Current Genetics (1982) 6:87-90 © Springer-Verlag 1982
Short Communication
A Regulatory Phenotype Associated With the en-am 1 Mutant of Neurospora crassa John A. A. Chambers 1., and Stephanie A. Wilkins2 1 Department of Genetics, The University, Leeds LS2 9JT, United Kingdom 2 Department of Combined Studies, The University, Leeds LS2 9JT, United Kingdom
S u l m m r y . We have investigated the nature o f the en-aml m u t a n t o f Neurospora erassa and have found that it affects the regulation o f proline oxidase and utilisation of other nitrogen sources. This mutant is closely linked to the gln gene but not allelic with it. Data from crosses suggest that the two genes lie on opposite sides of the inl gene on linkage group VR.
Key words: Neurospora - Regulation - Nitrogen metabolism
entre1 and en-am2 (Dunn-Coleman et al. 1981). The second o f these mutants appears to be due to mutation in the structural gene for GOGAT thereby blocking the minor pathway; en-aml does not appear to have a simple enzymatic deficiency associated with it (Dunn-Coleman et al. 1981) although it cannot grow on proline as sole nitrogen source (Burk 1965). In this paper we present evidence to suggest that the en-aml gene product is involved in the regulation o f gene expression in some pathways of nitrogen metabolism. We also report on tests of the allelism o f the en-arnl mutant and the closely linked gln gene, the structural gene for glutarnine synthetase.
Introduction The am mutants of Neurospora crassa have a requirement for a-amino nitrogen because o f a lack o f the biosynthetic (NADPH dependent) glutamate dehydrogenase (EC 1.4.1.4., Fincham 1962). The auxotrophy is not absolute as am mutants will grow on NH~ as sole nitrogen source after prolonged lag phase. This appears to be due to the existence o f a minor pathway involving the enzymes glutamine synthetase (GS, EC 6.3.1.2.) and glutamate: oxoglutarate aminotransferase (GOGAT, EC 1.4.1.14). Both of these enzymes have been identified in and purified from N. crassa (Palacios 1976; H u m m e r and Mora 1981; see also Dunn-Coleman et al. 1981). The leakiness of the am phenotype can be overcome by including glycine in the selective medium or by the presence o f one o f two cryptic "enhancer" mutants called
* Present address: Department of Biochemistry, The Ohio State University, 494 West 12th. Avenue, Columbus, Ohio 43210, USA Offprint requests to: J. A. A. Chambers
Materials and Methods Strains. Wild type strain STA4 and the aml, en-aml double mutant were from stocks in the Department of Genetics, University of Leeds, en-aml was supplied by Professor J. R. S. Fincham (Dept. Genetics, University of Edinburgh U.K.). The gin single mutant (FGSC 1449) and gln, inos double mutant (FGSC 1450) were obtained from the Fungal Genetics Stock Center (Arcata, California). Genetic Methods. Genetic methods were as described by Davis and de Serres (1970). Because of the difficulty of predicting the phenotype of the en-aml/gln double mutant we assumed reciprocal recombination over the distances involved only scored for the recombinant that did not carry both these mutants. The en-aml phenotype was scored by resistance to parafluorophenylalanine as described by Fincham (1981). Preparation of Extracts and Enzyme Assays. Conidia were innoculated into 400 ml of the appropriate medium in a 11 Erlenmeyer flask and grown with shaking at 30 °C overnight. Mycelium was harvested by filtration under suction through Whatman No. 1 filter paper, washed with distilled water and lyophilised. Dried mycelium was ground with an equal mass of acid-washed sand and five volumes of assay buffer in a cold mor-
O172-8083/82/0006/0087/$ O1.00
J . A . A . Chambers and S. A. Wilkins: Regulation in en-aml ofN. erassa
88 Table la and b. Growth tests on homo- and heterokaryons
006 a
a
Homokaryons
Nitrogen Source
Wild type
en-aml
am/en-aml
glnl-a
NH4C1 Urea Glutamate Glutamine Proline B.S.A.
110 153 64.5 95 55.5 49.5
121 111 65 11.8 7 9.2
6 82 70 6.5 10 12.5
23 98 18 85.5 44 16
0'04
~ 4-0
0.02
& c5 E
b Heterokaryons
c
Nitrogen
Heterokaryon
SOUrCe
NH4C1 Urea Glutamate Glutamine Proline B.S.A.
E
1
2
3
4
99.5 111 80 66.3 66 34
119 130 103.5 67 69 29.5
105 125 136.5 72 61.5 36
125 128 139 90 70.5 38
=
o o
004 b
0'02
Heteroka~ons were forced between am/emaml and gln/inl double mutants on minimal medium. The same heterokaryons are used throughout these tests. Values are mg dry weight of mycelium per culture as described in materials and methods. Each value is the average of three determiantions B.S.A. = bovine serum albumin/1 mg/ml
tar and pestle. The homogenate was clarified by centrifugation (45,000 g, 20 min, 4 °C); the pellets were discarded and the supernatants used for enzyme assays. Proline oxidase was assayed by the method of Arst and MacDonald (1975). Glutamine synthetase was assayed by the transferase assay of Ferguson and Sims (1974). Protein was determined by the Biuret assay (Layne 1959) using bovine serum albumin as a standard.
0
i
i
I
I
I
1
2
3
4
5
6
Time (h)
Fig. la and b. Induction and Repression of Proline Oxidase. Mycelium was grown without shaking for two days, harvested by filtration washed with water and transferred to the new medium and samples were taken for assay of proline oxidase at the time shown, a Wild type A • transfer from 10 mM proline to 10mM NH4C1 as sole nitrogen source. , - - , transfer from 10 mM NH4C1 to 10 mM proline as sole nitrogen source, b en-aml Transfer from 10 mM NH4C1 to 10 mM proline as sole nitrogen source
Quantitative Growth Tests. Quantitative growth tests were made in 25 ml of Vogels nitrogen-free minimal medium containing the appropriate nitrogen source at a concentration of 10 mg atom nitrogen per litre and 2% (w/v) sucrose. Flasks were innoculated with 106 conidia and incubated at 30 °C for 66 h without shaking. Mycelium was harvested onto dried preweighed Whatman No 1 filter papers, washed with distilled water, dried at 65 °C overnight and weighed. All growth tests were performed in triplicate.
Results Levels o f Proline Oxidase In the wild t y p e proline oxidase is present w h e n proline is the sole nitrogen source b u t is absent w h e n amino-
n i u m chloride is the sole nitrogen source (Table 1). We also f o u n d t h a t in the wild type, the e n z y m e activity is rapidly lost u p o n transfer to an a m m o n i u m - c o n t a i n i n g m e d i u m b u t is only slowly induced b y transfer to a m e d i u m containing proline as sole nitrogen source (Fig. 1), in agreement w i t h the finding o f F a c k l a m and Marzl u f (1978). In the en-aml strain proline oxidase was present at levels higher than the u n i n d u c e d levels o f wild t y p e in the absence o f proline and dissapeared rapidly u p o n transfer to a proline-eontaining m e d i u m . In gln m u t a n t s levels o f proline oxidase were similar to or slightly higher t h a n those o f wild type.
J. A. A. Chambers and S. A. Wilkins: Regulation in e n - a m l ofN. erassa
89
Table 2a and b. Prohne Oxidase Levels in Homo- and Heterokaryons a
Homokaryons
Expt
b
Wild-type on proline
1 2 3
Wild-type on NH4C1
3.5 3.0 3.3
en-aml on proline
0.14 0.11 0.28
en-aml on NH4C1
0.4 0.4 0.5
1.3 1.24 n.d.
glnl-a on proline
5.3 5.4 4.9
Heterokaryonsgrown on proline
Heterokaryons 1
2
3
4
3.75 3.2 3.6
3.9 3.1 3.5
5 8.2 4.2
3.3 3.1 3.7
Specific activities are expressed as AA500 nm/min/mg protein. Each value is the average of three determinations, n.d. = not determined
Table 3a and b. Glutamine Synthetase Levels in Homo- and Heterokaryons a
Homokaryons
Expt
Wild-type on NH4C1
Wild-type glutamate
en-aml on NH4C1
glnl-a on glutamate
1 2 3
6.7 5.3 6.3
15.1 16.3 13.2
6.2 5.9 6.9
3.9 3.1 3.0
Quantitative Growth Tests The en-aml mutant grows as well as the wild type on Vogels minimal medium but is unable to grow on proline, exogenous protein or glutamine as a nitrogen source. The glnl-a mutant showed the behaviour typical of an auxotroph, growing well on a medium containing glutamine but not on minimal medium (Table 1).
Complementation Tests With glnl-a and en-aml b
Heterokaryonsgrown on ammonium chloride
Expt
Heterokaryons 1
1 2 3
6.3 5.9 6.95
2 7.2 6.6 6.1
3
4
6.4 6.1 6.2
6.3 5.6 6.0
Heterokaryons forced between glnl-a, inl and am, enaml showed growth responses typical of wild type (Table 1). Enzyme assays showed that both proline oxidase and glutamine synthetase were restored to wild type levels (Tables 2 and 3). All heterokaryons could be broken down to the original homokaryons (Data not presented).
Specific activity is expressed as picomoles -r-glutamyl hydroxamate formed/min/mg protein. Each value is the mean of three determina- Mapping o f glnl-a and en-aml tions
Table 4. Progeny from cross of am, en-aml, a and gln inos A Genotype
No. progeny.
am en-am-1 ++ gin inl ++ am inl ++ am +++ ++++ inl +++ gin +++ en-am-1 +++ en-am-1 inl ++
4,000 3,200 1,500 3,000 50 140 500 15 10
Numbers of progeny are the number of each type per ml of a suspension of ascospores estimated by plating onto appropriate selective media
The commonest recombinant progeny from a cross of am, en-aml and glnl-a, inos were am +++/+ en am1 gln inos and am inos ++/++ en-aml gln (Table 4). These recombinants are most consistent with the map order am - gln - i n l - en am1 i .
Discussion The cryptic behaviour of the enhancer mutant of the am phenotype may be due to them affecting a minor pathway of ammonia assimilation. This appears to be the case for the en-am2 mutant which may be in the structural gene for GOGAT (Dunn-Coleman et al. 1981). The behaviour of the en-aml is not so easily explained, particularly since the en-aml single mutant cannot use proline as a sole nitrogen source (Burk 1965). We inves-
90
J.A.A. Chambers and S. A. Wilkins: Regulation in en-aml ofN. crassa
tigated the possibility that this was due to a deficiency in proline oxidase activity by trying to induce and assay the activity in wild type and en-aml strains. We found what appears to be an example of a regulation reversal, a phenomenon which has been suggested to be a diagnostic phenotype for a regulatory gene (Arst and Cove 1969.). In parallel we found that the en-arnl mutant had lost the ability to use other nitrogen sources in addition to proline. Fincham (1981) has also reported that the single mutant is resistant to normally toxic levels of DLethionine and parafluorophenylalanine. This suggests that the mutant is lacking in one or more amino acid permeases (see Pateman and Kinghorn 1976) although this would not be enough to explain the altered regulation of regulation ofproline oxidase of the poor growth of the mutant on extraceUular protein or glutamine. This apparent loss or alteration of a number of activities suggests that the en-aml phenotype may be due to a mutation in a regulatory gene. The en-aml gene lies close to the gln gene, the structural gene for glutamine synthetase (Sanchez et al. 1979). Glutamine synthetase has been suggested to play a direct role in the regulation of gene expression in Neurospora (Dunn-Coleman and Garret 1980). Thus it seemed of considerable interest to test for complementation of en-arnl and gln. By M1 the criteria used: levels of proline oxidase and glutamine synthetase and growth on extracellular protein, the two mutant phenotypes complemented each other, suggesting that they are not alMic. We have also found that the en-aml mutant complements tight glutamine-requiring strains supplied by J. R. S. Fincham and J. A. Kinsey (pers. commun.). These two mutants lie close to the inl gene on linkage group VR of the N. crassa linkage map (Radford 1976). Fincham (1981) has found en-aml and gln to be 1.3 map units apart, which is large for alleles. We have found that the numbers of the recombinant progeny of a cross between am, en-aml and glnl-a, inl to be most easily explained by the two genes lying on opposites sides of the inl gene with the most likely order being am gln - inl - en-aml, the rarity of the en-aml single mutant in the recombinant progeny is explained by an apparent close linkage of the am and gln loci. We propose therefore that the en-aml phenotype is due to mutation in a gene encoding a regulatory product that has a general rather than a pathway-specific effect in catabolic nitrogen metabolism. It is important to consider this suggestion with respect to the nit-2 locus. This locus is suggested to encode a positively-acting regulatory protein that is required for the transcription of genes involved in catabolic nitrogen metabolism (Reinert and Marzluf 1975; Facklam and Marzluf 1978; Wang and Marzluf 1979; Coddington 1976). This locus may act in a manner complementary to nit2, similar to the way that
tamA may act with areA in Aspergillus nidulans (Pateman and Kinghorn 1976; Cove 1979). We also wish to note that the expression of proline oxidase is not affected by the presence of mutant alleles at the nit2 locus, suggesthag that proline oxidase is not under the control of nit2 (Facklam and Marzluf 1978). We conclude that the enam1 gene product may act independently of the nit2 locus although we do not believe that the two are mutually exclusive as shown by the fact that neither en-aml or nit2 can grow on extracellular protein as sole nitrogen source. We have also shown that the L-amino acid oxidase of N. crassa, which is involved in the nitrogen control circuit (Sikora and Marzluf, in press) no longer requires induction in an en-aml background although it is still sensitive to nitrogen metabolite repression (S. Griffon, G. A. Marzluf and J. A. A. C., unpublished results). Acknowledgements. Thanks are due to J. R. S. Fincham, J. A. Kinsey, G. A. Marzluf, A. Radford and J. C. Wootton for information, criticism, advice and strains.
References Arst HN Jr, CoveDJ (1969) J Bacteriol 98:1284-1293 Arst HN Jr, Cove DJ (1973) Mol Gen Genet 126:111-142 Arst HN Jr. Mac Donald DW (1975) Nature 254:26-31 Burk RR (1965) Ph.D. Thesis. University of Cambridge Coddington A (1976) Mol Gen Genet 145:195-206 Cove DJ (1979) Biol Rev 54:291-327 Davis RH, de Serres FJ (1970) Methods Enzymol 17:79-143 Dunn-Coleman NS, Garrett RH (1980) Mol Gen Genet 179: 25-32 Dunn-Colemann NS, Robey EA, Tomsett AB, Garrett RH (1981) Mol Cell Biol 1:158-164 Facklam TJ, Marzluf GA (1978) Biochem Genet 16:343-354 Ferguson AR, Sims AP (1974) J Gen Microbiol 80:173-185 Fincham JRS (1962) J Mol Biol 4:257-274 Fincham JRS (1981) Neurospora Newsl 28:11 Hummelt G, Mora J (1980a) Biochem Biophys Res Commun 92:127-133 Hummelt G, Mora J (1980b) Biochem Biophys Res Commun 94:1688-1694 Kinghorn JR, Pateman JA (1975)Mol Gen Genet 140:137-147 Layne E (1957) Methods Enzymol 3:447-454 Palacios R (1976) J Biol Chem 251:4784-4791 Pateman JA, Kinghorn JR (1976) The Filamentous Fungi, vol 2, Smith JE Berry DR (eds) John Wiley and Sons, New York London, p 159-237 Radford A (1976) Linkage maps of Neurospora crassa. In: Fasman GD (ed) CRC Handbook of Biochemistry and Molecular Biology, 3rd edn. Nucleic Acids, vol 2. CRC Press, Cleveland, p 739-761 Reinert WR, Marzluf GA (1975) Mol Gen Genet 139:39-55 Sanchez F, Davila G, Mora J, Palacios R (1979) J Baeteriol 139:537-343 Wang LC' Marzluf GA (1979) Mol Gen Genet 176:385-392 Communicated by F. Kaudewitz Received May 12, 1982
Short Communication
A Regulatory Phenotype Associated With the en-am 1 Mutant of Neurospora crassa John A. A. Chambers 1., and Stephanie A. Wilkins2 1 Department of Genetics, The University, Leeds LS2 9JT, United Kingdom 2 Department of Combined Studies, The University, Leeds LS2 9JT, United Kingdom
S u l m m r y . We have investigated the nature o f the en-aml m u t a n t o f Neurospora erassa and have found that it affects the regulation o f proline oxidase and utilisation of other nitrogen sources. This mutant is closely linked to the gln gene but not allelic with it. Data from crosses suggest that the two genes lie on opposite sides of the inl gene on linkage group VR.
Key words: Neurospora - Regulation - Nitrogen metabolism
entre1 and en-am2 (Dunn-Coleman et al. 1981). The second o f these mutants appears to be due to mutation in the structural gene for GOGAT thereby blocking the minor pathway; en-aml does not appear to have a simple enzymatic deficiency associated with it (Dunn-Coleman et al. 1981) although it cannot grow on proline as sole nitrogen source (Burk 1965). In this paper we present evidence to suggest that the en-aml gene product is involved in the regulation o f gene expression in some pathways of nitrogen metabolism. We also report on tests of the allelism o f the en-arnl mutant and the closely linked gln gene, the structural gene for glutarnine synthetase.
Introduction The am mutants of Neurospora crassa have a requirement for a-amino nitrogen because o f a lack o f the biosynthetic (NADPH dependent) glutamate dehydrogenase (EC 1.4.1.4., Fincham 1962). The auxotrophy is not absolute as am mutants will grow on NH~ as sole nitrogen source after prolonged lag phase. This appears to be due to the existence o f a minor pathway involving the enzymes glutamine synthetase (GS, EC 6.3.1.2.) and glutamate: oxoglutarate aminotransferase (GOGAT, EC 1.4.1.14). Both of these enzymes have been identified in and purified from N. crassa (Palacios 1976; H u m m e r and Mora 1981; see also Dunn-Coleman et al. 1981). The leakiness of the am phenotype can be overcome by including glycine in the selective medium or by the presence o f one o f two cryptic "enhancer" mutants called
* Present address: Department of Biochemistry, The Ohio State University, 494 West 12th. Avenue, Columbus, Ohio 43210, USA Offprint requests to: J. A. A. Chambers
Materials and Methods Strains. Wild type strain STA4 and the aml, en-aml double mutant were from stocks in the Department of Genetics, University of Leeds, en-aml was supplied by Professor J. R. S. Fincham (Dept. Genetics, University of Edinburgh U.K.). The gin single mutant (FGSC 1449) and gln, inos double mutant (FGSC 1450) were obtained from the Fungal Genetics Stock Center (Arcata, California). Genetic Methods. Genetic methods were as described by Davis and de Serres (1970). Because of the difficulty of predicting the phenotype of the en-aml/gln double mutant we assumed reciprocal recombination over the distances involved only scored for the recombinant that did not carry both these mutants. The en-aml phenotype was scored by resistance to parafluorophenylalanine as described by Fincham (1981). Preparation of Extracts and Enzyme Assays. Conidia were innoculated into 400 ml of the appropriate medium in a 11 Erlenmeyer flask and grown with shaking at 30 °C overnight. Mycelium was harvested by filtration under suction through Whatman No. 1 filter paper, washed with distilled water and lyophilised. Dried mycelium was ground with an equal mass of acid-washed sand and five volumes of assay buffer in a cold mor-
O172-8083/82/0006/0087/$ O1.00
J . A . A . Chambers and S. A. Wilkins: Regulation in en-aml ofN. erassa
88 Table la and b. Growth tests on homo- and heterokaryons
006 a
a
Homokaryons
Nitrogen Source
Wild type
en-aml
am/en-aml
glnl-a
NH4C1 Urea Glutamate Glutamine Proline B.S.A.
110 153 64.5 95 55.5 49.5
121 111 65 11.8 7 9.2
6 82 70 6.5 10 12.5
23 98 18 85.5 44 16
0'04
~ 4-0
0.02
& c5 E
b Heterokaryons
c
Nitrogen
Heterokaryon
SOUrCe
NH4C1 Urea Glutamate Glutamine Proline B.S.A.
E
1
2
3
4
99.5 111 80 66.3 66 34
119 130 103.5 67 69 29.5
105 125 136.5 72 61.5 36
125 128 139 90 70.5 38
=
o o
004 b
0'02
Heteroka~ons were forced between am/emaml and gln/inl double mutants on minimal medium. The same heterokaryons are used throughout these tests. Values are mg dry weight of mycelium per culture as described in materials and methods. Each value is the average of three determiantions B.S.A. = bovine serum albumin/1 mg/ml
tar and pestle. The homogenate was clarified by centrifugation (45,000 g, 20 min, 4 °C); the pellets were discarded and the supernatants used for enzyme assays. Proline oxidase was assayed by the method of Arst and MacDonald (1975). Glutamine synthetase was assayed by the transferase assay of Ferguson and Sims (1974). Protein was determined by the Biuret assay (Layne 1959) using bovine serum albumin as a standard.
0
i
i
I
I
I
1
2
3
4
5
6
Time (h)
Fig. la and b. Induction and Repression of Proline Oxidase. Mycelium was grown without shaking for two days, harvested by filtration washed with water and transferred to the new medium and samples were taken for assay of proline oxidase at the time shown, a Wild type A • transfer from 10 mM proline to 10mM NH4C1 as sole nitrogen source. , - - , transfer from 10 mM NH4C1 to 10 mM proline as sole nitrogen source, b en-aml Transfer from 10 mM NH4C1 to 10 mM proline as sole nitrogen source
Quantitative Growth Tests. Quantitative growth tests were made in 25 ml of Vogels nitrogen-free minimal medium containing the appropriate nitrogen source at a concentration of 10 mg atom nitrogen per litre and 2% (w/v) sucrose. Flasks were innoculated with 106 conidia and incubated at 30 °C for 66 h without shaking. Mycelium was harvested onto dried preweighed Whatman No 1 filter papers, washed with distilled water, dried at 65 °C overnight and weighed. All growth tests were performed in triplicate.
Results Levels o f Proline Oxidase In the wild t y p e proline oxidase is present w h e n proline is the sole nitrogen source b u t is absent w h e n amino-
n i u m chloride is the sole nitrogen source (Table 1). We also f o u n d t h a t in the wild type, the e n z y m e activity is rapidly lost u p o n transfer to an a m m o n i u m - c o n t a i n i n g m e d i u m b u t is only slowly induced b y transfer to a m e d i u m containing proline as sole nitrogen source (Fig. 1), in agreement w i t h the finding o f F a c k l a m and Marzl u f (1978). In the en-aml strain proline oxidase was present at levels higher than the u n i n d u c e d levels o f wild t y p e in the absence o f proline and dissapeared rapidly u p o n transfer to a proline-eontaining m e d i u m . In gln m u t a n t s levels o f proline oxidase were similar to or slightly higher t h a n those o f wild type.
J. A. A. Chambers and S. A. Wilkins: Regulation in e n - a m l ofN. erassa
89
Table 2a and b. Prohne Oxidase Levels in Homo- and Heterokaryons a
Homokaryons
Expt
b
Wild-type on proline
1 2 3
Wild-type on NH4C1
3.5 3.0 3.3
en-aml on proline
0.14 0.11 0.28
en-aml on NH4C1
0.4 0.4 0.5
1.3 1.24 n.d.
glnl-a on proline
5.3 5.4 4.9
Heterokaryonsgrown on proline
Heterokaryons 1
2
3
4
3.75 3.2 3.6
3.9 3.1 3.5
5 8.2 4.2
3.3 3.1 3.7
Specific activities are expressed as AA500 nm/min/mg protein. Each value is the average of three determinations, n.d. = not determined
Table 3a and b. Glutamine Synthetase Levels in Homo- and Heterokaryons a
Homokaryons
Expt
Wild-type on NH4C1
Wild-type glutamate
en-aml on NH4C1
glnl-a on glutamate
1 2 3
6.7 5.3 6.3
15.1 16.3 13.2
6.2 5.9 6.9
3.9 3.1 3.0
Quantitative Growth Tests The en-aml mutant grows as well as the wild type on Vogels minimal medium but is unable to grow on proline, exogenous protein or glutamine as a nitrogen source. The glnl-a mutant showed the behaviour typical of an auxotroph, growing well on a medium containing glutamine but not on minimal medium (Table 1).
Complementation Tests With glnl-a and en-aml b
Heterokaryonsgrown on ammonium chloride
Expt
Heterokaryons 1
1 2 3
6.3 5.9 6.95
2 7.2 6.6 6.1
3
4
6.4 6.1 6.2
6.3 5.6 6.0
Heterokaryons forced between glnl-a, inl and am, enaml showed growth responses typical of wild type (Table 1). Enzyme assays showed that both proline oxidase and glutamine synthetase were restored to wild type levels (Tables 2 and 3). All heterokaryons could be broken down to the original homokaryons (Data not presented).
Specific activity is expressed as picomoles -r-glutamyl hydroxamate formed/min/mg protein. Each value is the mean of three determina- Mapping o f glnl-a and en-aml tions
Table 4. Progeny from cross of am, en-aml, a and gln inos A Genotype
No. progeny.
am en-am-1 ++ gin inl ++ am inl ++ am +++ ++++ inl +++ gin +++ en-am-1 +++ en-am-1 inl ++
4,000 3,200 1,500 3,000 50 140 500 15 10
Numbers of progeny are the number of each type per ml of a suspension of ascospores estimated by plating onto appropriate selective media
The commonest recombinant progeny from a cross of am, en-aml and glnl-a, inos were am +++/+ en am1 gln inos and am inos ++/++ en-aml gln (Table 4). These recombinants are most consistent with the map order am - gln - i n l - en am1 i .
Discussion The cryptic behaviour of the enhancer mutant of the am phenotype may be due to them affecting a minor pathway of ammonia assimilation. This appears to be the case for the en-am2 mutant which may be in the structural gene for GOGAT (Dunn-Coleman et al. 1981). The behaviour of the en-aml is not so easily explained, particularly since the en-aml single mutant cannot use proline as a sole nitrogen source (Burk 1965). We inves-
90
J.A.A. Chambers and S. A. Wilkins: Regulation in en-aml ofN. crassa
tigated the possibility that this was due to a deficiency in proline oxidase activity by trying to induce and assay the activity in wild type and en-aml strains. We found what appears to be an example of a regulation reversal, a phenomenon which has been suggested to be a diagnostic phenotype for a regulatory gene (Arst and Cove 1969.). In parallel we found that the en-arnl mutant had lost the ability to use other nitrogen sources in addition to proline. Fincham (1981) has also reported that the single mutant is resistant to normally toxic levels of DLethionine and parafluorophenylalanine. This suggests that the mutant is lacking in one or more amino acid permeases (see Pateman and Kinghorn 1976) although this would not be enough to explain the altered regulation of regulation ofproline oxidase of the poor growth of the mutant on extraceUular protein or glutamine. This apparent loss or alteration of a number of activities suggests that the en-aml phenotype may be due to a mutation in a regulatory gene. The en-aml gene lies close to the gln gene, the structural gene for glutamine synthetase (Sanchez et al. 1979). Glutamine synthetase has been suggested to play a direct role in the regulation of gene expression in Neurospora (Dunn-Coleman and Garret 1980). Thus it seemed of considerable interest to test for complementation of en-arnl and gln. By M1 the criteria used: levels of proline oxidase and glutamine synthetase and growth on extracellular protein, the two mutant phenotypes complemented each other, suggesting that they are not alMic. We have also found that the en-aml mutant complements tight glutamine-requiring strains supplied by J. R. S. Fincham and J. A. Kinsey (pers. commun.). These two mutants lie close to the inl gene on linkage group VR of the N. crassa linkage map (Radford 1976). Fincham (1981) has found en-aml and gln to be 1.3 map units apart, which is large for alleles. We have found that the numbers of the recombinant progeny of a cross between am, en-aml and glnl-a, inl to be most easily explained by the two genes lying on opposites sides of the inl gene with the most likely order being am gln - inl - en-aml, the rarity of the en-aml single mutant in the recombinant progeny is explained by an apparent close linkage of the am and gln loci. We propose therefore that the en-aml phenotype is due to mutation in a gene encoding a regulatory product that has a general rather than a pathway-specific effect in catabolic nitrogen metabolism. It is important to consider this suggestion with respect to the nit-2 locus. This locus is suggested to encode a positively-acting regulatory protein that is required for the transcription of genes involved in catabolic nitrogen metabolism (Reinert and Marzluf 1975; Facklam and Marzluf 1978; Wang and Marzluf 1979; Coddington 1976). This locus may act in a manner complementary to nit2, similar to the way that
tamA may act with areA in Aspergillus nidulans (Pateman and Kinghorn 1976; Cove 1979). We also wish to note that the expression of proline oxidase is not affected by the presence of mutant alleles at the nit2 locus, suggesthag that proline oxidase is not under the control of nit2 (Facklam and Marzluf 1978). We conclude that the enam1 gene product may act independently of the nit2 locus although we do not believe that the two are mutually exclusive as shown by the fact that neither en-aml or nit2 can grow on extracellular protein as sole nitrogen source. We have also shown that the L-amino acid oxidase of N. crassa, which is involved in the nitrogen control circuit (Sikora and Marzluf, in press) no longer requires induction in an en-aml background although it is still sensitive to nitrogen metabolite repression (S. Griffon, G. A. Marzluf and J. A. A. C., unpublished results). Acknowledgements. Thanks are due to J. R. S. Fincham, J. A. Kinsey, G. A. Marzluf, A. Radford and J. C. Wootton for information, criticism, advice and strains.
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