Cnx Mutants Research Articles

Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in Aspergillus niger

We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV), 1, 2, 7, 8-Diepoxyoctane (DEO), phenols mixture (Phx)) and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS) strain, which may specify nitrite specific transporter) and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system). The most relevant and innovative findings of this research work involve the isolation of the RR ( cbrn) mutants (a new type of nitrate assimilation defective mutants), that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments) were of the brn type, whereas the remaining percentage (6.76%) was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains) were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants) with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants` yield of Aspergillus niger mutant strains representing nine gene loci ( niaD, cnx-6 loci , nrt and nirA) were selected on the bases of chlorate (600 mM) toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM). The niaD mutants showed the highest percentage (73.97%) of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9%) of niaD mutants, whereas, the least (61%) was obtained with Phx treatment. The highest percentage of cnx mutants (32%) was obtained with Phx treatment. The DEO treatment as compared to other tested treatments was the best to use for obtaining the highest ratios of either nrt (13.8%) mutants or nirA (1.9%) mutants.

Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in aspergillus niger

We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV), 1, 2, 7, 8-Diepoxyoctane (DEO), phenols mixture (Phx)) and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS) strain, which may specify nitrite specific transporter) and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system). The most relevant and innovative findings of this research work involve the isolation of the RR ( cbrn) mutants (a new type of nitrate assimilation defective mutants), that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments) were of the brn type, whereas the remaining percentage (6.76%) was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains) were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants) with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants` yield of Aspergillus niger mutant strains representing nine gene loci ( niaD, cnx-6 loci , nrt and nirA) were selected on the bases of chlorate (600 mM) toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM). The niaD mutants showed the highest percentage (73.97%) of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9%) of niaD mutants, whereas, the least (61%) was obtained with Phx treatment. The highest percentage of cnx mutants (32%) was obtained with Phx treatment. The DEO treatment as compared to other tested treatments was the best to use for obtaining the highest ratios of either nrt (13.8%) mutants or nirA (1.9%) mutants.

The maize Viviparous10/Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesis

Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele of vp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABA-biosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses.

Screening forArabidopsismutants affected in theNiigene expression using theGusreporter gene

A mutant screen was developed to isolate Arahidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii 1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A, thaliana, and shown to be properly regulated by nitrate. Moreover, β-glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves showing that the organ-specific expression of the tobacco Nii 1 gene was retained in Arahidopsis. M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.

Screening for Arabidopsis mutants affected in the Nii gene expression using the Gus reporter gene

A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana, and shown to be properly regulated by nitrate. Moreover, β-glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis. M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.

Characterization of four molybdenum cofactor mutants of rice, Oryza sativa L.

Four nitrate reductase-deficient (NR −) mutants, C25, C27, C32 and C33, were isolated from 100 000 M 2 seedlings of rice ( Oryza sativa L., ssp. indica, cv. IR30) by means of selection for chlorate-resistance. All mutations were monogenic and recessive. Biochemical analysis showed that both NADH- and NADPH-dependent activities of nitrate reductase (NR) in these four mutants were less than 25% of the wild type IR30. However, nitrite reductase activities were more than the wild type. Also, xanthine dehydrogenase (XDH) activities and molybdenum cofactor biosynthesis abilities (MoCo activities) were deficient indicating that these mutants were cnx and not nia type. The cnx mutants could be divided into two groups: one could recover NADH-NR activity by adding 0.5 mM molybdate to the growth medium (C27, C32 and C33) while the other could not (C25). The result of allelism test coincided with the phenotypic classification by adding molybdate. The growth response to tungstate was also investigated in the mutants. None of the mutants classifies to the tungstate-sensitive group as reported in Arabidopsis cnx mutants.

Molybdenum Cofactor Mutants, Specifically Impaired in Xanthine Dehydrogenase Activity and Abscisic Acid Biosynthesis, Simultaneously Overexpress Nitrate Reductase.

The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728-733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.

Improved Media for Selecting Nitrate-Nonutilizing Mutants in Aspergillus flavus

Selection and identification of nitrate-nonutilizing (nit) mutants of Aspergillus flavus was compared on six chlorate-containing agar media. Three isolates of A. flavus were used. Nit mutants isolated comprised three phenotypic classes, equivalent to niaD, nirA-, and cnx mutants of A. nidulans. There were significant differences among isolates and among media in number and types of nit mutants isolated. The most effective medium contained only nitrate as a nitrogen source, whereas most previously described media for isolation of nit mutants contain both nitrate and an amino acid. Morphology of nit mutants was more distinctive on this medium than on other media. The nitrate-only medium yielded mainly niaD mutants; other media yielded more nirA- mutants, which complement niaD mutants. Few cnx mutants were found. Use of a nitrate-only medium in conjunction with another medium should optimize isolation of nit mutants and complementary pairs of mutants.

Biochemical characterization of cnx nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

In cnx mutants of Nicotiana plumbaginifolia, the over-expression of nitrate reductase (NR)-associated NADH cytochrome c reductase (Ccr) activity appears to be correlated with increased levels of NR mRNA. However, in ELISA tests, cnxB, C, D, E and F extracts present low levels of cross-reacting material, whereas cnxA extracts exhibit very high levels, indicating an altered NR structure in all cnx mutants except cnxA, due to the absence of a functional molybdenum cofactor (MoCo). Detection of dimeric NR in all cnx mutants, either by gel filtration or native gel electrophoresis, shows that the cofactor is not required for enzyme dimerization, but probably for the stability of the dimer. Similarities between results obtained with cnxB through F mutants, and those nia mutants impaired in the molybdopterin domain, suggest a related NR structure.

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants.

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.

Complementation analysis of nitrate reductase deficient mutants of Nicotiana tabacum by somatic hybridization

Mutant cell lines lacking nitrate reductase activity were analyzed genetically. Protoplasts from one apoprotein defective (nia) and four cofactor defective (cnx) mutants were fused in all possible pairwise combinations with the aid of polyethylene glycol. Complementing hybrids were detected by their ability to grow with nitrate as sole nitrogen source and confirmed by measuring their nitrate reductase activity. Strong complementation was observed in all types of nia+cnx hybrids, whereas the cnx mutants failed to complement each other. From the results it can be concluded that the mutants studied are recessive and that the four cnx mutants are alleles of the same pair of duplicate loci (cnxA1, cnxA2).

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.

Molybdenum cofactor in nitrate reductase-deficient tobacco mutants. III. Induction of cofactor synthesis by nitrate

The molybdenum cofactor (Mo-co) content of cultured cells of Nicotiana tabacum was estimated by the ability of the acid and heat treated extract to restore NADPH-nitrate reductase (NR) of Neurospora crassa nit-1. Addition of nitrate to wild-type cells grown on amino acids caused a rapid increase in Mo-co activity concomitant with induction of NADH-NR. Time course experiments revealed both the constitutive and the induced levels of Mo-co activity to be dependent on culture age, the maximum occurring during log phase. According to their Mo-co activity, the 12 NR-deficient mutant cell lines tested were shown to fall into 3 groups: Group I nia mutants are characterized by a low Mo-co level, which does not increase either during log phase or after addition of nitrate. Group II nia mutants are similar to wild type. The four cnx mutants tested display a constitutive-high Mo-co level, being not further inducible by nitrate. Possible mechanisms for the common regulation of Mo-co and NR synthesis are discussed.

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.

Nitrate Reductase Deficient Mutants of Chlamydomonas reinhardii. Isolation and Genetics

Following u.v.-irradiation and plating on a selective chlorate medium, about 200 chlorate-resistant isolates of Chlamydomonas reinhardii were obtained. These were divided into three groups all of which grew well with ammonium as nitrogen source. One group grew well with nitrate under all conditions tested. The second, and largest group, grew well with nitrate only in the absence of acetate. The third group could not grow with nitrate as sole nitrogen source. Isolates in this group were further subdivided according to their ability to grow with hypoxanthine or nitrite as sole nitrogen source. Three mutants (14/15, 17/4 and 0/8) were examined genetically in some detail. Each differed from wild-type (strain 2192) by a mutation in a single gene. Isolates 14/15 and 17/4 could grow with hypoxanthine as a nitrogen source. The mutant genes in these two isolates were shown to be located in different linkage groups and have been designated nitA (in 17/4) and nitB (in 14/15). There is evidence that nitA may be in linkage group VI. Mutant 0/8 resembles the cnx mutants of Aspergillus in that it would not grow on hypoxanthine. The mutation in 0/8 is designated nitC; it, too, may be in linkage group VI. Strain 137c was also analysed. It appears to be unable to grow on nitrate because of mutations in two loci.

Reconstitution of NADH-nitrate reductase in vitro from nitrate reductase-deficient Nicotiana tabacum mutants

Two types of nitrate reductase-deficient mutant cell lines (nia and cnx) of Nicotiana tabacum have been used for in vitro reconstitution of NADH-nitrate reductase. The cnx mutants simultaneously lack NADH-,FADH2-, red benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are interpreted to be defective in the molybdenum-containing cofactor necessary for nitrate reductase activity. In the nia lines xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities of nitrate reductase, including NADH-cytochrome c reductase. When cnx cells (induced by nitrate) were homogenized together with nia cells (induced by nitrate or uninduced), NADH-nitrate reductase activity was detectable in the cell extract. No nitrate reductase was observed when the cnx mutants were homogenized together, or after cohomogenization of the nia mutants. Thus, the inactive nitrate reductase molecule formed in the cnx mutants has been complemented in vitro with the molybdenum-containing cofactor supplied by nia extracts, thus giving rise to NADH-nitrate reductase activity. This result gives additional support to the interpretation that the active nitrate reductase of Nicotiana tabacum is composed of at least the NADH-cytochrome c reductase moiety and a molybdenum-containing cofactor which is formed by the action of the cnx gene product(s).

Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants.

Mutants of A. nidulans at several loci lack detectable NADPH-nitrate reductase activity. These loci include niaD, the structural gene for the nitrate reductase polypeptide, and five other loci termed cnxABC, E, F, G and H which are presumed to be involved in the formation of a molybdenum-containing component (MCC) necessary for nitrate reductase activity. When forzen mycelia from A. nidulans deletion mutant niaD26 were homogenized in a Ten Broeck homogenizer together with frozen mycelia from either cnxA6, cnxE29, cnsF12, cnxG4 or cnxH3 strains grown on urea + nitrate as the nitrogen source, nitrate reductase activity was detectable in the extract. Similar results were obtained by co-homogenizind niaD mycelia with Neurospora crassa nit-1 mycelia induced on nitrate. Thus, all A. nidulans cnx mutants are similar to the N. crassa nit-1 strain in their capacity to yield NADPH-nitrate reductase in the presence of the presumed MCC. As judged by the amounts of nitrate reductase formed, niaD26 mycelia grown on urea +/- nitrate contained much more available MCC than ammonium-grown mycelia. No NADPH-nitrate reductase activity was found in extracts prepared by co-homogenizing mycelia from all five A. nidulans cnx strains. Wild-type A. nidulans NADPH-nitrate reductase acid dissociated by adjustment to pH 2.0-2.5 AND RE-ADJUSTED TO PH 7 could itself re-assemble to form active nitrate reductase and thus was not a useful source of MCC for these experiments. These results are consistent with the conclusion that the active nitrate reductase complex is composed of polypeptide components which are the niaD gene product, plus the MCC which is formed through the combined action of the cnx gene products. Further, the production of MCC may be regulated in response to the nitrogen nutrition available to the organism.

A system for the study of interlocus specificity for both forward and reverse mutation in at least eight gene loci in Aspergillus nidulans

A newly-devised mutational system is described for the fungus, Aspergillus nidulans, which allows forward mutation to be assigned to at least 8 gene loci (10 complementation groups) in one and the same experiment, and reverse mutation to be studied separately at each of these same gene loci. The forward mutational system is based on an effect on conidial pigmentation which is produced by 2-thioxanthine (after its in vivo enzymatic conversion to 2-thiouric acid by xanthine dehydrogenase), and which discriminates between a normally green-conidiating wild type strain(s) (which conidiates yellow on 2-thioxanthine) and certain mutant strains which are resistant (conidiate green) to 2-thioxanthine. The mutants which respond in this way have been phenotypically characterised as lacking the enzyme, xanthine dehydrogenase (XDH), and being unable to grow on hypoxanthine as a sole of nitrogen. However, failure to grow on certain other sole nitrogen sources divides the XDH mutants into 3 classes: hx mutants which lack XDH activity, and fail to grow on hypoxanthine (covered by 2 gene loci); ua Y mutants which lack XDH and urate oxidase activities, and fail to grow on hypoxanthine or uric acid (covered by 1 gene locus); and cnx mutants which lack XDH and nitrate reductase activities, and fail to grow on hypoxanthine or nitrate (covered by 5 gene loci, 7 complementation groups). Mutants which show resistance to the effect of 2-thioxanthine on conidial pigmentation for reasons other than a lack of XDH activity (2-TxR mutants), are also discussed. Methods are described for the detection of 2-thioxanthine resistant mutants, the purification of the mutants, the categorising of mutants into their phenotypic classes, and the assignation of mutants (by heterocaryon complementation) to their particular gene loci. The distribution of diethyl sulphate induced XDH mutations is found not to be statistically significant from the distribution of spontaneous mutations; however, the spontaneous sample is based on few mutants. Using appropriate sole nitrogen sources, reverse mutation studies on a diethyl sulphate-induced representative from each of the 3 classes of XDH mutation have shown no evidence for their spontaneous reversion to a wild type condition. However, the cnx representative is found to be revertible by ultraviolet irradiation, and the ua Y representative is revertible by ultraviolet irradiation, and N-methyl- N-nitroso- N′-nitroguanidine treatments, but apparently not by treatment with diethyl sulphate. A method is described for the detection of suppressors of 2-thioxanthine resistant mutations. The mutational system is discussed in relation to its use for the study of interlocus specificity for both forward and reverse mutation.