genetic biochemical identification glutamate synthase

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JOURNAL OF BACTERIOLOGY, Sept. 1986, p. 1043-1047 Vol. 167, No. 3 0021-9193/86/091043-05$02.00/0 Copyright © 1986, American Society for Microbiology Genetic and Biochemical Identification of the Glutamate Synthase Structural Gene in Neurospora crassa DAVID ROMERO AND GUILLERMO DAVILA* Centro de Investigacion sobre Fijaci6n de Nitr6geno, Universidad Nacional Aut6noma de Mexico, Cuernavaca, Morelos, Mexico Received 31 January 1986/Accepted 2 June 1986 Neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions. Despite the physiological importance of glutamate synthase, little is known about the genetics of its expression. To identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. All mutations are recessive to the wild-type allele and belong to the same complementation group as the previously described en(am)-2 (C24) mutation. Two lines of evidence indicate that en(am)-2 is the structural gene for glutamate synthase in N. crassa. (i) The en(am)-2+ gene shows a gene dosage effect on enzyme activity, and (ii) some mutants lacking glutamate synthase activity have cross-reacting material. These data suggest that the mutations are located in the structural gene for N. crassa glutamate synthase. In the fungus Neurospora crassa as well as in enteric microorganisms, glutamate can by synthesized via glutamate dehydrogenase (EC 1.4.1.3) or through the concerted action of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (GOGAT) (EC 1.4.1.14) (24). Physiological studies of this fungus indicate that the main pathway for glutamate biosynthesis under conditions of ammonium excess is through glutamate dehydrogenase. However, when the fungi are growing under ammonium- limiting conditions, glutamate biosynthesis is achieved mainly through a glutamine synthetase-GOGAT pathway (18). In plants, the assimilation of low amounts of ammonia by the glutamine synthetase-GOGAT pathway is very well established (23). Moreover, GOGAT appears to play an important role in glutamine degradation in N. crassa. A mutant strain devoid of GOGAT activity is partially impaired in glutamine degradation (3). This enzyme is repressed by glutamate and to a lesser extent by glutamine (14). The isolation and characterization of mutants lacking glutamate dehydrogenase or GOGAT activities had contrib- uted to the understanding of the role of both pathways in glutamate biosynthesis. Several mutations that affect the glutamate dehydrogenase structural gene (am mutants) have been described in N. crassa. The am mutants require glutamate for optimal growth but grow, albeit poorly, on excess ammonia (9). Hummelt and Mora (13) have found that an am mutant grows as well as the wild type on batch-fed ammonium-limited cultures, due to the presence of GOGAT activity (14). A mutation affecting the en-am locus counteracts the leakiness of the am mutants on excess ammonia (M. Shields, M.S. thesis, University of Utah, 1968). The only phenotypic effect of the en-am mutation on an otherwise wild-type background is an inability to grow under ammonium-limited conditions (14). Hummelt and Mora (14) and later Dunn- Coleman et al. (8) reported that the en-am mutant was devoid of GOGAT activity. The en-am locus has been renamed en(am)-2 (8), and this is the designation that we will use in this paper. Some mutations that affect GOGAT activity in bacteria have been described (1, 6, 11); in two of them there are clear * Corresponding author. demonstrations of the structural nature of these mutations (6, 11). A few mutations have been described that affect GOGAT activity in eucaryotic organisms such as N. crassa (8, 14) and Arabidopsis thaliana (28), but none has been characterized enough to determine whether the mutation affects a regulatory or a structural gene. We report here the isolation and characterization of three new N. crassa mutants lacking GOGAT activity. These mutations are allelic to the en(am)-2 locus. We also present genetic and biochemical evidence that indicates that the en(am)-2 locus is the structural gene for GOGAT in N. crassa. MATERIALS AND METHODS Strains. All strains came from the collection of J. Mora or came from the Fungal Genetics Stock Center at the Depart- ment of Microbiology, University of Kansas Medical School, Kansas City. N. crassa 74-A and 73-a were used as wild types. Strains fi A and fl a were used as mating type testers. Other basic strains were am (allele number 132), en(am)-2 (allele number C24), inl (allele number 89601), ad-8 (allele number Y112M343), and T (II-I) NM177. Other double mutants were obtained from the appropriate crosses. Media and growth conditions. All strains were grown for 24 h at 25°C on Vogel minimal medium N (5) supplemented with 1.5% sucrose. Other nitrogen sources in place of or in addition to ammonium nitrate were used as stated below. Adenine (2 mM) or inositol (100 ,ug/ml) was added when needed. Mycelium was grown from a conidial inoculum in liquid medium bubbled with hydrated air. Preparation of cell extracts and determination of GOGAT activity. All steps were performed at 4°C. Mycelium was filtered through Whatman no. 41 filter paper and washed with twice-distilled water. Acetone powders were prepared from the mycelium, ground with dry ice, and suspended in 0.1 M potassium phosphate buffer (pH 7.6) plus 0.5% 2- mercaptoethanol. The suspensions were centrifuged for 15 min at 12,500 x g, and the supernatants were used as crude extracts. GOGAT activity was measured by NADH con- sumption at 25°C by the method of Boland and Benny (2), but with pH 7.75 buffer as the assay buffer. Protein was determined by the method of Lowry et al. (20) with bovine serum albumin as the standard. 1043 Downloaded from https://journals.asm.org/journal/jb on 19 November 2021 by 125.175.128.223.

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Page 1: Genetic Biochemical Identification Glutamate Synthase

JOURNAL OF BACTERIOLOGY, Sept. 1986, p. 1043-1047 Vol. 167, No. 30021-9193/86/091043-05$02.00/0Copyright © 1986, American Society for Microbiology

Genetic and Biochemical Identification of the Glutamate SynthaseStructural Gene in Neurospora crassa

DAVID ROMERO AND GUILLERMO DAVILA*

Centro de Investigacion sobre Fijaci6n de Nitr6geno, Universidad Nacional Aut6noma de Mexico, Cuernavaca,Morelos, Mexico

Received 31 January 1986/Accepted 2 June 1986

Neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions.Despite the physiological importance of glutamate synthase, little is known about the genetics of its expression.To identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. Allmutations are recessive to the wild-type allele and belong to the same complementation group as the previouslydescribed en(am)-2 (C24) mutation. Two lines of evidence indicate that en(am)-2 is the structural gene forglutamate synthase in N. crassa. (i) The en(am)-2+ gene shows a gene dosage effect on enzyme activity, and (ii)some mutants lacking glutamate synthase activity have cross-reacting material. These data suggest that themutations are located in the structural gene for N. crassa glutamate synthase.

In the fungus Neurospora crassa as well as in entericmicroorganisms, glutamate can by synthesized via glutamatedehydrogenase (EC 1.4.1.3) or through the concerted actionof glutamine synthetase (EC 6.3.1.2) and glutamate synthase(GOGAT) (EC 1.4.1.14) (24).

Physiological studies of this fungus indicate that the mainpathway for glutamate biosynthesis under conditions ofammonium excess is through glutamate dehydrogenase.However, when the fungi are growing under ammonium-limiting conditions, glutamate biosynthesis is achievedmainly through a glutamine synthetase-GOGAT pathway(18). In plants, the assimilation of low amounts of ammoniaby the glutamine synthetase-GOGAT pathway is very wellestablished (23). Moreover, GOGAT appears to play animportant role in glutamine degradation in N. crassa. Amutant strain devoid of GOGAT activity is partially impairedin glutamine degradation (3). This enzyme is repressed byglutamate and to a lesser extent by glutamine (14).The isolation and characterization of mutants lacking

glutamate dehydrogenase or GOGAT activities had contrib-uted to the understanding of the role of both pathways inglutamate biosynthesis. Several mutations that affect theglutamate dehydrogenase structural gene (am mutants) havebeen described in N. crassa. The am mutants requireglutamate for optimal growth but grow, albeit poorly, onexcess ammonia (9). Hummelt and Mora (13) have foundthat an am mutant grows as well as the wild type onbatch-fed ammonium-limited cultures, due to the presence ofGOGAT activity (14).A mutation affecting the en-am locus counteracts the

leakiness of the am mutants on excess ammonia (M. Shields,M.S. thesis, University of Utah, 1968). The only phenotypiceffect of the en-am mutation on an otherwise wild-typebackground is an inability to grow under ammonium-limitedconditions (14). Hummelt and Mora (14) and later Dunn-Coleman et al. (8) reported that the en-am mutant wasdevoid of GOGAT activity. The en-am locus has beenrenamed en(am)-2 (8), and this is the designation that we willuse in this paper.Some mutations that affect GOGAT activity in bacteria

have been described (1, 6, 11); in two of them there are clear

* Corresponding author.

demonstrations of the structural nature of these mutations(6, 11). A few mutations have been described that affectGOGAT activity in eucaryotic organisms such as N. crassa(8, 14) and Arabidopsis thaliana (28), but none has beencharacterized enough to determine whether the mutationaffects a regulatory or a structural gene.We report here the isolation and characterization of three

new N. crassa mutants lacking GOGAT activity. Thesemutations are allelic to the en(am)-2 locus. We also presentgenetic and biochemical evidence that indicates that theen(am)-2 locus is the structural gene for GOGAT in N.crassa.

MATERIALS AND METHODSStrains. All strains came from the collection of J. Mora or

came from the Fungal Genetics Stock Center at the Depart-ment of Microbiology, University of Kansas MedicalSchool, Kansas City. N. crassa 74-A and 73-a were used aswild types. Strains fi A and fl a were used as mating typetesters. Other basic strains were am (allele number 132),en(am)-2 (allele number C24), inl (allele number 89601), ad-8(allele number Y112M343), and T (II-I) NM177. Otherdouble mutants were obtained from the appropriate crosses.Media and growth conditions. All strains were grown for 24

h at 25°C on Vogel minimal medium N (5) supplemented with1.5% sucrose. Other nitrogen sources in place of or inaddition to ammonium nitrate were used as stated below.Adenine (2 mM) or inositol (100 ,ug/ml) was added whenneeded. Mycelium was grown from a conidial inoculum inliquid medium bubbled with hydrated air.

Preparation of cell extracts and determination of GOGATactivity. All steps were performed at 4°C. Mycelium wasfiltered through Whatman no. 41 filter paper and washedwith twice-distilled water. Acetone powders were preparedfrom the mycelium, ground with dry ice, and suspended in0.1 M potassium phosphate buffer (pH 7.6) plus 0.5% 2-mercaptoethanol. The suspensions were centrifuged for 15min at 12,500 x g, and the supernatants were used as crudeextracts. GOGAT activity was measured by NADH con-sumption at 25°C by the method of Boland and Benny (2),but with pH 7.75 buffer as the assay buffer. Protein wasdetermined by the method of Lowry et al. (20) with bovineserum albumin as the standard.

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Page 2: Genetic Biochemical Identification Glutamate Synthase

1044 ROMERO AND DAVILA

TABLE 1. Effect of en(am)-2+ gene dosage on GOGAT specificactivity

Control strain and partial diploidsa Gene dosag+ GOGAT sp actbof en(am) -2 ±

am(132) 1 23.3 ± 1.13am(132);en(am)-2 (C24)1en(am)-2+ 1 19.7 ± 0.79am(l32);en(am)-2+1en(am)-2+ 2 30.38 ± 1.58

a The second and third strains are partial diploids carrying a duplication ofthe en(am)-2 region of linkage group II; the first strain is euploid and has thenormal sequence of genes.

I GOGAT specific activity was determined after 24 h of growth at 25°C on25 mM ammonium nitrate as the sole nitrogen source. Specific activity (mean± standard deviation for duplicate determinations) is expressed as nanomolesof NADH oxidized per minute per milligram of extract protein at 25°C.

Mutagenesis and mutant selection. Conidia from theam(132) strain or from the am(132);inl mutant strains weremutagenized and enriched on N medium supplemented with1.5% sucrose and 25 mM NH4NO3 at 37°C as previouslydescribed (4). The enriched population of both strains wasplated on N agar medium supplemented with glucose andfructose (0.02% each), sorbose (1%), 1.36 mM glutamic acid,and 25 mM alanine. Isolates were maintained on slants of thesame medium with 1.5% sucrose as the sole carbon source.

Genetic techniques. Crosses, progeny analysis, spot test-ing, complementation, and mating type and fertility testswere carried out as previously described (5). All the muta-tions were transferred to a standard genetic background bytwo successive crosses with the am(132) mutant strain.

Construction of partial diploids. Strains diploid for seg-ments of the right arm of linkage group II were constructedas described previously (22, 25, 26). The heterozygouspartial diploid [i.e., am(132);en(am)-2 (C24)1en(am)-2+] wasobtained by crossing the am(132);en(am)-2 (C24), matingtype A mutant strain with T (II-4) NM177 mating type a.Progeny were screened for isolates of phenotype am, matingtype a and barren. These were used as heterozygous partialdiploids. The homozygous partial diploid came from a crossof am(132) mating type A mutant strain with T (II-I)NM177, mating type a. The progeny were screened forisolates of phenotype am, mating type a, and barren andused as the homozygous partial diploid. Nomenclature forpartial diploids was according to Metzenberg et al. (22).

Preparation of anti-GOGAT antibodies. GOGAT fromam(J32) ammonium-limited cultures was partially purified(150-fold) as described previously (2, 14), with the followingmodifications. The ammonium sulfate cut was from 30 to45% of saturation, and the dimensions of the DEAE-Sephadex A-25 column were 2.0 by 10.5 cm. Anti-GOGATantibodies were raised in rabbits and purified as previouslydescribed (27), but omitting the affinity chromatographystep.

Direct immunoprecipitation of GOGAT. Crude fractionscontaining a constant amount of total protein were incubatedwith various quantities of the anti-GOGAT total gammaglobulin fraction.

Incubation was performed overnight at 4°C in the presenceof buffer G (0.1 M potassium phosphate, 25 mM sodiumbisulfite, 6 mM 2-mercaptoethanol, pH 7.6) containing 1 mgof bovine serum albumin per ml. The final volume was 0.15ml. After incubation, the reactions were centrifuged througha discontinuous sucrose gradient (0.5 to 1.0 M sucrose inbuffer G plus 1 mg of bovine serum albumin per ml) at 7,500rpm for 30 min in an HB-4 Sorvall rotor. GOGAT activitywas measured in samples of the supernatants when indi-

cated, and the pellets were processed for sodium dodecylsulfate (SDS)-gel electrophoresis as described below.

Electrophoresis. The immunoprecipitates were suspendedin 0.1 M Tris (pH 6.8)-1% SDS-1% 2-mercaptoethanol andboiled for 3 min. Samples were subjected to polyacrylamidegel electrophoresis in slabs of 7.5% polyacrylamide contain-ing 0.5% SDS as described by Laemmli (17) and stained withCoomassie blue R-250. Molecular weight markers were fromSigma Chemical Co., St. Louis, Mo.

RESULTS

Phenotypic characterization of glutamate auxotrophs. Theglutamate auxotrophs described in this paper were isolatedas second-site mutations that suppress the leaky growth onexcess ammonia (25 mM ammonium nitrate) of the am(132)mutant strain. Three tight glutamate auxotrophs were iso-lated from ca. 600 survivors after mutagenesis and selectionfrom am(132) (alleles DR-i and DR-2) or from am(132);inlmutant strain (allele DR-3) as described in Materials andMethods. None grew on ammonium nitrate as the nitrogensource. All the mutants were able to grow as well asthe parental strain when glutamate, alanine, or glutamateplus ammonia were present in the growth medium. Thesephenotypic characteristics match exactly those of theam(132);en(am)-2 (C24) mutant strain.GOGAT specific activity in the mutants was measured and

compared with the activity in the am(132) mutant strain incultures grown for 24 h on 0.5 mM glutamate as the solenitrogen source at 25°C. This condition allows half-maximalinduction of GOGAT activity. All of the mutants exhibitedless than 10% of the GOGAT relative activity in am(132)cells. The GOGAT specific activity (mean ± standard devi-ation) of the am(132) mutant was 11.25 ± 1.05 nmol ofNADH oxidized per min per mg of extract protein; thespecific activities of the am(J32);en(am)-2 (C24),am(J32);en(am)-2 (DR-1), am(132);en(am)-2 (DR-2), andam(J32);en(am)-2 (DR-3) mutants were all <1.05 nmol min-mg'1. As reported previously (8, 14), the am(132);en (am)-2(C24) mutant strain is devoid of any detectable GOGATactivity. Extracts of these mutants neither inhibit nor en-hance the activity of wild-type extracts (data not shown).

Genetic analysis of glutamate auxotrophs. The results of thegenetic analysis indicate that the mutations responsible fortight glutamate auxotrophy behave as single mendelian fac-tors. The mutations, in an otherwise wild-type background,were cryptic on minimal medium, like the en(am)-2 (C24)mutant strain (14). The mutations described in this paper donot recombine with each other or with the previously de-scribed en(am)-2 (C24) mutation (no wild-type recombinantsamong 5 x 104 viable ascospores). These mutations, as wellas the en(am)-2 (C24) mutation, were recessive to thewild-type allele in forced heterokaryons. Heterokaryonsobtained by mixing the mutants in all pairwise combinationsdid not show complementation for growth on ammoniumnitrate, indicating that they belong to a single complementa-tion group and are alleles of the previously isolated en(am)-2(C24) mutation.

Effect of en(am)-2+ gene dosage on growth and GOGATactivity. Testing the effect of en(am)-2+ gene dosage onGOGAT activity is a valuable approach for identification ofen(am)-2 as the structural gene for GOGAT in N. crassa.Therefore, two partially duplicated strains bearing one ortwo en(am)-2 wild-type alleles were constructed as de-scribed in Materials and Methods. The partially duplicatedstrains were in an am(132) mutant background. These partial

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GLUTAMATE SYNTHASE STRUCTURAL GENE 1045

diploids were grown on minimal medium with ammoniumnitrate as the sole nitrogen source, and GOGAT specificactivity was measured in the cell extracts. A slight differencewas found between the GOGAT activities of the am(132)haploid strain and those in the heterozygous partial diploid(Table 1). However, when the partial diploid carried twoen(am)-2+ gene copies, the GOGAT specific activity was50% higher than that in the heterozygous partial diploid(Table 1). Thus, the en(am)-2+ gene shows a gene dosageeffect on GOGAT specific activity.The differences found in GOGAT specific activity in

partial diploids had a strong influence on the growth rate ofthese strains in ammonium nitrate-supplemented medium.After an initial lag phase of 8 h, strains am(132),am(132);en(am)-2 (C24)Ien(am)-2+, and am(132);en(am)-2+1en(am)-2+ grew with duplication times of 7, 9, and 4 h,respectively. Thus, there is an inverse relationship betweenGOGAT activity levels and duplication time. This is consis-tent with the demonstration that the leaky growth of an ammutant strain on ammonium was due to the presence ofGOGAT activity (14).CRM in mutant extracts. A partially purified preparation of

GOGAT (150-fold) was used to raise an anti-GOGAT anti-body in rabbits. The antibody preparation neutralized theGOGAT activity of am(132) cell extracts (Fig. 1). Thepresence of cross-reacting material (CRM) in mutant ex-tracts was shown by pretreating the antibody with mutantextracts and then titrating the GOGAT activity of theam(132) strain with the remaining antibody (Fig. 1). Curve 1shows the inactivation of am(132) GOGAT activity withincreasing concentrations of antibody. Pretreatment of theantibody preparation with 1 mg of extract protein from strainam(132);en(am)-2 (C24) slightly shifted the neutralizationprofile (Fig. 1, curve 2). Another mutant, am(132);en(am)-2(DR-2), had enough CRM in 1 mg of extract protein todisplace the neutralization profile strongly, so that theam(132) GOGAT activity was only 30% neutralized at thehighest antibody concentration tested (Fig. 1, curve 3).

-50

0%2 2.5 5 10MICROLITRE ANTIBODY

FIG. 1. Titration of GOGAT antibody in double mutants and theam(132) mutant strain. To each tube, various amounts of theantibody and 1 mg of protein from a cell extract of am(132);en(am)-2(C24) (0) or from am(132);en(am)-2 (DR-2) (A) were added. Thetubes were incubated for 12 h at 4°C and centrifuged. One milligramof protein from an am(132) cell extract was added to the superna-tants of each tube. After 5 h at 4°C, GOGAT activity was determinedin the supernatants. The titration of GOGAT activity in 1 mg ofprotein from an am(132) cell extract (0) is also shown.

A B C D E

-205K

-116 K

- 97.4K

W* _ F F * 4 _66 K

FIG. 2. Electrophoresis in the presence of SDS of im-munoprecipitated GOGAT from wild-type and mutant strains. Foreach strain, 500 ,ug of extract protein was immunoprecipitated asdescribed in Materials and Methods. Immunoprecipitates weresubjected to acrylamide gel electrophoresis in the presence of SDSfollowed by Coomassie blue staining. Lanes: (A) am(132), (B)am(J32);en(am)-2 (C24), (C) am(132);en(am)-2 (DR-1), (D)am(132);en(am)-2 (DR-2), (E) am(132);en(am)-2 (DR-3). The posi-tion of molecular weight markers is indicated in the right part of thefigure (Ks indicate thousands of molecular weight units). The bandat 66,000 molecular weight on all lanes is bovine serum albumin,which was used as an internal molecular weight marker.

These data clearly show the presence of an enzymaticallyinactive antigen on both mutants, albeit at different levels.GOGAT polypeptides in mutant strains. To search for

structural differences between the GOGAT polypeptide fromen(am)-2 mutants and the am(132) mutant strains, theirimmunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis.

It was previously reported that GOGAT from N. crassa iscomposed of a single polypeptide of 200,000 molecularweight (14). The antibody precipitated a 200,000-molecular-weight polypeptide from cell extracts of the am(J32) mutantstrain (Fig. 2, lane A). Some en(am)-2 mutant strains (allelesDR-I and DR-2; Fig. 2, lanes C and D) also show the 200 KGOGAT polypeptide. Thus, it appears that CRM detected inthe am(l32);en(am)-2 (DR-2) mutant strain by the assay ofFig. 1 (curve 3) is a polypeptide of the same molecularweight as that of wild-type GOGAT. In addition, thesestrains present a faint band at 180,000 molecular weight, notfound in the am(J32) cell extracts. The relationship of thispolypeptide with the GOGAT polypeptide remains unsettled(see Discussion).The remaining en(am)-2 alleles, C24, and DR-3, do not

show the 200,000-molecular-weight polypeptide or lower-molecular-weight polypeptides. However, in the en(am)-2(C24) mutant strain, low levels of CRM have been detectedwith immunochemical procedures (Fig. 1, curve 2). Thereason for this discrepancy remains unknown.

DISCUSSION

We have isolated three new N. crassa mutants lackingGOGAT activity. Complementation and mapping data indi-cate that these mutations are allelic to the previously de-scribed en(am)-2 locus (8, 14). All of these mutations are

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1046 ROMERO AND DAVILA

recessive to the wild-type allele in forced heterokaryons.The en(am)-2 (C24) allele is recessive to the wild-type alleleeven when present in the same nucleus (Table 1).We present two lines of evidence that suggest that en(am)-

2 is the structural gene for GOGAT. First, en(am)-2 shows agene dosage effect on GOGAT activity. A strain carryingtwo en(am)-2+ gene copies possesses GOGAT levels 50%higher than the heterozygous partial diploid (Table 1). Theduplication of the en(am)-2+ gene does not entail an exactdoubling in GOGAT activity. Therefore, we cannot excludethe presence of compensating factors limiting the GOGATlevels in the homozygous diploid. However, increases inactivity similar to that reported in this paper have been foundin Saccharomyces cerevisiae (15) and Drosophila melano-gaster (7) gene loci, where gene dosage has been suggested.Therefore, we think that the above result is entirely consis-tent with the idea that en(am)-2 is the GOGAT structuralgene, but would be hard to explain if the gene were regula-tory rather than structural. A direct relationship betweengene dosage and the corresponding enzyme activity is gen-erally assumed to exist, as indicated in the structural geneloci of S. cerevisiae for alkaline phosphatase (15), adenylatecyclase (21), and proteinase B (30). In contrast, a lack ofgene dosage effect has been observed in two S. cerevisiaepositive regulatory loci, GAL4 (16) and PH09 (15). Modifi-cations in the gene number have been used previously in thestudy of phosphate-regulatory genes in N. crassa (22).The second line of evidence indicating that en(am)-2 is the

structural gene for GOGAT is the finding of high levels ofCRM in some mutant strains. The en(am)-2 (DR-2);am(132)mutant strain had high amounts of CRM, whereas theen(am)-2 (C24);am(132) mutant strain had low amounts ofCRM (Fig. 1). However, both mutant strains are totallydevoid of GOGAT enzyme activity. The existence of CRMconcomitant with a low or null enzyme activity has beenused as a criterion of mutations in structural genes (10, 19,27); accordingly, our results suggest that mutations en(am)-2(C24) and en(am)-2 (DR-2) are located within the GOGATstructural gene.The molecular weight of the GOGAT polypeptide in at

least two of the en(am)-2 mutant strains (DR-I and DR-2;Fig. 2) is similar to that of the wild-type enzyme as indicatedby acrylamide gel electrophoresis in the presence of SDS ofimmunoprecipitates from cell extracts. This is further evi-dence for the presence of enzymatically inactive protein inthese strains. We believe that the faint band at 180,000molecular weight in both mutant strains could be due toproteolytic degradation from 200,000-molecular-weight un-stable mutant proteins, as reported for mutant proteins inEscherichia coli (29) and wild-type proteins in N. crassa(12). This could also account for our present inability tovisualize a mutant polypeptide in the en(am)-2 (C24) mutantstrain, despite its detection by immunochemical procedures(Fig. 1, curve 2). However, more experiments are needed toclarify this point.A lack of GOGAT activity in mutant strains could be due

to (i) mutations in the structural gene, (ii) mutations in otherloci responsible for transcriptional regulation, such as acti-vators or repressors, or (iii) mutation in a locus encoding aposttranslational processing enzyme. Since all of the muta-tions analyzed in this paper were affecting the same locusand two of them showed high levels of CRM, possibility (ii)becomes unlikely. If two allelic mutations were affecting alocus responsible for transcriptional activation or repres-sion, both mutants should be devoid of CRM, which is notthe case. Moreover, the en(am)-2 locus shows a gene dosage

effect, whereas loci encoding transcriptional regulators gen-erally do not show this property (15, 16). Possibility (iii) isalso unlikely, since mutations in a processing enzyme shouldlead to high levels of CRM, whereas two of the mutantsshow low or null levels of CRM (i.e., alleles C24 and DR-3).Therefore, the above results suggest that the mutationsanalyzed in this paper are affecting the GOGAT structuralgene. However, a definitive assignation of en(am)-2 as thestructural gene for GOGAT must wait for more directapproaches, such as gene or protein sequencing (or both)and the isolation of mutants carrying specific substitutions.

ACKNOWLEDGMENTS

We thank A. Gonzalez, J. Mora, R. Palacios, and L. Segovia fortheir critical reviews of the manuscript. We also thank A. Gutierrezfor the typing of the manuscript.D.R. was the recipient of a scholarship from the Consejo Nacional

de Ciencia y Tecnologfa (Mexico).

LITERATURE CITED1. Berberich, M. A. 1972. A glutamate-dependent phenotype in E.

coli K12: the result of two mutations. Biochem. Biophys. Res.Commun. 47:1498-1503.

2. Boland, M. J., and A. G. Benny. 1977. Enzymes of nitrogenmetabolism in legume nodules. Purification and properties ofNADH-dependent glutamate synthase from lupin nodules. Eur.J. Biochem. 79:355-362.

3. Calder6n, J., and J. Mora. 1985. Glutamine cycling in Neuros-pora crassa. J. Gen. Microbiol. 131:3237-3242.

4. Davila, G., S. Brom, Y. Mora, R. Palacios, and J. Mora. 1983.Genetic and biochemical characterization of glutamine synthe-tase from Neurospora crassa glutamine auxotrophs and theirrevertants. J. Bacteriol. 156:993-1000.

5. Davis, R. H., and F. J. De Serres. 1970. Genetic and microbio-logical research techniques for Neurospora crassa. MethodsEnzymol. 17A:79-143.

6. Desphande, K. L., and J. F. Kane. 1980. Glutamate synthasefrom Bacillus subtilis: in vitro reconstitution of an activeamidotransferase. Biochem. Biophys. Res. Commun. 93:308-314.

7. Devlin, R. H., D. G. Holm, and T. A. Gigliatti. 1982. Autosomaldosage compensation in Drosophila melanogaster strainstrisomic for the left arm of chromsome 2. Proc. Natl. Acad. Sci.USA 79:1200-1204.

8. Dunn-Coleman, N. S., E. A. Robey, A. B. Tomsett, and R. H.Garret. 1981. Glutamate synthase levels in Neurospora crassamutants altered with respect to nitrogen metabolism. Mol. Cell.Biol. 1:158-164.

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