Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa

Mol Gen Genet (1993) 238:81-90

© Springer-Verlag 1993

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa Patricia M. Okamoto 1,,, Reginald H. Garrett 2, George A. Marzluf 1

1 Department of Biochemistry, The Ohio State University, Columbus, OH, USA 2 Department of Biology, University of Virginia, Charlottesville, VA, USA

Received: 1 July 1992 / Accepted: 2 October 1992

Abstract. Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heine, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-l-suppressed nonsense mutants showed restoration of the wild-type, fulMength nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.

Key words: Neurospora crassa - Nitrate reductase - nit-3 mutant genes - Repeat-induced point mutation

Introduction

The filamentous fungus, Neurospora crassa, can utilize nitrate as a secondary nitrogen source when favored

* Present address:Worcester Foundation for Experimental Biology, Shrewsbury, MA, USA

Communicated by C.A.M.J.J. van den Hondel

Correspondence to: G.A. Marzluf, Department of Biochemistry, Bi- ological Sciences, Room 776, The Ohio State University, 484 West 12th Avenue, Colombus, OH 43210, USA

compounds such as ammonia, glutamate, and glutamine are not available (Marzluf 1981). The use of nitrate requires the de novo synthesis of nitrate reductase and nitrite reductase, encoded by nit-3 and nit-6, respectively. The expression of the nit-3 gene is highly regulated and requires simultaneous nitrogen limitation and nitrate induction (Fu and Marzluf 1987a). Nitrogen derepression appears to be mediated by a positive-acting global regulatory protein NIT2, which contains a single Cysi/Cysz type zinc finger DNA-binding domain (Fu and Marzluf 1987b, 1990a). NIT2 has been demonstrated to bind to elements bearing two or more copies of a core GATA sequence found in the 5' upstream promoter regions of relevant structural genes; the promoter region of the nit-3 gene contains three such NIT2 binding sites (Fu and Marzluf 1990b).

The expression of the nit-3 structural gene is also completely dependent upon a pathway-specific, positive- acting regulatory protein NIT4, which is believed to mediate nitrate induction. The NIT4 protein has a GAL4-1ike Cys6-Zn2 motif, which is essential for function and is believed to represent a sequence-specific DNA binding domain (Yuan et al. 1991). NIT4 displays considerable homology, particularly in the putative DNA binding domain, with NIRA, the corresponding control protein ofAspergillus nidulans (Burger et al. 1991); moreover, the Neurospora nit-4 gene can substitute via transformation for a mutant nirA gene (Hawker et al. 1991).

The enzyme nitrate reductase is a dimer of two large identical subnnits, each with three separate domains that comprise individual redox centers (Le and Lederer 1983; Kinghorn and Campbell 1989). The carboxyl-terminal domain contains a flavin cofactor, the central domain contains a heme, and the amino-terminal domain contains an unusual molybdenum-containing pterin cofactor; electrons derived from NADPH are believed to be passed in turn to the flavin, heine, and molybdenum domains, and finally used to reduce nitrate to nitrite. It is possible to assay for the function of the holoenzyme, nitrate reductase, and also to conduct partial assays for each of the three separate domains (Garrett and Nason 1967; Campbell 1986).

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Many genetic and biochemical studies have been carried out with nitrate reductase and conventional nit-3 mutants of N. crassa. These include studies of enzyme synthesis and stability, genetic and metabolic regulation, amber nonsense mutants, autogenous control, and mRNA content (Premakumar et al. 1978, 1979; Su- bramanian and Sorger 1972; Tomsett and Garrett 1980; Perrine and Marzluf 1986; Fu and Marzluf 1988). How- ever, although many nit-3 mutants were isolated and studied over two decades ago, no molecular characterization of them has been possible. The recent cloning and sequencing of the nit-3 gene now makes it possible to identify the mutational lesions in various nit-3 mutants and to correlate the changes with alterations in partial and complete enzyme activities (Fu and Marzluf 1987a; Okamoto et al. 1991). Similarly, it is now possible to determine the nature of transformants obtained when a wild-type or deleted variant of the nit-3 gene is introduced into mutant host strains. We report here an analysis of several conventional mutants and also de- scribe the creation and characterization of new null nit-3 mutants obtained by repeat-induced point mutation (RIP). We also demonstrate that homologous recombination can occur at a high frequency when a defective or deleted nit-3 gene is introduced into a strain with a mutant resident nit-3 gene.

Materials and methods

Neurospora crassa strains and growth conditions. The N. crassa wild-type strains, 74OR23-1A) (74A) and 74OR23-1a (74a), and nit-3 mutant strains, 14789A and 14789a, were obtained from the Fungal Genetics Stock Center (Kansas City, Kan., USA). N. crassa nit-3 mutant strains V 1M 16 and V 1 M4 have been described (Tomsett and Garrett 1981). Isolation of four nit-3 amber mutant strains (alleles 026, 0213, 1211, and 1222) and the corresponding suppressor Ssu-1 nit-3 strains were previously reported (Perrine and Marzluf 1986). Routine genetic manipulations and maintenance of stock cuttures were performed as described by Davis and deSerres (1970). The growth medium consisted of Vogel's minimal medium lacking the usual nitrogen source plus 2% su- crose and 0.01 lag/ml biotin to which was added as the sole nitrogen source, either 20 mM L-glutamine or 20 mM potassium nitrate; the former corresponded to nitrogen-repressed and the latter to nitrogen-derepressed, nitrate-induced conditions, respectively.

Flasks of liquid medium were inoculated with conidia and after growth at 30 ° C with shaking at 150 to 180 rpm for at least 15 h, the mycelia were harvested, rinsed with water and quickly frozen in liquid nitrogen. The various nit-3 mutant strains cannot grow on nitrate and thus were only grown on medium containing glutamine. When induction of cultures grown under repressing nitrogen conditions was desired, the mycelia were harvested, rinsed several times with water, and transferred to nitrate medium for an additional 3 h of incubation. Chlorate medium, which was routinely used to select

for the nit-3 mutant phenotype, is 1 × Vogel's minimal medium without nitrogen, containing 5 mM urea and 100 mM potassium chlorate. Neurospora homokaryons were isolated from microconidia purified by passage through a Millipore Durapore Millex-SV 0.5 gm filter according to the method of Ebbole and Sachs (1990).

Plasmids and transformation of N. crassa. Nested deletion subclones were made by using either convenient restriction sites or exonuclease III and mung bean nuclease as previously described (Fu and Marzluf 1988). Transformation of N. crassa spheroplasts was described elsewhere (Akins and Lambowitz 1985; Cherniack et al. 1990). Cotransformation assays were performed with a nit-3 plasmid plus a vector carrying the hygromycin resistance gene (pCSN44; Staben et al. 1989) at a 1 : 5 ratio selection for hygromycin resistance in medium containing 0.2 gg/ml filter-sterilized hygromycin B.

Nucleic acid techniques and the polymerase chain reaction. N. crassa genomic DNA was isolated using a modified method of Metzenberg and Baisch (1981). Plasmid DNA was isolated by the small-scale method of Birnboim and Doly (1979). Other DNA techniques such as Southern analysis were carried out as described in Sambrook et al. (1989). The polymerase chain reaction (PCR; Saiki et al. 1988) mixture consisted of 25-50 ng of genomic DNA, 1.5 mM MgCI2, 1.5 mM of each dNTP, 250 ng of each primer, 1 × reaction buffer (10 mM TRIS, pH 8.3, 50 mM KC1) and 2.5 Taq polymerase (Bethesda Research Laboratories, Gaithersburg, Md.) in a total volume of 50 gl overlaid with an equal volume of mineral oil. The sample was placed in an automated thermal cycler (Eri- comp, San Diego, Calif.) which was set for the following parameters 94 ° C for 30 s, 45 ° C for 1 rain, 72 ° C for 3 rain. After 30 cycles, an additional cycle at 94 ° C for 30 s, 45 ° C for 1 rain, and 72 ° C for 7 rain was run to complete the amplification process. Amplified nit-3 fragments were cloned into the pBluescript vector (Stra- tegene, LaJolla, Calif.) and sequenced via the dideoxy- nucleotide chain termination method (Sanger et al. 1977) using the modified T7 polymerase, Sequenase (United States Biochemical Co., Cleveland, Chio). In each case, three independent PCR-amplified DNA fragments were cloned and sequenced to confirm that the mutational changes identified were authentic (and not introduced by the PCR reaction).

Western blot analysis. Expression of the heine domain of the Neurospora nitrate reductase protein in Escherichia coli and isolation of this protein for polyclonal antibody production will be described in detail elsewhere. For Western blot experiments, crude Neurospora cell extracts were prepared by grinding on ice 500 mg of mycelia with 0.5 ml of extraction buffer (20 mM TRIS-Hel, pH 7.4, 100 mM NaC1, 1 mM EDTA, 1 mM phenylmethylsul- fonyl fluoride). The extracts were centrifuged at 14000 rpm, 4 ° C for 10 rain to remove cellular debris. The protein samples were resolved by discontinuous SDS- polyacrylamide gel electrophoresis (Laemmli 1970), and

83

electroblotted onto two layers of BA-85 nitrocellulose filter (Schleicher and Schuell, Keene, N.H.) as described in Sambrook et al. (1989). Protein transfer was usually for 2.5 to 3 h at 55 V and room temperature.

For detection of the NIT-3 protein with the anti-NIT3 polyclonal antibody, either the Immunoselect kit (Beth- esda Research Laboratories, Gaithersburg, Md), which employs a biotinylated goat anti-rabbit immunoglobulin (IgG), streptavidin-alkaline phosphatase system, or the enhanced chemiluminescence kit (Amersham Corp., Ar- lington Heights, Ill.), which utilizes a donkey anti-rabbit IgG, horseradish peroxidase system, were used according to the manufacturers' recommendations. With each detection system, specificity was enhanced by preincubating the primary polyclonal antibody with 5 10 gl of Neuro- spora extract from nitrogen-repressed wild-type mycelia per 10 ml hybridization buffer.

Enzyme assays. All enzyme and protein concentration assays were performed in duplicate, and absorbance readings were made on a Gilford 250 spectrophotometer. Full nitrate reductase enzyme activity was assayed as described previously (Garrett and Cove 1976). Specific activity was calculated from measurements taken at reaction times of 10 and 20 rain. Partial enzyme activities were assayed by the procedures of Garrett and Nason (1967). For the methyl viologen-nitrate reductase partial activity, each assay consisted of 50 ~tl crude enzyme extract, 50 gl 200 mM KNO3, 25 gl 10 mM methyl viologen (Sigma Chemical Co., St Louis, Mo.) and 0.85 ml 30 mM phosphate buffer, pH 7.4, with 10 mM EDTA. The reaction mixtures were prewarmed for 5 rain at 30 ° C after which freshly prepared sodium dithionite reductant (25 gl 100 mM solution in 0.1 N NaOH) was added to start the redox reaction. Assays were carried out for 5 and 10 rain, and stopped by vortexing vigorously for about 20 s followed by precipitation with 50 gl 1 M zinc acetate. The supernatant was analysed for nitrite formation using the diazo reaction as in the full nitrate reductase assay.

Partial activity of the combined heme-flavin domain was assayed by the method of Garrett and Nason (1967) and involved measuring cytochrome c reductase activity, determined as an increase in absorbance at 550 nm. All measurements were made at room temperature. For partial activity assay of the flavin domain, the reaction

composition was identical to that for the heme-flavin assay except that 100 gl of 10 mM potassium ferricyanide replaced cytochrome c, and the decrease in absorbance at 420 nm was determined (Garrett and Nason 1967). For both heme-flavin and flavin partial assays, it was sometimes necessary to dilute the crude enzyme extract with phosphate buffer to obtain a linear absorbance curve over a 2 min period. The reaction rates were followed on a Gilford chart recorder (model 6051; chart speed, 5 cm/min) which was interfaced to the spectrophotometer. Protein concentrations were assayed by the Bradford method (1976) using bovine serum albumin as the protein standard.

Results

Western analysis of nit-3 amber nonsense mutant strains. Four amber nonsense mutants of nit-3 were previously isolated and shown to be suppressible by Ssu-1, which inserts tyrosine in response to the UAG nonsense codon (Perrine and Marzluf 1986). It was of interest to determine whether these mutants accumulate any detectable nitrate reductase protein. Crude cellular extracts of these four nit-3 nonsense mutants and their suppressed mutant strains (alleles KGP1222, KGP0213, KGP026, KGP1211) were prepared from mycelia that had been grown under both nitrogen repressed and nitrogen derepressed, nitrate induced conditions. The samples were loaded in duplicate onto 7.5% denaturing polyacrylamide gels along with extracts from the wild-type strain 74A and, in some cases, nit-3 mutant strain 14789A. One gel was stained for protein with Coomassie Brilliant blue to show that equal amounts of sample had been loaded per lane. The other gel was electroblotted onto two layers of nitrocellulose filters which were then probed with the anti-NIT3 antibody, with detection via a secondary antibody conjugated to horseradish peroxidase. Figure 1 shows that three of the amber mutants possess truncated nitrate reductase proteins of distinct-sizes, while no protein was detected in extracts of the fourth mutant (0213). Furthermore, the levels of expression of full-length nitrate reductase protein in the suppressor strains are in good agreement with prior nitrate reductase activity and growth studies (Perrine and Marzluf 1986). An additional smaller protein band, of about 85-90 kDa, was ob-

200kDa--

M

14789 SSU-1 SSU-1 74Ar_L.~, 1222 1222 026 026 DI gI a gI ' r -R-- -~ t-R----~r--R----.~

200kDa

SSU-1 SSU-1 74A 1211 1211 0213 0213 r - ~ ~ r - ~ - - - - ~ M 'R gl'WR gl ~

Fig. 1. Western analysis of nit-3 amber mutant and suppressed mutant strains. Crude extracts of four nit-3 amber mutant strains (KGP1222, KGP026, KGP0213, and KGP1211) and their corresponding suppressor strains (prefixed with SSU-1 in the figure) were prepared from repressed (R) and induced (DI) mycelia and analyzed

by Western blot experiments. Nitrogen-derepressed, nitrate- induced (DI) samples of wild-type strain 74A and the conventional nit-3 mutant strain 14789A were used as controls and as size markers. Lane M represents a marker lane for protein size, with the position of the 200 kDa protein indicated

84

served in all but one of the suppressed mutant strains. This smaller protein may result from enzyme instability due to the insertion by the suppressor of a tyrosine residue at a site normally occupied by other amino acids, thereby resulting in an altered enzyme that may undergo partial degradation. Interestingly, the truncated mutant proteins that were detected (i.e., alleles KGP1222, KGP1211, and KGP026) appear to be synthesized constitutively; that is, unlike the wild type, these mutant proteins are synthesized without a requirement for nitrate induction. Upon suppression, however, the normal mode of regulation is restored. This result is consistent with the suggestion that nitrate reductase negatively autoregulates its own synthesis and that of nitrite reductase.

Analysis of nit-3 mutant alleles. Although many nit-3 mutants have been isolated and analyzed genetically and biochemically, no studies have previously been under- taken to characterize the nature of the mutational changes. To determine directly the type of mutation that occurs in each of three conventional nit-3 mutants (alleles 14789A, V1M16 and V1M4), the mutant genes were cloned via the polymerase chain reaction (PCR) and sequenced. Partial enzyme activity assays had previously indicated which domains of the enzyme were affected in different mutants and thus tentatively localized the mutant changes to particular coding regions of the nit-3 gene. The mutations of alleles V1M16 and 14789A were estimated to be in the 3' region of the gene while the mutation for V1M4 was believed to be in the 5' end (Tomsett and Garret t 1981). Due to the relatively large size (about 3 kb) of this gene, which made it difficult to amplify in its entirety by PCR, the nit-3 coding region was amplified into approximately 1.1 kb of partially overlapping fragments using three sets of primers and subcloned into pBluescript. Since Taq polymerase misincorporates nu- cleotides at a relatively high rate, e.g., 2 x 1 0 - 4 (Saiki et al. 1988), at least three independently isolated colonies from independent PCR reactions were sequenced for each mutant allele.

In Figure 2, the nature of each mutation and also their relative locations in the nit-3 gene are shown. These results agree very well with biochemical data in that the previously observed lack of partial activity for the flavin adenine dinucleotide (FAD) domain for mutant alleles 14789A and V1M16 correlates with two different de-

MUTANT nit-3 ALLELES V1ML 14789 V1M16

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V 1 N14 822 TTA~TAA 14789A 2331 AAG~AXG V1M16 2858 ATG-ATX

Fig. 2. Localization of the mutation in nit-3 mutant alleles. The localization of each mutation is presented in relation to the three different cofactor-binding domains. The position of altered or missing bases is given relative to the first transcriptional start site which is designated as + 1. X represent the deletion of one base

letions of a single base found in the 3' end of these mutant genes. Likewise the absence of any partial activities, as shown earlier for the mutant allele V1M4, is consistent with the molecular results; namely, a leucine codon (TTA), which occurs close to the amino-terminus of the nit-3 protein has been mutated to a nonsense codon (TAA), thereby producing the observed null phenotype.

Crude cellular extracts of wild-type 74A and nit-3 mutant 14789, V1M16 and V1M4 Neurospora strains were prepared from mycelia grown in both nitrogen- repressed and in nitrogen-derepressed, nitrate-induced medium and electrophoresed in duplicate on SDS 7.5% polyacrylamide gels. The 7.5% polyacrylamide concentration resolved the fairly large full-length nitrate reductase subunit and also permitted the detection of the smallest possible polypeptide (about 66 kDa) that could be detected with the anti-NIT3 antibody. One gel was stained for protein to insure that equal amounts were present in each lane, and the other gel was analyzed via

M 1 2 3 4 5 6 7 8

200kDa

1 2 3 4 5 6 7 8

Fig. 3. Western blot analysis of nitrate reductase in three nit-3 mutants. Upper panel: Crude extracts of nitrogen-repressed, and nitrogen-derepressed, nitrate-induced mycelia, respectively, from wild-type (lanes 1 and 2) and three nit-3 mutant strains 14789A (lanes 3 and 4), V1M16 (lanes 5 and 6) and V1M4 (lanes 7 lane 8) were electrophoresed in 7.5% denaturing polyacrylamide gel. The proteins were electroblotted onto two nitrocellulose filters and probed with the anti-NIT3 antibody. The polyclonal antibody was diluted 1:1000 in TBST buffer and preabsorbed with nitrogen- repressed Neurospora wild-type extract to increase its specificity. Detection of the proteins was by chemiluminescence using horseradish peroxidase-linked secondary antibody as described in the Materials and methods. The nitrate reductase subunit sizes for V1M16 and 14789A were estimated to be 100 and 85 kDa in comparison to the wild-type monomer size and with the protein Rainbow markers (lane M). The additional smaller bands observed with the V1M16 samples are postulated to result from partial protein degradation. Lower panel: A gel run in parallel and containing the same extracts was stained for protein to confirm that identical amounts of protein had been loaded onto each lane; the lane positions are identical to those of the upper panel

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pN3RVK pN3XK pN3PA33 pN3RA205 pN3BA317 pN3ZX508 pN3A620 pN3A3UT pN3A67 pN3A127 pN3&287 Fig. 4. Transformation assays of nit-3 nested deletion subclones with two different nit-3 mutant host strains. A set of plasmids containing nested 5' and 3' deletions of the nit-3 gene was transformed into the nit-3 mutant strain 14789A to determine if targeting occurs to the nit-3 gene. A restriction map of the nit-3 gene and relative positions of the cofactor-binding sites are shown at the top. The three asterisks represent NIT-2 binding sites as determined from previous footprinting experiments (Fu and Marzluf 1990a). The region of the gene remaining in each deleted form is shown

85

14789 RIP22

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aligned with the physical map. The single mutation in 14789A is shown by the dot in the flavin adanine dinucleotide (FAD) domain. The same set of deleted nit-3 genes was also transformed into a second mutant host, containing the repeat-induced point mutation (RIP)-induced RIP22 nit-3 allele. Plus (+) and minus ( - ) signs indicate the presence or absence of transformants with each of the deleted nit-3 genes. In all transformation assays, 1 ~tg of DNA was used

a Western blot. The subunit size of the wild-type Neuro- spora nitrate reductase is estimated to be 108 kDa, as predicted from the nit-3 nucleotide sequence.

As shown in Fig. 3, the anti-NIT3 antibody speci- fically detects a protein of of the estimated size but only in the nitrogen-derepressed, nitrate-induced wild-type Neurospora cellular extract. As expected, no protein was detected in the nitrogen-repressed, wild-type extract. These results agree with previous studies, which showed that the steady-state level of the nitrate reductase protein is highly controlled by the prevailing nitrogen source. Moreover, Western analyses of the mutant strains 14789A and V1M16 revealed mutant nitrate reductase proteins truncated to approximately 84 kDa and 100 kDa, respectively, which correlate very well with the position of the mutat ion identified in each of these strains by PCR and sequencing. Interestingly, these mutations representing a single base deletion in each strain, also affect the regulation of nitrate reductase synthesis such that the mutant proteins are expressed constitutively (Fig. 3). Not surprisingly, the anti-NIT3 antibody did not detect any V1M4 protein (Fig. 3), as anticipated from the molecular analysis which identified a stop codon occurring very early in the protein sequence.

Transformation o f host strain 14789A with partially deleted nit-3 genes. Previous results (Okamoto et al. 1991) suggested that even a deleted form of the nit-3 gene could transform to protot rophy a host strain carrying the nit-3 allele, 14789, which has a point mutat ion in the 3' end of the gene as shown above. Even though most transformants in Neurospora arise from integration at ectopic sites, these results suggested that an introduced nit-3 gene might undergo homologous recombination with the mutant resident gene at an unusually high frequency. To investigate this possibility, nested deletion subclones of

nit-3 were made in both directions and transformed into the strain 14789A; transformants were selected for their ability to grow in nitrate-containing medium. As shown in Fig. 4, all but one of the partially deleted nit-3 genes were able to transform the host strain to prototrophy. The number of transformants decreased dramatically from approximately 400 transformants per pmole D N A obtained with a complete nit-3 gene, to less than 25 per pmole D N A for subclones that contained any deletion into the nit-3 coding region. As anticipated, pN3A287, the deletion in which overlaps the mutant site of allele 14789, failed to transform this mutant host. Thus, these results imply that a nit-3 gene introduced by transformation targets to and recombines with the resident locus in the host strain 14789A. These results also strongly sup- port our previous identification of a single mutation site in nit-3 allele 14789. However, the possibility that some other event might also be involved such as protein- protein complementation, could not be completely ex- cluded.

Characterization o f the deletion transformants. In order to verify that the acquired nitrate-utilizing ability of the deletion transformants was due to the synthesis of an active nitrate reductase, a representative group of transformants was assayed for nitrate reductase activity. Ni- trate reductase activity was readily detected in each transformant assayed, regardless of the size of the deletion in the tranformed nit-3 gene, at levels that were approximately 50% of the wild-type strain. The strains transformed with the deleted nit-3 genes actually had slightly more nitrate reductase activity than a control in which the same host strain was transformed with a wild- type gene. The fact that considerable nitrate reductase activity was present in these transformants suggests that homologous recombination between the introduced de-

86

leted genes and the mutant resident gene gave rise to a wild-type nit-3 gene.

Western analyses of the deletion transformants. If homologous recombination has generated a wild-type nit-3 gene in Neurospora cells transformed with deleted nit-3 genes, this can be verified directly by determining the size of the enzyme protein by Western analysis. Since both the host mutant nit-3 gene and the deleted forms used for transformation can encode only a truncated protein, only homologous recombination between them could allow the formation of a full-size nitrate reductase subunit. Crude extracts were prepared from nitrate-induced mycelia of the wild-type strain, the host strain 14789A, and the representative transformants pN3RVK, pN3PA33, pN3RA205, and pN3BA317. The extracts were electrophoresed in duplicate on SDS -7 .5% polyacrylamide gels, of which one was stained with Coomas- sie brilliant blue to show that uniform amounts of protein were loaded in each lane (data not shown). The other

1 2 3 4 5 6

200kDa

M 1 2 3 4

Fig. 5. Western blot analysis of nitrate reductase. Upper panel: Western blots of the transformants of nit-3 mutant 14789A with deleted nit-3 genes. Extracts of nitrogen derepressed, nitrate induced mycelia of: lane 1, wild-type 74A (lane 2), mutant strain 14789A; and lanes 3-6, deletion transformants pN3RVK, pN3PA33, pN3RA205, and pN3A317 respectively were resolved in a 7.5 % denaturing polyacrylamide gel, then blotted to nitrocellulose membranes, and probed with the anti-NIT3 antibody. The secondary antibody used for detection was linked to streptavidin-alkaline phosphatase, which accounts for the cross-reacting band observed in all of the lanes. The upper arrow points to the position of wild- type nitrate reductase protein, whereas the lower arrow shows the location of the mutant 14789A NIT3 protein. The presence of the mutant 14789A protein in the deletion transformants is due to the use of heterokaryons for analysis. Lower panel: Western analysis of the RIP22 mutant. Extracts from nitrogen repressed and nitrogen derepressed, nitrate induced mycelia from wild-type 74A and nit-3 mutant allele RIP22A were examined by a Western blot experiment. Lanes 1 and 2 are repressed and induced wild-type extracts, respectively; lanes 3 and 4 are repressed and induced RIP22A extracts, respectively. The polyclonal anti-NIT3 antibody detected a nitrate reductase protein only in the lane containing the induced wild-type extract. As expected, no nitrate reductase was present in the mutant strain RIP22A. Lane M, size marker lane with the 200 kDa and indicated

gel was electroblotted and probed with the polyclonal anti-NIT3 antibody for detection of the NIT3 protein. A protein of approximately 95 kDa was found to cross- react with the streptavidin-alkaline phosphatase-linked secondary antibody in a control experiment (data not shown). This cross-reacting protein has also been observed with Neurospora crude extracts from cells grown under completely different conditions and is apparently a biotin-containing protein, which thus binds avidin and which is conjugated to the second antibody used for detection (Hurlburt and Garret t 1988; Jarai and Marzluf 1991a). This 95 kDa protein was useful as an internal standard to show that approximately equal quantities of protein had been transferred to the filter for each lane.

Figure 5 reveals that all of the deletion transformants examined contain a protein of the same size as the wild- type nitrate reductase subunit. Because heterokaryotic cells were used, the smaller endogenous mutant nitrate reductase monomer was also detected. Southern blot analyses of EcoRI-digested genomic DNA of two independent isolates (one for 205) of transformants that were obtained with four of the deleted nit-3 (RVK, 33, 205, and 317) was also conducted. The results (not shown) revealed that most of these transformants had only a single 3.8 kb band as found in the Wild type when probed with the 5' half of the nit-3 gene, as expected if homologous recombination had taken place. In two cases, a second band was also observed, which is not unusual since multiple integration events at ectopic sites frequently occur during transformation. The combined results clearly imply that an introduced nit-3 gene under- goes significant homologous recombination when transformed into the nit-3 mutant 14789A to yield a wild-type gene that encodes a full-length, functional nitrate reductase protein.

Generation of a nit-3 mutant host strain by the RIP phenomenon. To construct a new nit-3 mutant strain in which the resident nit-3 locus was so badly damaged that targeting could not occur, we took advantage of the recently discovered phenomenon called RIP, or repeat- induced point mutation (Selker et al. 1987; Selker and Garret t 1988; Selker 1990; Marathe et al. 1990). A nit-3 plasmid containing the entire nit-3 coding region plus 3'- and Y-flanking regions was cotransformed with the hygromycin-resistant gene into spheroplasts of wild-type strain 74A, resulting in a large number of hygromycin- resistant transformants. To enhance the mutation frequency in the ensuing cross with the wild type, homokaryons were isolated from several of the heterokaryotic transformants. The isolation of homokaryons prior to crossing has previously been shown to be an efficient method for increasing the frequency of RIP mutants (Jarai and Marzluf 1991b). Thirty hygromycin-resistant homokaryotic colonies were crossed with the wild type. Random ascospores were activated and spread onto plates containing minimal medium; from a total of 144 colonies that were tested, 12 displayed the nit-3 mutant phenotype. Since it has been shown that additional rounds of crossing result in even more mutagenesis by RIP, several mutant F1 progeny from the first cross

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Tyr Stop Lys 200 220 240

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Ser Asn Tyr Asn Stop Asn 280 300

AAT GAC ATT GAC AAC GAC CTC GCC AGC ACT CGC ACC GCC AGC CCG ACT ACA ACG Asn Asp l le Asp Asn Asp Leu Ala Ser Thr Arg Thr Ala Ser Pro Thr Thr Thr

Asn Val Asn 340 360 380

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Tyr Tyr Ire lle 400 420 440

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Lys kys Tyr 460 480 500

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Gin Tyr 540 560

ATC CGC GAC CCG CGC CTC ATT CGC CTC ACG GGA TCC CAC CCC TTC AAC lle Pro Asp Pro Arg keu lle Arg Leu Thr Gly Ser His Pro Phe Asn

His Asn lle 580 600 620 640

CTT TTC GAA CAC GGA TTC CTA ACC CCG CAA AAC CTC CAT TAC GTG CGC AAC CAC GGG CCC ATC CCT TCA Leu Phe Glu His Gly Phe Leu Thr Pro Gin Asn Leu His Tyr Val Arg Asn His Gly Pro lle Pro Ser

Tyr Lys His Tyr Arg 660 680 700

TCT GTC GCT ACC CCT CCC GCT ACC ATC AAT AAG GAG GAA GAC GAC TCG CTA CTA AAC TGG GAA TTC ACC Ser Val Ala Thr Pro Pro Ala Thr lle Asn kys Glu Glu Asp Asp Ser Leu keu Asn Trp Glu Phe Thr Phe lle

720

GTC GAA GGA CTC GTC GAG CAC CCC CTA Vat Glu Gly Leu Val Glu His Pro Leu

Tyr 780 800

AAC GTC ACG TAC CCC GTC ACT TTA GTG Asn Val Thr Tyr Pro Val Thr keu Val

l le Met l le 840

TCG AAG GGG TTC TCA TGG GGC GCG GGC Ser Lys Gly Phe Ser Trp Gly Ala Gly

Leu Stop Ser

Leu Leu 52O

CTCAAA ACC CCC GAC CAC CTC Leu Lys Thr Pro Asp His Leu

Asn Tyr 580

GTC GAA CCG CCG CTC ACT GCG Vat Glu Pro Pro Leu Thr Ala

Lys Leu

Leu Stop 740 760

AAA ATC AGC GTG CGG GAG TTG ATG GAC GCT TCC AAA TGG GAC kys lle Ser Val Arg Glu leu Met Asp Ala Ser kys T~-p Asp

Asn lle Stop lle Vat Stop 820 840

TGC GCG GGC AAC AGG AGG AAA GAA CAA AAC GTG CTC CGA AAA Cys A[a Gly Asn Arg Arg Lys Glu Gin Asn Va~ keu Arg Lys Tyr Asn Lys kys Stop

860 880 900

GGG CTA TCG ACT GCG TTG TGG ACG GGG GTG GGA CTT TCT GAG Gly keu Ser Thr Ala leu Trp Thr Gly Va~ Gly Leu Ser Glu Arg keu lle Vat keu Stop Lys

Fig. 6. Sequence analysis of the newly generated nit-3 mutant allele RIP22A. An abbreviated restriction map of nit-3 is shown above the sequence, and the positions of the polymerase chain reaction (PCR) primers used to amplify the region analyzed are indicated. The nucleotide and corresponding amino acid sequences are given for a 790 nucleotide region that begins with the ATG translational start codon. All G and C residues that have been mutated by the RIP . process are underlined. Amino acid residues that have been altered as a result of the induced point mutations are shown directly below the wild- type one. Nonsense mutations are de- picted as Stop

which retained at least two copies of the nit-3 gene were crossed again with the wild type. Progeny from this second cross were examined for their ability to grow on nitrate or in the presence of chlorate.

Table 1 gives the results of crosses of representative F1 and F2 mutant progeny to the established nit-3 mutant (14789A). The progeny from these crosses all failed to utilize nitrate, i.e., no wild-type recombinants were obtained. When the new RIP-induced mutants were crossed with the the wild-type strain, about one-half of the progeny tested were nitrate non-utilizing, as expected (Table 1). These results suggest that new mutants of the

nit-3 gene had been generated by the RIP process. These RIP-induced nit-3 mutants would be expected to lack nitrate reductase enzyme activity. Several RIP mutants, along with a wild-type strain and the standard nit-3 mutant (14789A) as controls, were assayed for complete and partial activities of nitrate reductase. No nitrate reductase activity or any partial activities at all could be detected in any of the RIP mutant strains (data not shown), suggesting that the RIP-induced mutagenesis was extensive throughout the nit-3 gene. Moreover, no nitrate reductase protein at all could be detected in the RIP22A strain by Western blot experiments (Fig. 5).

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Table 1. Results of crosses of repeat-induced nitrate non-utilizing (RIP) mutants with a conventional nit-3 mutant and with the wild type

RIP mutant crossed with

Wild-type 74A nit-3 (14789A)

RIPll 10/24 12/12 RIP17 15/24 12/12 RIP54 15/24 12/12 RIP66 11/24 12/12 RIP70 11/24 12/12 RIP22 5/12 12/12

Wild-type spheroplasts were transformed with an XbaI-KpnI clone of nit-3 as described in the Results. Homokaryons possessing multiple copies of the nit-3 gene were crossed with wild-type strain 74A, and the putative nit-3 RIP mutants were identified by their failure to grow on nitrate. These putative nit-3 mutants were crossed with the wild type and with the conventional nit-3 mutant strain (14789A), and the progeny were tested for growth on nitrate. RIP22 is an F2 nit-progeny obtained from a backcross of RIP17 to the wild type. The values in the table represent the number of nitrate non- utilizing isolates/total progeny tested

Southern blot analysis with genomic DNA digested with EcoRI and with PstI (not shown) revealed that all of the RIP mutants differed from the wild type, with the loss or gain of restriction sites. Two nit-3 RIP mutants, 22A and 24A, failed to respond to a wild-type probe (not shown), presumably because the sequence had diverged sufficient- ly to preclude hybridization.

Further to ascertain the extent of damage caused by the RIP process, one of the mutant alleles (RIP22A) was partially cloned by PCR and sequenced. A 790 nucleotide sequence of the 5' region was analyzed and found to have multiple GC to AT transition mutations, which give rise to many missense codons and multiple stop codons (Fig. 6). The primary goal in generating a strain with a heavily damaged nit-3 gene was to prevent formation of a wild-type gene via homologous recombination with any nit-3 gene introduced into the host during transformation. Thus, it was important to determine whether the new RIP22A mutant strain would be a suitable host for transformation assays. The RIP22A mutant was transformed with high efficiency by vectors carrying the full- length, wild-type nit-3 gene. The same deleted nit-3 subclones used in the studies described above with the nit-3 mutant strain (14789A) were then transformed into the RIP22A strain with selection on nitrate medium. As indicated in Fig. 4, all of the subclones that were missing any part of the nit-3 coding region completely failed to transform the new nit-3 mutant strain, suggesting that homologous recombination had been totally eliminated with this mutant strain.

Discussion

Amber nonsense mutants of nit-3, the structural genc that encodes nitrate reductase, were isolated previously. The mutants were identified by the fact that they were suppressed by the tRNA suppressor Ssu-1, which inserts

tyrosine in response to the UAG amber nonsense codon (Perrine and Marzluf 1986). In this study we demonstrated that three of these nonsense mutants each accumulate a truncated nitrate reductase protein, due to pre- mature chain termination as expected. The fourth mutant (0213) lacks any detectable cross-reacting protein, possibly because its nonsense fragment is unstable or because it accumulates a truncated protein lacking all of the heme domain, for which the antibody is specific. Nitrate reductase possesses three domains, which presumably are folded separately into globular units. Therefore it might be expected that even if the mutation led to protein instability, mutants in the 3' end of the gene which affect the third domain would accumulate a truncated protein, since probably only the defective domain would be degraded. However, the finding that each of these three mutants accumulated a different-sized truncated protein suggests that each retains more of the nitrate reductase protein than is represented by intact domains.

A feature of particular interest is the finding that these truncated proteins are expressed constitutively without any requirement for nitrate induction, whereas nitrate reductase expression in the wild type requires induction (Bahns and Garrett 1980). This finding is in agreement with the long-standing suggestion that nitrate reductase expression is subject to negative autogenous regulation in Aspergillus (Cove and Pateman 1969) and Neurospora (Tomsett and Garrett 1981; Fu and Marzluf 1988). A full-length nitrate reductase protein is present in all four of the Ssu-1 suppressed nit-3 mutants, the suppression level being consistent with the amount of enzyme activity previously detected (Perrine and Marzluf 1986). More- over, normal regulation was re-established in the suppressed amber mutant strains, in which the protein was only detected in extracts from cells that had been subject to simultaneous nitrogen limitation and nitrate induction. Although the precise mechanism by which autogenous regulation occurs is unknown, it has been spec- ulated that nitrate reductase may bind to NIT4, the pathway-specific positive-acting protein, and prevent the entry of NIT4 into the nucleus or inhibit its trans-activa- tion function. The various mutant nitrate reductase proteins thus appear not only to lack enzyme activity but also to be incapable of mediating autogenous control, perhaps because they cannot bind to NIT4. In three cases, the Ssu-1 nit-3 strains also accumulate a smaller protein, which was clearly detected via the anti-NIT3 antibody; this suggests that the protein in the suppressed mutant strains is less stable than the wild-type enzyme since such a degradation product was not observed in extracts from the wild-type strain.

We have reported here the nature of the mutation in three different classic nit-3 mutant alleles and showed that the site of the mutation agreed with the observed partial enzyme activities. Two of the mutants have a single base deletion that occurs in the 3' region of the nit-3 gene, which encodes the flavin domain, consistent with their possessing activity for the other two domains. The third mutant (V1M4) is due to a UAA nonsense mutation and should lack most of the amino-terminal

89

domain and all of the second and third domains of nitrate reductase; in complete agreement with this finding, this mutant lacks all partial enzyme activities and no protein can be detected with the anti-NIT3 antibody. The defective nitrate reductase protein observed in nit-3 mutants 14789A and V1M16 is expressed without nitrate induction, and, as discussed above, indicates that autogenous regulation may play a role in controlling nitrate reductase synthesis.

Convincing evidence was presented to demonstrate that homologous recombination occurs between a nit-3 gene introduced by transformation with exogenous D N A and the mutant nit-3 gene resident in the host, in the case of allele 14789, which has a single point mutation, i.e., a deleted base, in the 3' end of the gene. nit-3 genes deleted for the coding region for the entire molybdenum- containing domain and part of the heme domain recom- bined with the resident mutant gene to give a wild-type nit-3 gene, which encoded a full-length nitrate reductase monomer. That this explanation was correct was verified not only by demonstrating the presence of the protein in transformants but also by showing that the same set of nit-3 deleted genes absolutely failed to transform a new, severely damaged nit-3 allele created by repeat-induced point mutation, although a wild-type gene transformed this host with high efficiency. This new nit-3 allele (RIP22) will be a valuable host strain to test the function of in vitro manipulated nit-3 genes, since it completely eliminates the possibility that a wild-type gene can be formed by homologous recombination with the resident gene or any potential complication of complementation that might arise by protein-protein interactions.

It is possible that homologous recombination per se is strongly reduced or eliminated between a gene that has suffered multiple RIP mutations and a wild-type sequence although this possibility was not examined. In this context, it was recently demonstrated that sequence divergence between two mouse strains in the retinoblas- toma susceptibility gene (Rb) drastically reduced homologous recombination when one strain was transformed with an Rb construct derived from the other (Riele et al. 1992). The RIP22 allele created by RIP (Selker et al. 1987) clearly represents a null mutation and a severely damaged nit-3 gene, as indicated by several lines of evidence. The new RIP22 mutant fails to yield wild-type recombinants when crossed with a conventional nit-3 mutant, lacks all nitrate reductase activity and protein, and is transformed by a wild-type nit-3 gene but not by any of the deleted versions. Moreover, sequence analysis of the RIP22 mutant nit-3 allele revealed a significant number of point mutations throughout the duplicated region. With respect to the coding strand, at the 5' end, 42% of guanosines (71 of 171) and 20% of cytidines (60 of 307) were converted exclusively to adenosine and thymidine residues, respectively, in a stretch of 790 nu- cleotides (Fig. 6). These mutational changes caused the modification of 104 codons, which led to 71 amino acid substitutions and the creation of 8 stop codons. Interes- tingly, it appeared that there was a preference for G-to-A over C-to-T changes in one strand, as evidenced by the higher number of mutated guanosine residues at the 5'

end (Fig. 6). This apparently nonrandom difference in mutagenesis by the RIP process of one strand over another has been observed with other Neurospora genes (Cambareri et al. 1989; Selker 1990). An extreme exam- ple of strand selectivity was shown to occur with a fragment of the nmr-1 gene in which G to A changes were exclusively present on only one strand (Jarai and Marzluf 1991b).

Acknowledgements. This research was supported by Public Health Service grant GM-23367 (to G.A.M.) from the National Institutes of Health. We thank Dr. Gabor Jarai and Dr. Wilbur H. Campbell for helpful suggestions.

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Documents

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa