The ]ulB7 and ]uIC8 mutations neither prevent the repair of pyrimidine auxotrophies by exogenous pyrimidines, nor suppress pyrA mutations. They are unlikely to lead to fluoropyrimidine resistance either by blockage of the uptake or salvage pathways, or by a breakdown in the channelling of carbamoyl phosphate for arginine synthesis. They most probably confer resistance by having increased pyrimidine pool sizes arising from alterations in regulation of the biosynthetic pathway. This explanation is supported in the case of fulB7 by the demonstration that aspartate carbamoyl transferase in this strain is less sensitive to repression by uridine, than it is in the wild-type. The close linkage of ]ulC8 to the pyrA, B, C, N locus, and the failure to detect aspartate carbamoyl transferase activity in [ulC8 mutant strains, suggests that this mutation m a y lead to a uracil-specific carbamoyl phosphate synthetase/aspartate carbamoyl transferase complex which is both insensitive to inhibition by uridine, and more labile than norma]. The main conclusions arrived at this in section are summarised in Table 2.

Acknowledgements. We wish to thank the Science Research Council for a research grant to D. J. Cove, which partially supported this work. L. M. Palmer thanks the Science Research Council, and Trinity Hall, Cambridge for Research Studentships.

References Adelberg, E. A., Mandel, M., Chen, G. C. C. : Optimal conditions for mutagenesis by N-methyl N'-nitro N-nitrosoguanidine in Escherichia cell K12. Biochim. biophys. Res. Commun. 18, 788-795 (1965) Clutterbuck, A. J., Cove, D. J. : The genetic loci of Aspergillus nidulans. In: CRC handbook of microbiology, (Laskin, A.I. and Lechvalier, M. A., eds.), vol. IV, p. 665-675. Microbial metabolism, genetics and immunology. Cleveland, Ohio: Chemical Rubber Co. 1974 Cove, D. J. : The induction and repression of nitrate reductase in the fungus AspergiUus nidulans. Biochim. biophys. Acta. (Amst.) 113, 51-56 (1966) Furth, J. J., Cohen, S. S. : Inhibition of mammalian DNA polymerase by the 5" triphosphate of 1-D-arabino furanosyl cytosine and the 5' triphosphate of 9-D-arabino furanosyl adenine. Cancer Res. 28, 2061-2067 (1968) Jund, R., Lacroute, F. : Genetic and physiological aspects of resistance to 5-fluoropyrimidines in Saccharomyces cerevisiae. J. Bact. 102, 607-615 (1970) Mackintosh, M.E., Pritohard, R. H. : The production and replica plating of micro colonies of Aspergillus nidulans. Genet. Res. 4, 321-322 (1963) McKully, 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.: Pyrimidine metabolism in micro organisms. Bact. Rev. ~4, 278-343 (1970)

Fluoropyrimidine Resistant Mutants of Aspergillus

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Palmer, L.M., Cove, D. J. : Pyrimidine Biosynthesis in Aspergillu8 nidulans. Isolation and Preliminary Characterisation of Auxotrophic Mutants. Molec. gen. Genet. 188, 243-255 (1975) Pontceorvo, G., Roper, J. A., Hemmons, L.M., Macdonald, K. D., Bufton, A. W. J. : The genetics of Aspergillus nidulans. Advanc. Genet. 5, 141-238 (1953) Prevost, G. : Etude de la biosynthese de l'uracile chez le Coprln. C. R. Soc. Biol. (Paris) 160, 915-919 (1966) C o m m u n i c a t e d b y W. G a j e w s k i Dr. L. M. Palmer Department of Energy Thames House South Millbank London, S.W.1. England

Dr. C. Scazzocchio Dep~rtment of Biology University of Essex Wivenhoe Park Colchester, C04 3S Q England

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