allele specific, gene unspecific suppressors in aspergillus nidulans

Molec. gen. Genet. 177, 57-64 (1979) © by Springer-Verlag 1979

Allele Specific, Gene Unspecific Suppressors in Aspergillus nidulans

Timothy Roberts 1, Sylvia Martinelli 1, and Claudio Scazzocchio 2 1 Department of Botany, Birkbeck College, University of London, London, WC1E 7HX, England 2 Department of Biology, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, Essex, England

Summary. Seven suppressor mutations have been isolated in Aspergillus nidulans by coreversion of alleles in physiologically unrelated genes namely, alX, sB, alcA, putative structural genes for allantoinase, sulphate permease and alcohol dehydrogenase respectively. The suppressors are allele specific, gene unspecific. Those described map in four loci, suaA, B, C, D. suaA and suaB are on linkage group III, suaC and suaD on VII. suaBl l l , suaDl03 and suaDl08 are semi-dominant in their suppression of alX4 and sB43. suaAlO1, suaAl05 and suaCl09 are recessive and have a pleiotropic effect on morphology. SuaCl09 is cold sensitive for growth as is suall5, an unmapped mutation on linkage group III which is similar in morphology to suaCl09. The two mutations, suaAlO1 and suaAl05 have different spectra of suppression and morphologies, suaAl05 weakly suppresses alX4 and sB43 whereas suaAlO1 strongly suppresses these and alcA125, suaDl03 and suaDl08 have the same spectrum of suppression. The properties of these suppressors are consistent with their being informational suppressors of the nonsense type.

Introduction

Informational suppressors, and particularly suppressors of nonsense mutations, have been a most useful tool in the molecular genetics of the prokaryotes. Among the eukaryotes, nonsense suppressors have been identified and thoroughly studied in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (see Hawthorne and Leupold, 1974 for review) and in Neurospora crassa (Seale, 1972, 1976; Seale etal. , 1977). Informational suppressors have also

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been identified in Podospora anserina (Picard, 1973) and Coprinus lagopus (Todd and Casselton, 1973; Sealy-Lewis and Casselton, 1977). Putative nonsense suppressors have been described most recently in the nematode Caenorhabditis elegans (Waterston and Brenner, 1978). This communication reports the identification of allele specific, gene unspecific, informational suppressors in the ascomycete Aspergillus nidulans. While this work was in progress, suppressors of this type were obtained independently in A. nidulans by Bal and co-workers (Bal, Maciejko, Kajtaniak and Gajewski, 1978; Bal, personal communication).

The method used to obtain allele specific, gene unspecific suppressor mutations was chosen following the observation by one of us (Scazzocchio, unpub- lished) that alX4, a non-leaky allele mapping in the putative allantoinase structural gene (alX, Scazzoc- chio and Darlington, 1968) reverts extragenically and that the suppressors thus obtained are allele specific. Thus, forward mutations at a variety of genes, for which efficient selective techniques are available, can be induced in an alX4 background and each allele obtained checked for coreversion with atX4. Corever- sion has been successfully employed by Hawthorne and Mortimer (1963) in S. cerevisiae and by Seale (1972) in Neurospora crassa to obtain allele specific suppressors. The work described here has been reported briefly elsewhere (Roberts, Martinelli and Scazzocchio, 1978).

Materials and Methods

Strains. Strains of the following genotypes were used for mutagenesis :

yA1 pyroA4 alX4 ; JivA1 pabaA1 alX4 ; biA1 alX4.

y (yellow) and fw (fawn) are conidial colour markers and pyro, paba, bi represent aucotrophy for pyridoxine, p-aminobenzoic acid and biotin respectively.

0026-8925/79/0177/0057/$01.60

58 T. Roberts et al. : Allele Specific Suppressors in A. nidulans

Medium. Complete and minimal solid media are described in Cove (1966) and liquid minimal medium in Martinelli (1976). These additions were made to solid media, when required, to give the following final concentrations: 10 g/1 glucose or 10 ml/1 ethanol or 23.9 g/l sodium acetate as carbon sources; 0.85 g/1 NaNO3 or 0.46 g/1 NH4C1 or 0.1 g/1 allantoin as nitrogen sources; 1 g/1 Na2S203 • 5 H 2 0 as sulphur source; for resistance tests, Na2SeO4.10 H 2 0 0.8 g/l, K2CrO 4 1.9 g/l, altyl alcohol (propen-1- ol-3) 0.2 ml/1 added with 5 g/1 L-arabinose; for haploidisation benlate (du Pont, Commercial grade) was added to complete medium as a filtered aqueous extract, about 1 ml/l of a saturated solution.

Genetical Techniques. These are described in Pontecorvo et al. (1953), Pontecorvo and Kfifer (1958), McCully and Forbes (1965). Haploidisation was induced by benlate (Hastie, 1970).

Mutagenesis. All experiments were designed to kill 70 95% conidia used. Diethyl sulphate treatment: an ethanolic solution of diethyl sulphate was added to 5 ml conidia suspended in buffered saline (pH 7) at 30 ° to give a final concentration of 0.4%. The suspension was shaken for 25 rain then the mutagen destroyed by adding 1 ml of 0.5 M Tris-HC1 buffer (pH 9.5) and shaking vigorously for 1 h. Before plating, 0.08 ml of 5 M HC1 was added, to neutralise the suspension.

N-methyl-N'-nitro-N-nitrosoguanidine t reatment: crystals were added to 2 ml conidial suspension buffered with 0.1 m Tris- HC1 (pH 9.5). The final concentration was about 0.1%. The suspension was shaken at 35 ° for 2 h before plating.

Photodynamic mutagenesis : 8-methoxypsoralen (9 mg/ml ethanolic solution) was added to 5 ml aqueous conidial suspension to give a concentration of 90 gg/ml. The suspension was incubated in the dark for 0.5-1 h before exposure to a 125 watt Wood ' s light for 0.5 h at 25 cm distance.

Mutant Selection. Mutagenised suspensions were added to molten medium for overlaying, sB and sC mutat ions were selected by growth on minimal medium containing selenate, to which they are resistant (Arst, 1968). The genotypes were confirmed by their lack of growth on medium containing sulphate as sole sulphur source, sB mutat ions were distinguished from sC by the former conferring resistance to chromate.

alcA mutat ions were selected by their resistance to allyl alcohol and confirmed by their lack of utilisation of ethanol as sole carbon source whilst retaining the ability to ntilise acetate (Page, 197i).

Preparation of Mycelial Extracts. Mycelia for allantoinase assays were grown at 28 °, 100 r.p.m, for 23 h in 250 ml minimal medium per 21 flask. The enzyme was induced by adding allantoin 50 mg/1 and 2-thioxanthine 5 mg/1 at 18 h. Extraction was performed in 100 m M sodium pyrophosphate (pH 8.5). Mycelia were washed and soaked in buffer at 0 ° for 0.5 h then pressed dry and weighed. The mycelia were ground with 2 X wet weight of sand and 5 ml buffer per g mycelium. The extract was centrifuged at 4 ° for 20 rain at 50,000 g to remove debris.

Mycelia for alcohol dehydrogenase assays were grown in liquid minimal medium with 4 g/1 L-arabinose as carbon source and 1.5 ml/100 ml ethanol as inducer (Bailey and Arst, personal communication). The enzyme was extracted as above, but using 20 m M phosphate buffer (pH 6.5).

Enzyme Assays. Alcohol dehydrogenase (EC 1.1.1.1) was assayed as described by Page (1971). N A D H production was followed at 25 ° and 340 nm.

Allantoinase was assayed by the method of Lee and Roush (1964) as modified by D. Gor ton (personal communication), The hydrolysis of 3 4 m M allantoin was followed in 100mM Tris-HC1 buffer (pH 8.0) in the presence and absence of the mycelial extract.

The difference between these two determinations was taken as the amoun t of allantoin hydrolysed by enzyme catalysis. 1 ml sam- ples taken over a 20 min period at 26 ° were mixed with 1 ml cold HCI (150 mM) and 3 ml water and heated at 100 ° for 4 rain before cooling to 26 °. 1 ml phosphate buffer (40 mM, pH 7) and 1 ml phenyl hydrazine HC1 (3.3 g/1 filtered, freshly prepared, stored in dark) were added and the mixture incubated for 15 min before cooling on ice and adding 5 ml concentrated HC1 with 1 ml K3Fe(CN)6 (16.7 mg/ml, freshly prepared, in dark bottles). The mixture was incubated for a further 23-26 min at 26 ° and the optical density measured at 522 nm.

The molar concentration of allantoate formed was obtained from a s tandard curve constructed with varying concentrations of potassium allantoate.

Protein Estimation. Protein was determined by the method of Lowry et al. (1951).

Results

Isolation of Strains Carrying Cosuppressible Mutations

24spontaneous mutations and 100 induced by photodynamic treatment were isolated as selenate resistant strains in an alX4 background. These mutations map in the sB or sC loci, leading to defective utilisation of sulphate as sole sulphur source (Arst, 1968). When a sB mutation clearly cosuppressible with alX4 was identified (sB43, vide infra), 43 alcA mutations (lacking alcohol dehydrogenase, see materials and methods and Page, 1971) were isolated in an alX4 sB43 background after mutagenesis by either diethyl sulphate or N-methyl-N'-nitro-N-nitrosoguanidine.

Identification of Corevertible Mutations

Non-leaky mutations in the sB or sC loci were tested for coreversion with alX4 by plating on to three different media (i) containing thiosulphate as sulphur source and allantoin as sole nitrogen source, thus testing the reversion of the alX4 allele (ii) containing sulphate as sulphur source and nitrate as nitrogen source, thus testing the reversion of the sB43 allele (iii) containing sulphate as sulphur source and allantoin as nitrogen source, thus testing for coreversion. The number of colonies on (iii) gives an approximate estimate of the coreversion frequency. When a given allele was shown to corevert, strains carrying putative suppressors were identified by replica plating from medium (i) to (ii).

Similarly, non-leaky mutations in the alcA gene, obtained in an alX4, sB43 background, were tested for coreversion by plating mutagenised conidia on (i) thiosulphate as sulphur source, ammonium as ni-

T. R o b e r t s et al. : Allele Specif ic S u p p r e s s o r s in A. nidulans 59

Tab le 1. R e v e r s i o n o f sB, sC or alcA s ingly a n d co reve r s ion o f these m u t a n t s wi th alX4

F o r w a r d I so l a t i on m u t a g e n N o . o f R e v e r s i o n m u t a g e n R e v e r s i o n fo r

m u t a t i o n alleles sB, sC o r alcA a tes ted

N o . o f alleles

a p p a r e n t l y

c o r e v e r t i n g wi th alX4

sB s p o n t a n e o u s 9 p h o t o d y n a m b b - -

3 _+ -

2 + -

sB p h o t o d y n a m i c 12 p h o t o d y n a m i c - -

1 + -

2 2 4- 4 15 n o t tes ted -

sC s p o n t a n e o u s 6 p h o t o d y n a m i c - - 3 + -

5 4- -

sC p h o t o d y n a m i c 8 p h o t o d y n a m i c - -

7 _+ : - 19 + 4 21 n o t tes ted -

alcA d i e t h y l s u l p h a t e 22 d i e t h y l s u l p h a t e 4- 1 p h o t o d y n a m i c 4- -

alcA n i t r o s o g u a n i d i n e 2 d i e t h y l s u l p h a t e - - p h o t o d y n a m i c 4- -

5 d i e t h y l s u l p h a t e - - p h o t o d y n a m i c - -

6 d i e t h y l s u l p h a t e + -

p h o t o d y n a m i c + -

a - ind ica tes n o r eve r s ion ; + low revers ion , i.e. r eve r s ion a t a f r e q u e n c y be tween 0 % a n d 10% t h a t o f alX4; + h i g h revers ion ,

i.e. a t a level s imi la r to t h a t o f alX4 (see T a b l e 2) b P h o t o d y n a m i c m u t a g e n e s i s u s ing n e a r u l t r av io l e t l ight a n d 8 - m e t h o x y p s o r a l e n

F o r each allele, 3 x 10 a v iable c o n i d i a were m u t a g e n i s e d , t he r e to r e 5 x 107 v iable c o n i d i a were p la ted . (See m a t e r i a l s a n d m e t h o d s )

trogen source and ethanol as carbon source thus testing the reversion of the alcA allele (ii) sulphate as sulphur source, nitrate as nitrogen source and ethanol as carbon source, thus testing for coreversion of sB43 and the alcA allele (iii) sulphate as sulphur source, glucose as carbon source and allantoin as nitrogen source thus providing an estimate of the coreversion of sB43 and alX4. Colonies isolated on media (i) and (iii) were checked for coreversion of the other alleles on the appropriate media. Only one of thirty-six alcA alleles tested, alcA125, coreverted with alX4 and sB43. In Table 1, the results of the reversion studies are summarised and in Table 2 the frequencies of reversion and coreversion of alX4, sB43, alcA125 with two different mutagenic treatments are given. It should be noted that the frequency of coreversion was within an order of magnitude of the single reversion frequency. This implies that coreversion can occur by a single event.

Cosegregation of the Suppressor Phenotype

Strains containing all relevant suppressible mutations and presumed to contain a suppressor mutation were crossed to a wild-type strain. A minimum of

50 progeny were tested. The appearance of alX-, sB and alcA- progeny, when appropriate, confirmed the presence of a suppressor mutation. The absence of progeny suppressed for one allele but still displaying the mutant phenotype for at least one gene constitutes evidence that only one suppressor mutation segregated in the cross. Where suppression is partial, it is possible to distinguish between the wild-type and suppressed phenotypes for each mutation. In the event (vide infra) some suppressor mutations have pleiotropic effects thus allowing the scoring of the presence of the suppressor mutation. Strains containing the suppressible mutations alX4 and sB43 and a presumed suppressor were also crossed to aIX4 containing strains. In these crosses, homozygous for the alX4 mutation, the absence of progeny able to grow on allantoin but still displaying the mutant phenotype for sB43 and/or alcA125 constitutes evidence for cosuppression.

Of the eight revertants of other sB and sC alleles thus investigated three contained a suppressor for alX4 but a back mutation in sB or sC, three others contained two independent suppressor mutations and two had back mutated at both alX4 and sB. Five mutations designated sual03, sual08, sual09, sual l l

60

Table 2. Reversion of alX4, sB43 and alcA125

Alleles reverted Revertants as a proportion of viable conidia after mutagnesis

Photodynamic Diethyl sulphate

(1) Single reversion

alX4 2 x 10- 5 5 × 10 - 6

sB43 2x 10 -5 4 x 10 - 6

alcA125 1 x 10 .6 3 x 10 .6

(2) Coreversion

alX4, sB43 1.5 x 10 -6 2x 10 6 alX4, sB43, alcA125 1.0 x 10-6 1.5 x 10-6

3 x 108 viable conidia of an fwA pabaA alX4 sB43 alcAi25 strain were mutagenised with diethyl sulphate or by photodynamic mutagenesis (see materials and methods). Survival rates were 13% with diethyl sulphate and 18 % with photodynamic treatment. Therefore about 5 x 107 viable conidia were plated. The following media were used to select revertants and corevertants (i) glucose as carbon source, thiosulphate as sulphur source and allantoin as nitrogen source for reversion of alX4 (ii) glucose as carbon source, sulphate as sulphur source and nitrate as nitrogen source for reversion of sB43 (iii) ethanol as carbon source, thiosulphate as sulphur source and ammonium as nitrogen source for .reversion of alcA125 (iv) glucose as carbon source, sulphate as sulphur source and allantoin as nitrogen source for coreversion of alX4 and sB43 (v) for alX4 sB43 alcA125 coreversion rate, 100 colonies were repli- cated from (iv) to (iii). It was not possible to estimate directly coreversion of alcAI25 and alX4 owing to poor growth on ethanol with allantoin medium or of alcA125 and sB43 owing to excessive heterokaryon formation between singly reverted strains. The data come from one experiment. Similar reversion rates for alX4 were found in experiments summarised in Table 1

a n d s u a l l 5 w e r e s h o w n u n e q u i v o c a l l y to s u p p r e s s

b o t h alX4 a n d sB43. A n o t h e r s u p p r e s s o r sual05 pre -

v i o u s l y i s o l a t e d ( S c a z z o c c h i o , u n p u b l i s h e d ) as a s u p -

p r e s s o r o f alX4 w a s a l so s h o w n t o s u p p r e s s w e a k l y

sB43. sual09 i s o l a t e d in a n alX4 sB43 b a c k g r o u n d

a l so s u p p r e s s e s w e a k l y alcA125, sualOl, i s o l a t e d in

a n alX4 sB43 alcA125 b a c k g r o u n d s u p p r e s s e s s t r o n g l y

al l t h r e e al le les . A s t r a i n c a r r y i n g alX4 sB43 alcA125 a n d p r e s u m a b l y t h i s s u p p r e s s o r , sualO1, w a s c r o s s e d

to a w i l d - t y p e s t r a i n . 584 p r o g e n y w e r e s c o r e d fo r

t h e m o r p h o l o g i c a l e f fec t o f t h e s u p p r e s s o r o n

c o m p l e t e m e d i u m (v ide i n f r a ) a n d c o s u p p r e s s i o n o f

t he t h r e e al le les . T h e f o u r p h e n o t y p e s s h o w e d

c o m p l e t e c o s e g r e g a t i o n ( T a b l e 3). T h e s e a n d o t h e r

c h a r a c t e r i s t i c s o f t h e s u p p r e s s o r m u t a t i o n s a re s u m -

m a r i s e d in T a b l e 4 w h i l e F ig . 1 s h o w s t h e p h e n o t y p e s

o f a s a m p l e o f s u p p r e s s o r m u t a t i o n s .

Number and Location o f Suppressor Genes

M i t o t i c h a p l o i d i s a t i o n s h o w e d sual05, s u a l l l a n d s u a l l 5 t o b e o n l i n k a g e g r o u p I I I a n d t h a t sual03,

T. Roberts et al. : Allele Specific Suppressors in A. nidulans

Table 3. Phenotypes segregating in a cross between wild type and a strain carrying three suppressible mutations, alX4, sB43 and alcAI25 and their co-suppressor suaAlO1 which has a pleiotropic effect on morphology. Total progeny= 584

Phenotypes Morphology

alX4 sB43 alcA125 Normal Altered

+

+ sup

+ 47 83 sup 0 76 - 6 4 0

+ 0 71 sup 0 65 - 0 0

+ 52 0 sup 0 0 - 4 6 0

+

sup

+ 20 0 sup 0 0 - 2 4 0

+ 0 0 sup 0 0 - 0 0

+ 17 0 sup 0 0 - 1 9 0

The progeny have been scored for utilisation of allantoin as nitrogen source, of sulphate as sulphur source and of ethanol as carbon source and for the presence of the morphological alteration asso- ciated with suaAlOl. On sulphate plates, three phenotypes are distinguishable, the mutant (sB43), the wildtype (sB +) and the suppressed phenotype (sB43, suaAIO1) referred to as - , + and sup. in the table, respectively. The ethanol plates are analogous. On allantoin plates only two phenotypes are distinguishable. One cor- responds to the mutant (alX4) and the other is non mutant and comprises the alX + sua + , alX + suaAlOI and alX4 suaAlO1 classes.

In no case is a mutant suppressed for any one allele unsuppressed for another allele. All suppressed colonies had an altered morphology on complete medium and there were no cases in which mutant colonies had altered morphology

sual08 a n d sual09 w e r e o n l i n k a g e g r o u p VI I . A c ross

m a d e b e t w e e n sual05 a n d sualO1 in a h o m o z y g o u s

alX4 b a c k g r o u n d g a v e n o u n s u p p r e s s e d p r o g e n y in

t h e 274 e x a m i n e d , i n d i c a t i n g t he p o s s i b l e a l l e l i sm o f

t h e m u t a n t s . T h e s e m u t a t i o n s w e r e d e s i g n a t e d

suaAlO1 a n d suAl05, suaAlOl w a s f o u n d t o b e l i n k e d

b y 28 c e n t i m o r g a n s to alX4 in a c ross in w h i c h

584 p r o g e n y w e r e a n a l y s e d , s u a B l l l , a lso in l i n k a g e

g r o u p I I I , w a s l oose ly l i n k e d to suaAl05 i n c r o s s e s

( a p p r o x i m a t e l y 18 c e n t i m o r g a n s ) a n d t h e r e f o r e des ig -

n a t e d s u a B l l l . T h e m u t a t i o n s o n l i n k a g e g r o u p V I I

de f ine t w o u n l i n k e d loci in c rosses , C a n d D. sual09 was d e s i g n a t e d suaCl09 a n d t h e a l le l ic m u t a t i o n s

sual03 a n d sual08 p u t i n t o suaD. s u a l l 5 w a s l o c a t e d

o n l i n k a g e g r o u p I I I b u t w a s n o t i n v e s t i g a t e d f u r t h e r .

T. Roberts et al. : Allele Specific Suppressors in A. nidulans

Table 4. Properties of suppressor mutations

61

Suppressor mutation and mutagen used to induce it

Growth and suppression of suppressible alleles on test media

S.C. alX4 sB43 alcA 125 (DES) (PD) (DES)

Dominance of Cold suppression sensitive in diploids for growth

Morphological phenotype cosegregating with suppressor

suaAl01 (DES) 4 4 3 3 recessive No Yes suaAl05 (NTG) 5 2 1 - recessive No Yes suaBlll (PD) 5 4 4 n.t semi-dominant No No suaCl09 (PD) 3 3 3 2 recessive Yes Yes suaDl03 (PD) 5 4 4 - semi-dominant No No suaDl08 (PD) 5 4 4 n.t semi-dominant No No sua115 (PD) 4 3 3 n.t n.t Yes Yes

The mutagen used to produce each mutation is placed in brackets beside it, viz. : PD = photodynamic mutagenesis, DES = diethyl sulphate, NTG=N-methyl-N'-nitro-N-nitrosoguanidine. Growth of strains carrying suppressible alleles with a specific suppressor is given for synthetic complete medium (S.C.), allantoin medium (for alX4 suppression), sulphate medium (for sB43 suppression) and ethanol medium (for alcA125 suppression). Growth is expressed quantitatively from 0 (unsuppressed mutant) to 5 (wild-type). Growth on test medium indicates suppression of the relevant allele, n.t. =not tested

Allele Specificity o f Suppressor Mutations

N o n e of the suppressors of alX4 also suppressed alXl and alX2, nei ther o f which c o m p l e m e n t alX4 in a d ip lo id and are t ight ly l inked to it ( < 0.5 cM). Those mu ta t i ons suppress ing sB43 did no t suppress the stan- d a r d allele a t this locus sB3. sB43 and sB3 do no t c o m p l e m e n t and in a cross no wi ld- type r ecombinan t s were found in 183 progeny, alcA20, an allele of alcA125, was not suppressed by suaAlO1, the only suppressor i so la ted in a r ever tan t of alcA125, alcA20 and alcA125 do no t c o m p l e m e n t and in a cross gave no wi ld- type r ecombinan t s ou t o f 1 ,500progeny tested. (Table 4).

Apparent Efficiency of the Suppressor Mutations

The suppressor m u t a t i o n s had different efficiencies o f suppress ion o f the three suppress ib le alleles judg- ing by rad ia l g rowth and hypha l densi ty o f the suppressed mu tan t s c o m p a r e d to the same mutan t s unsuppressed , tes ted on the a p p r o p r i a t e m e d i u m (Fig. 1). The relat ive efficiencies are summar i sed be- l o w : s u p p r e s s i o n o f alX4 : suaDl03 =suaDl08 =suaB- 111 > suaAlO1 > sua115 = suaCl09 > suaAl05; suppress ion o f sB43 ." suaDl03 =suaD108=suaB- 111 >suaAlOl=suaClO9=sua115>suaA105; suppress ion of alcA125." suaAlOl>suaCl09, suaDl03 and suaAl05 do no t suppress alcA125. Other suppressors have no t been tested.

Pleiotropic Effects o f the Suppressor Mutations

suaAlO1, suaAl05, suaCl09 and the u n m a p p e d sual l5 have an a l te red m o r p h o l o g y af ter two day ' s g rowth on comple te m e d i u m at 37 ° . Af t e r one day ' s g rowth colonies car ry ing these suppressor mu ta t i ons

are clear ly smal ler than sua + strains. These charac te rs segregated with suppress ion in every cross and pro- vide an unequivoca l m e t h o d for scor ing the presence o f the suppressor muta t ion , suaB l l l , suaDl03 and suaDl08 have no a p p a r e n t m o r p h o l o g i c a l effects on comple te or synthet ic comple te media . Al l the suppressor mu ta t i ons were tested for g rowth at 37 ° , 30 ° and 20 ° on comple te med ium, suaCl09 and sua115 are c lear ly cold sensitive. Strains car ry ing these mu ta - t ions are comple te ly unab le to g row at 20 ° and grow very weakly at 30 °. In a cross be tween suaCl09 alX4 sB43 and suaC + alX4 sB ÷, the presence o f the suppressor was scored by suppress ion o f alX4. In every case, suppress ion o f aIX4 cosegrega ted with suppression o f sB43, the m o r p h o l o g i c a l effect a t 37 ° and the inabi l i ty to grow at 20 ° .

Dominance o f the Suppressor Mutations

The suppressor m u t a t i o n s were tested for d o m i n a n c e in d ip lo ids he te rozygous for the suppressor and homozygous for alX4 and sB43 and for alcA125 when appropr i a t e . The h o m o z y g o u s d ip lo id sua + aIX4 sB43/sua + alX4 sB43 and each h o m o z y g o u s d ip lo id sua- alX4 sB43/sua- alX4 sB43 were inc luded in the same plates as controls . The d ip lo ids were tested for suppress ion o f alX4 on a l l an to in as sole n i t rogen source and of sB43 on su lphate as sole su lphur source and in the case o f alcA125 on e thano l as sole ca rbon source.

The results showed suaAIO1, suaAl05, suaCl09 and sual l5 to be comple tey recessive, suaBlI1, suaDl03 and suaDl08 showed par t i a l suppress ion in- d ica t ing dose dependence o f the suppress ion process. The character is t ics o f the suppressor m u t a t i o n s are summar i sed in Table 4.


Table 5. Allantoinase activity in wild-type, mutant and suppressed strains

Genotype nMoles allantoate %of produced per min wild-type per mg protein activity

Wild-type 20 100 alX4 0 0 alX4, sua AIO1 0.9 4.5 alX4, suaAl05 2 10 aIX4, suaBl l l 0.97 4.9 alX4, suaCl 09 3.9 19.5 alX4, suaDl03 0 0 alX4, suaDl08 1.9 9.5

Strains were grown in liquid minimal medium at 28 ° with nitrate as nitrogen source. 50 mg/1 allantoin and 5 mg/l 2-thioxanthine were added at 18 h to fully induce allantoinase. Mycelium was harvested after further 5h growth. Duplicate assays were performed on duplicate mycelia and an average enzyme activity is given

locus showed a degree o f suppress ion in te rmedia te be tween tha t a f fo rded by suaAlO1 and by suaAl05.

N o c o m p l e m e n t a t i o n , e i ther posi t ive or negat ive was f o u n d be tween the two suaA alleles.

Fig. 1. Growth of suppressed and unsuppressed strains on various media. The plates from top to bottom are minimal media with (i) ethanol as carbon source, thiosulphate as sulphur source, ammonium as nitrogen source ; (ii) glucose as carbon source, thiosulphate as sulphur source, allantoin as nitrogen source; (iii) glucose as carbon source, sulphate as sulphur source, nitrate as nitrogen source; (iv) glficose, thiosulphate and nitrate. The ten spots have been inoculated as follows (relevant genotype given) from left to right and top to bottom: 1. suaCl09 aleA125 alX4 sB43, 2. suaBlI1 alX4 sB43, 3. suall5 alX4 sB43, 4. suaAl05 alcA125 alX4 sB43, 5. alcA125 alX4 sB43, 6. suaDl08 alX4 sB43, 7. suaA101 alcA125 alX4 sB43, 8. not inoculated, 9. suaDl03 alcA125 aIX4 sB43, 10 wild-type. Spots 2, 3, 6 have not been inoculated on medium (i)

Complementation at the suaA Locus

A d ip lo id h o m o z y g o u s for alX4 and he te rozygous for the two recessive suppresso r alleles a t the suaA

Restoration o f Enzyme Activities

In the case o f each m u t a t i o n used in this s tudy alX4,

sB43, and alcA125 the p h e n o t y p e was non- l eaky on the test me d ium. However , b a c k g r o u n d g rowth occurs owing to the type of pa thways involved and the presence of c on t a mina t i ng a l te rna t ive sources of n i t rogen, su lphur and ca rbon in the med ium. This is negl igible for alX4 and alcA125 but cons iderab le in the case o f s B m u t a t i o n s (see Fig. 1) where res idual su lpha te up t ake occurs via o ther permeases (Arst , 1968). This leakiness is locus specific and for each m u t a t i o n used the res idual g rowth is the least obse rved for the locus in quest ion.

Su lpha te up t ake has no t been measu red in the m u t a n t and suppressed strains. St ra ins car ry ing aleA125 have 1% of the wi ld - type a lcoho l dehydro - genase act ivi ty when induced with e thano l on L-a rab i - nose as c a r b o n source. Stra ins wi th bo th alcA125

and suaAlO1 did no t show a de tec tab le increase in a lcoho l dehyd rogenase activity.

The a l lan to inase activi t ies o f wi ld- type , alX4 and suppressed s t ra ins are shown in Table 5. In each case except for suaDl03, a r e s to ra t ion o f a l l an to inase ac- t ivi ty is appa ren t . I t is no t e w or thy tha t the enzyme levels do n o t c o r r e s p o n d to the efficiency o f suppression, in vivo, descr ibed above. Whi l e the levels of a l l an to inase show conclusively tha t enzyme act ivi ty is res tored , it is diff icult to assess the signif icance o f differences at low levels o f activity. I f these differ-

T. Roberts et al. : Allele Specific Suppressors in A. nidulans 63

ences prove significant they may reflect differences of stability between the variously suppressed enzyme species.

Discussion

The suppressor mutat ions described in this publica- tion are allele specific and gene unspecific. Thus they are informational rather than physiological suppressors. While in the absence of characterised mutational changes in at least one of the proteins coded by alX, sB or alcA it is not possible to reach definitive conclu- sions on the nature of the suppressor mutations, some discussion of their properties is merited here. Statisti- cally, it would be unlikely to obtain cosuppression of three different missense mutations. Frameshift suppressors would superficially resemble nonsense suppressors. However, it is unlikely that alX4 and alcA125 are frameshift mutations as they have been isolated after diethyl sulphate treatment. It could also be argued that coreversion experiments are likely to expose general suppressors which alter translational fidelity.

The situation in which both semi-dominant suppressors and recessive suppressors mapping at different loci suppress the same alleles is similar to that found for nonsense suppressors in Saccharomyces cerevisiae. In this organism there are amber and ochre specific dominant or semidominant suppressors some of which have been shown to map in transfer R N A genes (Capecchi, Hughes and Wahl, 1975; Gesteland et al., 1976; Piper et al., 1976). There is also a class of recessive amber/ochre non-specific suppressors (Smirnov et al., 1974; Gerlach, 1975; Hawthorne and Leupold, 1974). It has been proposed that the last class of suppressors map in a gene coding for a protein involved in chain termination (Smirnov et al., 1976) or coding for an essential r ibosomal protein (Gerlach, 1975, 1976). It is suggestive that the semi-dominant suppressors described in this communication do not seem to have obvious pleiotropic effects, while all the recessive suppressors result in slow growth, changes in colony morphology and in two cases, in cold sensitivity. These effects suggest that the mis- reading caused by the recessive suppressor mutat ion is more widespread than that caused by the dominant suppressor mutations. The cold sensitivity could suggest the participation of the product of the suaC gene in a multimeric aggregate.

A striking property of the two mutations mapping at the suaA locus is their different spectrum of suppression, suaAl05 significantly suppresses alX4 and weakly sB43 while not suppressing alcA125, suaAlO1, a noncomplementary, tightly linked suppressor muta-

tion, suppresses strongly all three mutations. Either the two alleles are inserting different amino acids at the same mutated sites or they insert the same amino acid with different efficiency, the number of suppressed molecules of alcohol dehydrogenase being in the latter case below the threshold of detection in strains carrying suaAl05.

The most likely explanation is that all these suppressors act on nonsense mutations. Paromomycin has been found to suppress phenotypically alX4 and a new niaD mutant cosuppressible with alX4, sB43 and alcA125. This suppression is allele specific and does not affect sB43 and alcA125. Paromomycin acts on many but not all nonsense mutations in yeast (Singh et al., 1979; Palmer et al., 1979).

The semidominant suppressor mutations may map in transfer R N A genes and the recessive ones may be mutations in genes coding for proteins involved in the translation of proteins or in termination of this process.

The recent isolation of suppressible mutations in the structural gene for nitrate reductase, niaD, (Rob- erts, Scazzocchio, Martinelli, in preparation) where the protein is well characterised and monospecific anti- bodies are available (Lewis, 1975; Tomsett , 1977) might clarify the nature of the mutations described here.

Please note that in our previous communication (1978), the sua genes were referred to as sup.

sua ÷ is the wild-type, nonsuppressing phenotype while sua indicates the suppressor mutation.

Acknowledgement. T. Roberts is grateful to the Science Research Council for the award of a post-graduate studentship. We are all grateful to Tania Cresswell and Richard Packer for technical assistance and to Herbert Arst and Heather Sealy-Lewis for their critical reading of the manuscript.

References

Arst, H.N., Jr.: Genetic analysis of the first steps of sulphate metabolism in Aspergillus nidulans. Nature 219, 268 270 (1968)

Bal, J., Maciejko, D.M., Kajtaniak, E.M., Gajewski, W.: Super- suppressors in Aspergillus nidulans. Mol. Gen. Genet. 159, 22%228 (1978)

Capecchi, M.R., Hughes, S.H., Wahl, G.M. : Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro. Cell 6, 269 277 (1975)

Cove, D.J.: The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113 51 56 (1966)

Gerlach, W.L.: Genetic properties of some amber-ochre super- suppressors in Saccharomyces cerevisiae. Mol. Gen. Genet. 138, 58-63 (1975)

Gerlach, W.L.: Mutation properties of sup P amber-ochre super- suppressors in Saccharomyces cerevisiae. Mol. Gen. Genet. 144, 213-215 (1976)

Gesteland, R.F., Wolfner, M., Grisafi, P., Fink, G., Botstein, D.,


Roth, J.R.: Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA. Cell 7, 381 390 (1976)

Hastie, A.C. : Benlate-induced instability of Aspergillus diploids. Nature 226, 771 (1970)

Hawthorne, D.C., Leupold, U.: Suppressor mutations in yeast. Curt. Top. Microbiol. Immunol. 64, 147 (1974)

Hawthorne, D.C., Mortimer, R.K.: Super-suppressors in yeast. Genetics 48, 61%620 (1963)

Lee, K.W., Roush, A.H. : Allantoinase assays and their application to yeast and soybean allantoinases. Arch. Biochem. Biophys. 108, 460467 (1964)

Lewis, N.J. : Immunological and biochemical studies of the genetic determination of xanthine dehydrogenase and nitrate reductase of Aspergillus nidulans. Ph.D. thesis, University of Cambridge (1975)

Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951)

Martinelli, S.D. : Conidiation of Aspergillus nidulans in submerged culture. Trans. Br. Mycol. Soc. 67, 121-128 (1976)

McCully, K.S., Forbes, E.: The use ofp-fluorophenyl alanine with 'master strains' of Aspergillus nidulans for assigning genes to linkage groups. Genet. Res. (Cambridge) 6, 352-359 (1965)

Page, M.M. : Genetical and biochemical studies on the catabolism of amines and alcohols in Aspergillus nidulans. Ph .D. thesis, University of Cambridge (1971)

Palmer, E., Wilhelm, J.M., Sherman, F.: Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics. Nature 277, 148 150 (1979)

Picard, M. : Genetic evidence for a polycistronic unit of transcrip- tion in the complex locus "14" in Podospora anserina. II. Genetic analysis of informational suppressors. Genet. Res. (Cambridge) 21, 1-15 (1973)

Piper, W.P., Wessertein, M., Engbaek, F., Kaltoft, K., Celis, J.E., Zeuthen, J., Liebman, S., Sherman, F. : Nonsense suppressors of Saccharomyces cerevisiae can be generated by mutation of the tyrosine tRNA anticodon. Nature 262, 757-761 (1976)

Pontecorvo, G., K/ifer, E.: Genetic analysis based on mitotic re- combination. Adv. Genet. 9, 71-104 (1958)

Pontecorvo, G., Roper, J.A., Hemmons, L.M., Macdonald, K.D., Burton, A.W.J. : The genetics of Aspergillus nidulans. Adv. Genet. 5, 141-238 (1953)

Roberts, T.J., Martinelli, S.D., Scazzocchio, C. : Allele-specific suppressor mutations in Aspergillus nidulans. Heredity 41, 129 (1978)

Scazzoechio, C., Darlington, A.J. : The induction and repression of the enzymes of purine breakdown in Aspergillus nidulans. Biochim. Biophys. Acta 166, 557-568 (1968)

Seale, T.W. : Super-suppressors in Neurospora crassa. I. Induction, genetic localisation and relationship to a missense suppressor. Genetics 70, 385 396 (1972)

Seale, T.W.: Super-suppressor action spectrum in Neurospora. Mol. Gen. Genet. 148, 105-108 (1976)

Seale, T.W., Brett, M., Baron, A.J., Fincham, J.R.S. : Amino acid replacements resulting from suppression and missense reversion of a chain-terminator mutation in Neurospora. Genetics 86, 261 274 (1977)

Sealy-Lewis, H., Casselton, L.A. : Dominant and recessive informational suppressors of a missense mutation in Coprinus. Mol. Gen. Genet. 151, 189 195 (1977)

Singh, A., Ursic, D., Davies, J. : Phenotypic suppression and mis- reading in Saccharomyces cerevisiae. Nature 277, 146-148 (1979)

Smirnov, V.N., Kreier, V.G., Lizlova, L.V., Andrianova, V . M . , Inge-Vechtomov, S.G.: Recessive super-suppression in Yeast. Mol. Gen. Genet. 129, 105-121 (1974)

Smirnov, V.N., Surguchov, A.P., Fominykch, E.S., Saprygina, T.V., Inge-Vechtomov, S.G.: Recessive nonsense-suppression in yeast: further characterisation of a defect in translation. FEBS Lett. 66, 12-15 (1976)

Todd, N.K., Casselton, L.A. : Non-complementation and recessive- ness as properties of missense suppressor genes in the fungus Coprinus. J. Gen. Microbiol. 77, 197 207 (1973)

Tomsett, A.B. : Structure and function in the niiA and niaD gene region of Aspergillus nidulans. Ph.D. thesis, University of Cambridge (1977)

Waterston, R.H., Brenner, S. : A suppressor mutation in the nematode acting on specific alleles of many genes. Nature 275, 715-719 (1978)

Communicated by W. Gajewski

Received March 14/July 31, 1979

allele specific, gene unspecific suppressors in aspergillus nidulans

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