Mol Gen Genet (1992) 231:485-488 © Springer-Verlag 1992
Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation Kim L. Hawker, Paul Montague, and James R. Kinghorn Plant MolecularGenetics Unit, Sir Harold Mitchell Building,Universityof St. Andrews, Fife KY16 9TH, UK
Summary. The regulation of the expression of the A. nidulans niiA and niaD genes (encoding nitrite reductase and nitrate reductase activities, respectively) was investigated by Northern blotting. It was demonstrated that expression of the niiA and niaD genes is controlled at the level of mRNA accumulation and that mutations within the nirA and areA regulatory genes, as well as certain mutations within niaD itself or cnxE (for its molybdenum cofactor), markedly affect niiA and niaD transcript levels. Key words: Filamentous fungi trate assimilation
Gene regulation - Ni-
Introduction The genetics of the nitrate assimilation pathway of the filamentous fungus Aspergillus nidulans has been extensively investigated (see Cove 1979; Scazzocchio and Arst 1989; Kinghorn 1989; Tomsett 1989 for reviews). The genes, niaD and niiA, encode the enzymes nitrate reductase (NR) and nitrite reductase (NiR) respectively (Cove and Pateman 1963 ; Pateman et al. 1967). Recent analysis by molecular cloning confirms evidence from classical genetics that the niiA and niaD genes are contiguous and are divergently transcribed from a relatively short (1262 bp) intergenic region (Tomsett and Cove 1979; Johnstone et al. 1990). Furthermore a third gene, crnA, thought to be involved with nitrate uptake is situated adjacent to the niiA gene (Tomsett and Cove 1979; Brownlee and Arst 1983; Unkles et al. 1991); the gene cluster is located on chromosome VIII. Finally, there are eight cnx loci, necessary for the formation of the molybdenum-containing co-factor which is inserted into the NR apoenzyme (Cove and Pateman 1963). The cnxE gene product is thought to be required for the incorporation of molybdenum into the NR molecule (Arst et al. 1970). Offprint requests to: J.R. Kinghorn
Classical genetic experimentation has indicated that the expression of the niiA and niaD gene products is regulated by two positive-acting regulatory genes, namely areA and nirA. The nirA gene is pathway-specific and its product is required for nitrate induction of NR and NiR (Cove and Pateman 1969). The areA gene product is responsible for nitrogen metabolite repression (Cohen 1972; Arst and Cove 1973; Hynes 1975). Furthermore, regulation of NR and NiR activities is considered to be exerted by NR itself (Cove and Pateman 1969; MacDonald et al. 1973; Cove 1979), requiring both functional niaD and cnx gene products. Biochemical analysis of mutants shows that an almost identical situation exists in Neurospora crassa (Tomsett and Garrett 1981). Using molecular techniques we further investigated the level at which these genes exert an effect on the regulation of niiA and niaD gene expression.
Materials and methods Strains. The A. nidulans strain biA1 was used as wild type. The mutant strains used had the following genotypes: yA1 wA3 niaDlO; biA1 niaD16; biA1 niaD21; biA1 niaD26; biA1 puA2 niaD42; biA1 niiA17; yA2 biA1 crnA1; biAl areA19; biA1 xprD1; biAI nirACl, biA1 cnxE17 and yA2 pyroA4 nirA1. Growth conditions. Mycelia from each strain were grown in liquid minimal media with the required supplements (Cove 1966) and the nitrogen sources indicated in the text. Incubation was carried out at 30° C, with 250 rpm orbital shaking, for a period of 12 h. Where a switch in culture conditions was necessary, mycelia were grown for a further 4 h. Northern blotting, hybridisation and DNA probes. Total cellular RNA was obtained by the method of Cathala et al. (1983) and l0 gg samples were electrophoresed on a 1.2% formaldehyde denaturing agarose gel. RNA was transferred to nylon membrane (Amersham International) by Northern blotting and UV irradiated. Hybridisa-
486
don was carried out at 42 ° C in 40% formamide to 32p_ hexaprime labelled D N A probes (Sambrook et al. 1989). A 0.9 kb EcoRI fragment of pSTA3 (Johnstone et al. 1990) was used as a niiA-specific probe. The niaD-specific probe was the 2.4 kb J(baI fragment of pSTA1 (Johnstone et al. 1990). Filters were washed at 55°C in 0.1 x SSC, 0.1% SDS. After autoradiography, filters were stripped of probe by submerging in 0.1% SDS for 1 h at 100° C. Rehybridisation to radiolabelled A. nidulans actA fragments, encoding actin (Fidel et al. 1988) was included as an internal control to assess RNA loading and transfer.
Results
Transcript levels in wild type
The 3.2 kb niiA and 2.8 kb niaD (Fig. 1) transcripts are observed in wild type cells when nitrate (lane 1) and nitrite (lane 2) are sole nitrogen sources. In contrast, no transcripts are seen when ammonium (lane 3), glutamate (lane 4), proline (lane 5) or glutamine (data not shown) are supplied. When the inducer (nitrate) and a neutral nitrogen source (proline) is used as combined nitrogen source (lane 6) synthesis of niiA and niaD transcripts occurs. However, neither transcript is visible when nitrate (inducer) and ammonium (lane 7) or glutamine (data not shown) (both nitrogen metabolite repressing substances) are supplied simultaneously.
1 2
3
4
5
~ ~
6
7
~
niiA
~
niaD
actA Fig. 1. Northern blot of niiA and niaD transcripts in wild-type cells grown in minimal media with 10 m M nitrogen source; Lane 1, nitrate; lane 2, nitrite; lane 3, a m m o n i u m ; lane 4, glutamate; lane 5, proline; lane 6, nitrate (5 mM) and proline (5 m M ) ; and lane 7, nitrate (5 mM) and a m m o n i u m (5 mM). For hybridisation conditions and D N A probes, see Materials and methods. Filters were stripped of probe and re-hybridised using the A. nidulans actA gene (Fidel et al. 1988) in all experiments
1 2
3
4
5
!
niiA
niaD
Transcript levels in nirA and areA regulatory mutant strains
The nirA and areA pleiotropic loss-of-function mutants, nirA1 (Fig. 2, lane 2) (Cove and Pateman 1969) and areA19 (lane 4) (Hynes 1975) are shown to be incapable of niiA or niaD m R N A synthesis under inducing conditions, in contrast to the wild-type strain (compare with lane 1). The mutation nirACl (lane 3) (Cove and Pateman 1969) results in the constitutive synthesis of niiA and niaD transcripts when glutamate is available as the nitro-
actA Fig. 2. Northern blot of
niiA and niaD transcripts in regulatory gene m u t a n t backgrounds. Lane 1, wild-type grown on minimal medium with 5 m M a m m o n i u m and transferred to 10 m M nitrate; lane 2, nirA1, 5 m M a m m o n i u m and transferred to 10 m M nitrate, lane 3, nirAC1, I0 m M glutamate; lane 4, areAl9, 5 m M ammonium and transferred to I0 m M nitrate and lane 5, xprD1, 5 m M a m m o n i u m and 5 m M nitrate
Table 1. Chlorate resistance, NiR activities and niiA and niaD transcript levels Strain
Chlorate resistance a' b Glutamate c
Urea ~
Proline °
Inducible (I) or constitutive (C) NiR activities
Wild type
S
S
S
I
I
niaD 10 niaD16 d niaD26 a niaD21 d niaD42 cnxE17
R R R
R R R
R R R
C C C
C C C
S S
S/R S/R
S/R S/R
I I
I I
R
R
R
C
C
Inducible (I) or constitutive (C) niiA and niaD transcript levels
Minimal medium with 200 m M sodium chlorate. Plates were incubated at 37 ° C for 4 days b R (resistance) indicates considerable growth; S (sensitivity) no growth; S/R (intermediate resistance) low level of growth c The concentration of the nitrogen source was 10 m M d Taken from Cove 1976
487
1
2
3
4
5
6
7 niiA
niaD
actA Fig. 3. Northern blot of niiA and niaD transcripts in structural gene mutant strains grown on 10 mM glutamate: lane 1, wild type; lane 2, niaD21; lane 3, niaD26; lane 4, niaD42; lane 5, niiA17; lane 6, cnxE17 and lane 7, crnA 1 gen source. N o transcripts are observed in wild-type cells u n d e r this condition (Fig. 1, lane 4). A g a i n in contrast to wild-type (Fig. 1, lane 7), niiA and niaD transcripts are observed w h e n xprD1 (an allele o f the area gene), which results in depression o f a n u m b e r o f nitrogen metabolite repressible systems ( C o h e n 1972; Arst and Cove 1973), is g r o w n in the presence o f nitrate and a m m o n i u m (Fig. 2, lane 5).
Transcript levels in structural gene mutant strains The data presented in Fig. 3 show that b o t h niiA and niaD transcripts are synthesised in the absence o f inducer in m u t a n t strains niaD26 (lane 3), niaDlO and niaD16 (K. Hawker, unpublished results) and cnxE17 (lane 6). In contrast, niiA and niaD transcripts are n o t observed in m u t a n t strains niaD21 (lane 2) and niaD42 (lane 4), similar to wild type (lane 1), w h e n cells are g r o w n in n o n - i n d u c i n g conditions, and they show n o r m a l inducibility by nitrate (K. H a w k e r , unpublished results). Cove (1976, M a c D o n a l d et al. 1973) showed that niaD and cnx m u t a n t strains which show normal, i.e. wild-type, regulation o f N i R activity a n d the m u t a n t N R protein are m o d e r a t e l y sensitive to chlorate whilst strains that are constitutive for N i R are highly chlorate resistant. N o r t h e r n blotting o f R N A f r o m the m u t a n t s previously tested by Cove (1976, M a c D o n a l d et al. 1973) as well as m u t a n t s tested here (Table 1) shows that transcript levels m i r r o r e n z y m e activities and the level o f chlorate resistance. Finally, it is n o t e w o r t h y that strains having mutations in niiA (niiA17) or crnA (crnA1) result in wild-type niiA and niaD transcript levels (Fig. 3, lanes 5 and 7 respectively); niiA and niaD expression in these m u t a n t s is dependent on nitrate induction (K. Hawker, u n p u b lished results).
Discussion This molecular analysis o f representative m u t a n t s defective in nitrate assimilation confirms previous formal biochemical genetic results and shows that control is re-
flected in the steady-state level o f transcript. T h a t a functional niaD p r o d u c t is required for wild-type regulation o f niiA and niaD m R N A synthesis is suggested by the fact that certain m u t a t i o n s within niaD (e.g. niaDlO, niaD16 and niaD26) a n d cnxE result in constitutive niiA and niaD gene expression. Such m u t a t i o n s also lead to resistance to higher concentrations o f chlorate. H o w e v e r other m u t a n t strains in niaD (e.g. niaD21 and niaD42), which are significantly less chlorate resistant, are wildtype with regard to transcript synthesis.
Acknowledgements. We wish to thank Drs. B. Tomsett and A.J. Clutterbuck, Professors H.N. Arst and M.J. Hynes for providing mutant strains. J.R.K. acknowledges funds from the Science and Engineering Research Council (grant number GR/D/48496). J.L.H. is indebted to the same research council for a postgraduate research studentship (Quota Award).
References Arst HN Jr, Cove DJ (1973) Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet 176 : 111-141 Arst HN Jr, MacDonald DW, Cove DJ (1970) Molybdate metabolism in Aspergillus nidulans. I. Mutations affecting nitrate reductase and/or xanthine dehydrogenase. Mol Gen Genet 108:129145 Brownlee AG, Arst HN Jr (1983) Nitrate uptake in Aspergillus nidulans and the involvement of the third gene of the nitrate assimilation gene cluster. J Bacteriol 155 : 1138-1146 Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:32%335 Cohen BL (1972) Ammonium repression of extracellular protease in Aspergillus nidulans. J Gen Microbiol 71:293--299 Cooley RN, Tomsett AB (1985) Determination of the subunit size of NADPH-nitrate reductase from Aspergillus nidulans. Biochim Biophys Acta 831:89-93 Cove DJ (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113:51-56 Cove DJ (1976) Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation. Mol Gen Genet 146 : 147-159 Cove DJ (1979) Genetical studies of nitrate assimilation in Aspergillus nidulans. Biol Rev 54:291-303 Cove DJ, Pateman JA (1963) Independently segregating loci concerned with nitrate assimilation in Aspergillus nidulans. Nature 168:262-263 Cove DJ, Pateman JA (1969) Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. J Bacteriol 97:13741378 Fidel S, Doonan JM, Morris NR (1988) Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a actin. Gene 70 : 283-293 Hynes MJ (1975) Studies on the role of the area gene in the regulation of nitrogen catabolism in Aspergillus nidulans. Aust J Biol Sci 28:301 313 Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Brow MAD, Unktes SE, Clutterbuck AJ, Kinghorn JR, Innis MA (1990) Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90: 181192 Kinghorn JR (1989) Genetic, biochemical and structural organisation of the Aspergillus nidulans crnA-niiA-niaD gene cluster. In: Wray JL, Kinghorn JR (eds) Molecular and genetic aspects of nitrate assimilation. Oxford University Press, Oxford, pp 69-87
488 MacDonald DW, Cove D J, Coddington A (1973) Cytochrome c reductases from wild-type and mutant strains of Aspergillus nidulans. Mol Gen Genet 128:187-199 Pateman JA, Rever BM, Cove DJ (1967) Genetical and biochemical studies of nitrate assimilation in Aspergillus nidulans. Biochem J 104:103-111 Sambrook J, Fritsch EF, Maniatis T (1982) Molecular cloning: A laboratory manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbor, New York Scazzocchio C, Arst HN Jr (1989) Regulation of nitrate assimilation in Aspergillus nidulans. In: Wray JL, Kinghorn JR (eds) Molecular and genetic aspects of nitrate assimilation. Oxford University Press, Oxford, pp 314-363 Tomsett B, Cove DJ (1979) Deletion mapping of the niiA-niaD gene region of Aspergillus nidulans. Genet Res 34:19-32
Tomsett B, Garrett RH (1981) Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: Evidence for autogenous control by nitrate reductase. Mol Gen Genet 184:183-190 Tomsett B (1989) Nitrate assimilation in ascomycete fungi. In: Boddy L, Merchant R, Read DJ (eds) Nitrogen, Phosphorus and Sulphur Utilisation by Fungi. Cambridge Press, Cambridge Unkles SE, Hawker KL, Grieve C, Campbell EI, Montague P, Kinghorn JR (1991) crnA encodes a nitrate transporter in Aspergillus nidulans. Proc Nat Acad Sci USA 88 : 204-208
C o m m u n i c a t e d b y W. G a j e w s k i
Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation Kim L. Hawker, Paul Montague, and James R. Kinghorn Plant MolecularGenetics Unit, Sir Harold Mitchell Building,Universityof St. Andrews, Fife KY16 9TH, UK
Summary. The regulation of the expression of the A. nidulans niiA and niaD genes (encoding nitrite reductase and nitrate reductase activities, respectively) was investigated by Northern blotting. It was demonstrated that expression of the niiA and niaD genes is controlled at the level of mRNA accumulation and that mutations within the nirA and areA regulatory genes, as well as certain mutations within niaD itself or cnxE (for its molybdenum cofactor), markedly affect niiA and niaD transcript levels. Key words: Filamentous fungi trate assimilation
Gene regulation - Ni-
Introduction The genetics of the nitrate assimilation pathway of the filamentous fungus Aspergillus nidulans has been extensively investigated (see Cove 1979; Scazzocchio and Arst 1989; Kinghorn 1989; Tomsett 1989 for reviews). The genes, niaD and niiA, encode the enzymes nitrate reductase (NR) and nitrite reductase (NiR) respectively (Cove and Pateman 1963 ; Pateman et al. 1967). Recent analysis by molecular cloning confirms evidence from classical genetics that the niiA and niaD genes are contiguous and are divergently transcribed from a relatively short (1262 bp) intergenic region (Tomsett and Cove 1979; Johnstone et al. 1990). Furthermore a third gene, crnA, thought to be involved with nitrate uptake is situated adjacent to the niiA gene (Tomsett and Cove 1979; Brownlee and Arst 1983; Unkles et al. 1991); the gene cluster is located on chromosome VIII. Finally, there are eight cnx loci, necessary for the formation of the molybdenum-containing co-factor which is inserted into the NR apoenzyme (Cove and Pateman 1963). The cnxE gene product is thought to be required for the incorporation of molybdenum into the NR molecule (Arst et al. 1970). Offprint requests to: J.R. Kinghorn
Classical genetic experimentation has indicated that the expression of the niiA and niaD gene products is regulated by two positive-acting regulatory genes, namely areA and nirA. The nirA gene is pathway-specific and its product is required for nitrate induction of NR and NiR (Cove and Pateman 1969). The areA gene product is responsible for nitrogen metabolite repression (Cohen 1972; Arst and Cove 1973; Hynes 1975). Furthermore, regulation of NR and NiR activities is considered to be exerted by NR itself (Cove and Pateman 1969; MacDonald et al. 1973; Cove 1979), requiring both functional niaD and cnx gene products. Biochemical analysis of mutants shows that an almost identical situation exists in Neurospora crassa (Tomsett and Garrett 1981). Using molecular techniques we further investigated the level at which these genes exert an effect on the regulation of niiA and niaD gene expression.
Materials and methods Strains. The A. nidulans strain biA1 was used as wild type. The mutant strains used had the following genotypes: yA1 wA3 niaDlO; biA1 niaD16; biA1 niaD21; biA1 niaD26; biA1 puA2 niaD42; biA1 niiA17; yA2 biA1 crnA1; biAl areA19; biA1 xprD1; biAI nirACl, biA1 cnxE17 and yA2 pyroA4 nirA1. Growth conditions. Mycelia from each strain were grown in liquid minimal media with the required supplements (Cove 1966) and the nitrogen sources indicated in the text. Incubation was carried out at 30° C, with 250 rpm orbital shaking, for a period of 12 h. Where a switch in culture conditions was necessary, mycelia were grown for a further 4 h. Northern blotting, hybridisation and DNA probes. Total cellular RNA was obtained by the method of Cathala et al. (1983) and l0 gg samples were electrophoresed on a 1.2% formaldehyde denaturing agarose gel. RNA was transferred to nylon membrane (Amersham International) by Northern blotting and UV irradiated. Hybridisa-
486
don was carried out at 42 ° C in 40% formamide to 32p_ hexaprime labelled D N A probes (Sambrook et al. 1989). A 0.9 kb EcoRI fragment of pSTA3 (Johnstone et al. 1990) was used as a niiA-specific probe. The niaD-specific probe was the 2.4 kb J(baI fragment of pSTA1 (Johnstone et al. 1990). Filters were washed at 55°C in 0.1 x SSC, 0.1% SDS. After autoradiography, filters were stripped of probe by submerging in 0.1% SDS for 1 h at 100° C. Rehybridisation to radiolabelled A. nidulans actA fragments, encoding actin (Fidel et al. 1988) was included as an internal control to assess RNA loading and transfer.
Results
Transcript levels in wild type
The 3.2 kb niiA and 2.8 kb niaD (Fig. 1) transcripts are observed in wild type cells when nitrate (lane 1) and nitrite (lane 2) are sole nitrogen sources. In contrast, no transcripts are seen when ammonium (lane 3), glutamate (lane 4), proline (lane 5) or glutamine (data not shown) are supplied. When the inducer (nitrate) and a neutral nitrogen source (proline) is used as combined nitrogen source (lane 6) synthesis of niiA and niaD transcripts occurs. However, neither transcript is visible when nitrate (inducer) and ammonium (lane 7) or glutamine (data not shown) (both nitrogen metabolite repressing substances) are supplied simultaneously.
1 2
3
4
5
~ ~
6
7
~
niiA
~
niaD
actA Fig. 1. Northern blot of niiA and niaD transcripts in wild-type cells grown in minimal media with 10 m M nitrogen source; Lane 1, nitrate; lane 2, nitrite; lane 3, a m m o n i u m ; lane 4, glutamate; lane 5, proline; lane 6, nitrate (5 mM) and proline (5 m M ) ; and lane 7, nitrate (5 mM) and a m m o n i u m (5 mM). For hybridisation conditions and D N A probes, see Materials and methods. Filters were stripped of probe and re-hybridised using the A. nidulans actA gene (Fidel et al. 1988) in all experiments
1 2
3
4
5
!
niiA
niaD
Transcript levels in nirA and areA regulatory mutant strains
The nirA and areA pleiotropic loss-of-function mutants, nirA1 (Fig. 2, lane 2) (Cove and Pateman 1969) and areA19 (lane 4) (Hynes 1975) are shown to be incapable of niiA or niaD m R N A synthesis under inducing conditions, in contrast to the wild-type strain (compare with lane 1). The mutation nirACl (lane 3) (Cove and Pateman 1969) results in the constitutive synthesis of niiA and niaD transcripts when glutamate is available as the nitro-
actA Fig. 2. Northern blot of
niiA and niaD transcripts in regulatory gene m u t a n t backgrounds. Lane 1, wild-type grown on minimal medium with 5 m M a m m o n i u m and transferred to 10 m M nitrate; lane 2, nirA1, 5 m M a m m o n i u m and transferred to 10 m M nitrate, lane 3, nirAC1, I0 m M glutamate; lane 4, areAl9, 5 m M ammonium and transferred to I0 m M nitrate and lane 5, xprD1, 5 m M a m m o n i u m and 5 m M nitrate
Table 1. Chlorate resistance, NiR activities and niiA and niaD transcript levels Strain
Chlorate resistance a' b Glutamate c
Urea ~
Proline °
Inducible (I) or constitutive (C) NiR activities
Wild type
S
S
S
I
I
niaD 10 niaD16 d niaD26 a niaD21 d niaD42 cnxE17
R R R
R R R
R R R
C C C
C C C
S S
S/R S/R
S/R S/R
I I
I I
R
R
R
C
C
Inducible (I) or constitutive (C) niiA and niaD transcript levels
Minimal medium with 200 m M sodium chlorate. Plates were incubated at 37 ° C for 4 days b R (resistance) indicates considerable growth; S (sensitivity) no growth; S/R (intermediate resistance) low level of growth c The concentration of the nitrogen source was 10 m M d Taken from Cove 1976
487
1
2
3
4
5
6
7 niiA
niaD
actA Fig. 3. Northern blot of niiA and niaD transcripts in structural gene mutant strains grown on 10 mM glutamate: lane 1, wild type; lane 2, niaD21; lane 3, niaD26; lane 4, niaD42; lane 5, niiA17; lane 6, cnxE17 and lane 7, crnA 1 gen source. N o transcripts are observed in wild-type cells u n d e r this condition (Fig. 1, lane 4). A g a i n in contrast to wild-type (Fig. 1, lane 7), niiA and niaD transcripts are observed w h e n xprD1 (an allele o f the area gene), which results in depression o f a n u m b e r o f nitrogen metabolite repressible systems ( C o h e n 1972; Arst and Cove 1973), is g r o w n in the presence o f nitrate and a m m o n i u m (Fig. 2, lane 5).
Transcript levels in structural gene mutant strains The data presented in Fig. 3 show that b o t h niiA and niaD transcripts are synthesised in the absence o f inducer in m u t a n t strains niaD26 (lane 3), niaDlO and niaD16 (K. Hawker, unpublished results) and cnxE17 (lane 6). In contrast, niiA and niaD transcripts are n o t observed in m u t a n t strains niaD21 (lane 2) and niaD42 (lane 4), similar to wild type (lane 1), w h e n cells are g r o w n in n o n - i n d u c i n g conditions, and they show n o r m a l inducibility by nitrate (K. H a w k e r , unpublished results). Cove (1976, M a c D o n a l d et al. 1973) showed that niaD and cnx m u t a n t strains which show normal, i.e. wild-type, regulation o f N i R activity a n d the m u t a n t N R protein are m o d e r a t e l y sensitive to chlorate whilst strains that are constitutive for N i R are highly chlorate resistant. N o r t h e r n blotting o f R N A f r o m the m u t a n t s previously tested by Cove (1976, M a c D o n a l d et al. 1973) as well as m u t a n t s tested here (Table 1) shows that transcript levels m i r r o r e n z y m e activities and the level o f chlorate resistance. Finally, it is n o t e w o r t h y that strains having mutations in niiA (niiA17) or crnA (crnA1) result in wild-type niiA and niaD transcript levels (Fig. 3, lanes 5 and 7 respectively); niiA and niaD expression in these m u t a n t s is dependent on nitrate induction (K. Hawker, u n p u b lished results).
Discussion This molecular analysis o f representative m u t a n t s defective in nitrate assimilation confirms previous formal biochemical genetic results and shows that control is re-
flected in the steady-state level o f transcript. T h a t a functional niaD p r o d u c t is required for wild-type regulation o f niiA and niaD m R N A synthesis is suggested by the fact that certain m u t a t i o n s within niaD (e.g. niaDlO, niaD16 and niaD26) a n d cnxE result in constitutive niiA and niaD gene expression. Such m u t a t i o n s also lead to resistance to higher concentrations o f chlorate. H o w e v e r other m u t a n t strains in niaD (e.g. niaD21 and niaD42), which are significantly less chlorate resistant, are wildtype with regard to transcript synthesis.
Acknowledgements. We wish to thank Drs. B. Tomsett and A.J. Clutterbuck, Professors H.N. Arst and M.J. Hynes for providing mutant strains. J.R.K. acknowledges funds from the Science and Engineering Research Council (grant number GR/D/48496). J.L.H. is indebted to the same research council for a postgraduate research studentship (Quota Award).
References Arst HN Jr, Cove DJ (1973) Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet 176 : 111-141 Arst HN Jr, MacDonald DW, Cove DJ (1970) Molybdate metabolism in Aspergillus nidulans. I. Mutations affecting nitrate reductase and/or xanthine dehydrogenase. Mol Gen Genet 108:129145 Brownlee AG, Arst HN Jr (1983) Nitrate uptake in Aspergillus nidulans and the involvement of the third gene of the nitrate assimilation gene cluster. J Bacteriol 155 : 1138-1146 Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:32%335 Cohen BL (1972) Ammonium repression of extracellular protease in Aspergillus nidulans. J Gen Microbiol 71:293--299 Cooley RN, Tomsett AB (1985) Determination of the subunit size of NADPH-nitrate reductase from Aspergillus nidulans. Biochim Biophys Acta 831:89-93 Cove DJ (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113:51-56 Cove DJ (1976) Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation. Mol Gen Genet 146 : 147-159 Cove DJ (1979) Genetical studies of nitrate assimilation in Aspergillus nidulans. Biol Rev 54:291-303 Cove DJ, Pateman JA (1963) Independently segregating loci concerned with nitrate assimilation in Aspergillus nidulans. Nature 168:262-263 Cove DJ, Pateman JA (1969) Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. J Bacteriol 97:13741378 Fidel S, Doonan JM, Morris NR (1988) Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a actin. Gene 70 : 283-293 Hynes MJ (1975) Studies on the role of the area gene in the regulation of nitrogen catabolism in Aspergillus nidulans. Aust J Biol Sci 28:301 313 Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Brow MAD, Unktes SE, Clutterbuck AJ, Kinghorn JR, Innis MA (1990) Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90: 181192 Kinghorn JR (1989) Genetic, biochemical and structural organisation of the Aspergillus nidulans crnA-niiA-niaD gene cluster. In: Wray JL, Kinghorn JR (eds) Molecular and genetic aspects of nitrate assimilation. Oxford University Press, Oxford, pp 69-87
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C o m m u n i c a t e d b y W. G a j e w s k i
