Nit Mutants Research Articles

Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatase when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in the nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiated at the same sites as the nit1 endogenous gene, confirms the hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of the effects of nested deletions in the nit1 promoter region on the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.

Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.

Use of nitrate non-utilizing mutants to study vegetative incompatibility in Fusarium solani ( Nectria haematococca), especially members of mating populations I, V and VI

Nitrate non-utilizing (nit) mutants were obtained from 89 (out of a sample of 94) strains of Fusarium solani isolated from several plant hosts in Japan, New Zealand and North America. Nit1 (unable to utilize nitrate) and nit3 (unable to utilize nitrate or nitrite), mutants were readily recovered from all 89 strains when these were grown on a minimal medium containing KCLO3 at 30 g l−1 (MMC), but NitM (unable to utilize nitrate, hypoxanthine or uric acid) mutants were only obtained from 53 strains on this medium. Modified MMC containing l -threonine instead of l -asparagine enabled recovery of NitM mutants from an additional 11 strains. Sulphate non-utilizing mutants (sul) were generated for a few strains that did not produce NitM mutants. Based on complementation between nit1 (or nit3) and NitM (or sul) mutants, 57 strains were heterokaryon self-compatible, of which 31 were assigned to single-strain vegetative compatibility groups (VCGs). Nine multi-strain VCGs were identified among New Zealand strains: here members of each VCG came from the same host and usually from the same location even though they were often isolated at different times. Two pea strains from North America were in the same VCG, and a third North American pea strain was compatible wih a pea strain from New Zealand. Although the VCG results were consistent and repeatable the vigour of individual heterokaryons varied widely. Frequently the intra-strain complementations produced heterokaryons of greater vigour from those that formed between compatible strains. There was a high frequency of self-incompatibility due either to simple or complex genetic mechanisms among Nectria haematococca MPI strains.

Vegetative compatibility among isolates ofColletotrichum gloeosporioides from almond in Israel

Anthracnose, caused byColletotrichum gloeosporioides, is the major disease of almond in Israel. Pathogen attack of young fruit results in fruit rot and leaf wilting. Seventy isolates ofC. gloeosporioides were obtained from affected almond fruits collected at 11 sites during 1991–2 and 1994. Chlorate-resistant nitrate-nonutilizing (nit) mutants were generated from each isolate and used in complementation (heterokaryon) tests. The formation of complementary stable heterokaryons between mutants from different isolates showed that all the isolates belonged to a single vegetative compatibility group. Representative isolates ofC. gloeosporioides from almond did not form heterokaryons with local isolates ofColletotrichum from anemone and avocado, indicating that the almond isolates constitute a distinct subspecific group withinC. gloeosporioides.

Incidence and stability of infection by double-stranded RNA genetic elements inAspergillussectionflaviand effects on aflatoxigenicity

Ninety-two isolates belonging to Aspergillus sect, flavi were analyzed for double-stranded (ds) RNA via standard cellulose chromatography. Double-stranded RNA infection was detected in fungal isolates that had been in culture for long periods (5 of 26 were infected) and in those recently isolated (5 of 66 were infected). The number of dsRNA genetic elements differed among infected isolates and no two isolates contained identical dsRNAs on the basis of electrophoretic migration in agarose gels. Addition of micronutrients to culture media affected both the amount of dsRNA produced and the number of dsRNA genetic elements detected. Attempts to cure six fungal isolates of dsRNA by serial single conidial transfer, chlorate selection for nitrogen-metabolism mutants, and cycloheximide treatment, met with variable results. The frequency at which serial single conidial transfer and nitrogen-metabolism mutant (nit) selection successfully cured six Aspergillus sect, flavi isolates varied from 11 to 100% and 0 to 100%, respectively. The cycloheximide treatment was effective at curing 40% of the dsRNA-infected isolates. Comparison of aflatoxin production prior to and after dsRNA curing indicated that infection by dsRNA did not influence aflatoxin production. However, aflatoxin production by two isolates (91-031B and 91-184G) was reduced by both single conidial transfer and induction of nit mutants. Keywords: aflatoxin, cycloheximide, mycovirus, nitrogen-metabolism mutant.

Molecular Evidence for Chromosome Transfer Between Biotypes ofColletotrichum gloeosporioides

Two genetically distinct biotypes (A and B) of Colletotrichum gloeosporioides that cause different anthracnose diseases on the legumes of Stylosanthes spp. have been identified in Australia. All virulent isolates of these biotypes are anamorphic. Laboratory pairings of nitrate reductase (nit) mutants representing the two biotypes indicated that biotypes A and B form separate vegetative compatibility groups (VCGs). We have investigated the mechanisms leading to a major karyotype polymorphism in this fungus. All biotype A isolates studied to date carry a 2-Mb chromosome while some biotype B isolates carry an apparently dispensable but homologous 1.2-Mb chromosome. A biotype B-like field isolate termed Bx was recently obtained from 5. guianensis in Northern Australia. The Bx isolate was shown to carry two homologous chromosomes, 1.2 Mb and 2 Mb in size. These two chromosomes lacked two repeat sequences that were present on other chromosomes in biotype B and these repeats appeared to be monomorphic in biotype B and Bx. This suggests the 1.2-Mb and 2-Mb chromosomes may be recent additions to the Bx genome. Analysis of the DNA sequence organization of the 2-Mb and 1.2-Mb chromosomes in Bx by means of chromosome-specific DNA markers indicated that these chromosomes were each identical to the 2-Mb and 1.2-Mb sized homologous chromosomes in biotype A and some biotype B isolates, respectively. Extensive analysis of the rest of the genome of Bx by restriction fragment length polymorphisms, random amplified polymorphic DNAs, and minichromosome profiling indicated that the background genome of Bx was like that of biotype B. No other biotype A-like markers outside of the 2-Mb chromosome were identified in Bx. Pairing of nit mutants indicated that Bx was part of the biotype B VCG. The results therefore indicate that a dispensable 2-Mb chromosome in the biotype B isolate Bx most probably originated by a relatively recent horizontal transfer from biotype A. The results suggest that the occasional transfer of specific chromosomes may occur between apparently genetically isolated clonal lines of asexual pathogens. Copyright 1996 The American Phytopathological Society.

Use of Nitrate Nonutilizing Mutants in Ecological Studies of Fusarium Diseases. III. Growth, Benomyl Sensitivity, Pathogenicity, and Stability of nit Mutants of Fusarium oxysporum Compared to Wild-type Strains.

Properties of nitrate nonutilizing (nit) mutants of Fusarium oxysporum were compared to those of wild-type strains using F. oxysporum f. sp. raphani, f. sp. spinaciae, and a nonpathogenic strain of F. oxysporum (S-52). Most mutants showed the same growth as the wild-type strain on potato sucrose agar (PSA) medium, though some mutants from F. oxysporum f. sp. spinaciae showed slower growth. No difference was observed in the proliferation in liquid medium or sterilized soil between nit mutants and the wild-type strain for the strains tested. All of the 24 mutants from benomyl-sensitive F. oxysporum f. sp. raphani were also benomyl-sensitive. Most of the 43 mutants from f. sp. raphani and f. sp. spinaciae retained pathogenicity to their respective hosts, although two mutants from f. sp. spinaciae had clearly weakened pathogenicity. Among the total of 195 mutants from 10 formae speciales, most mutants retained their phenotypes at their formation even after three-year preservation by subculture on slanted PSA at room temperature, while 21 mutants (10.8%) changed their phenotypes and recovered the ability to utilize nitrate. When nit mutants of f. sp. raphani were inoculated into nursery soil and reisolated three years after inoculation by soil dilution plating, all of the reisolated F. oxysporum were still nit mutants and no phenotypic change was observed. Population densities of the mutants in the soil were at almost the same level as for the wild-type strain. When mutants of f. sp. spinaciae were inoculated to spinach, reisolated, and tested for their phenotypes, all of the mutants retained their original phenotypes. Thus, although some nit mutants had weakened pathogenicity or were unstable, most nit mutants had the same properties as wild-type strain and were stable over long periods of time. It is thus assumed that nit mutants can be used as markers in ecological studies of F. oxysporum.

Molecular cloning and analysis of nre, the major nitrogen regulatory gene of Penicillium chrysogenum

We have isolated the Penicillium chrysogenum nre gene which is homologous to the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa. Overall, nre shows 60% identity to areA and 30% identity to nit-2 at the amino-acid level. The gene encodes a protein of 835 amino-acid residues and contains a single Cys2/Cys2-type zinc finger with an adjacent basic region and a putative acidic activation region. In the DNA-binding domain, 98% of the amino-acid residues are identical in nre, areA and nit-2. The nre gene has been shown to be functional in N. crassa by heterologous complementation of a nit-2 mutant. Growth tests indicated that transformants could utilize nitrate, amino-acids, purines and amides as sole nitrogen sources. Nitrate reductase activity assays performed with transformants demonstrated that nitrogen control was completely normal. Complementation of N. crassa nit-2 mutants with 5'-deletion clones of nre suggests the possible presence of an internal promoter within the coding region. Northern analysis and ribonuclease protection assays of total cellular RNA indicated that nre encodes a 3.2-kb transcript which is reduced in content under conditions of nitrogen repression.

Competitiveness of Mutant and Wild-type Isolates ofColletotrichum gloeosporioidesf. sp.aeschynomeneon Northern Jointvetch

The competitiveness of mutant and wild-type isolates of Colletotrichum gloeosporioides f. sp. aeschynomene was determined. Experiments were conducted in growth chambers constructed within a greenhouse in which available free moisture and splash dispersal were used to control successive infection cycles. Isolates of nitrate non-utilizing (nit) mutants or benomyl-resistant mutants were introduced with a wild-type isolate onto susceptible northern jointvetch plants in the growth chambers at initial population ratios of 1 :1 or 9 :1. The proportion of each isolate infecting northern jointvetch was determined at each of seven to 10 subsequent infection cycles. From initial lesion populations, benomyl-resistant lesions decreased to an apparent equilibrium level of 10 to 20% of the total lesion population after three to five infection cycles. In experiments with a nit mutant and a wild-type isolate, the population of lesions caused by the nit mutant decreased to an apparent equilibrium of 10 to 30% of the total population in two experiments when initial ratios were 1 :1. Comparison of infection-cycle components of mutants with their wild-type parent suggest that the decrease in competitiveness of mutants was associated with a decrease in infectivity, longer latent periods, reduced lesion size, and reduced sporulation.

The Use of Vegetative Compatibility and Heterokaryosis to Determine Potential Asexual Gene Exchange in Colletotrichum gloeosporioides

Abstract Auxotrophic and nitrate nonutilizing (nit) mutants were used to characterize vegetative compatibility, heterokaryosis, and asexual gene exchange in the fungal biological control agent Colletotrichum gloeosporioides f.sp. aeschynomene (Cga). Several paired auxotrophic strains produced heterokaryotic colonies, but conidia recovered from the colonies comprised only the parent phenotypes. Analysis of mycelial plugs removed from along the colony radius demonstrated that heterokaryosis was limited to the colony center. In host inoculation studies, only parental phenotypes were recovered from cocolonized lesions of the weed host Aeschynomene virginica. Based on pairings with nit mutants, 13 Cga isolates were vegetatively compatible but were vegetatively incompatible with 13 C. gloeosporioides isolates obtained from other hosts. Although heterokaryosis was demonstrated with Cga, no putative diploids or recombinant phenotypes were detected. Host specialization, vegetative compatibility, and lack of a demonstrated parasexual cycle may serve to genetically isolate Cga from other populations of C. gloeosporioides. Evidence of asexual gene exchange can be used to assess potential risks associated with the importation of an exotic biocontrol fungus or release of a genetically modified strain for biological control.

Vegetative compatibility in populations of Fusarium oxysporum from wild carnation

Wild carnation plants (Dianthus strictus) were collected from undisturbed, non-cultivated soils at four sites in Israel. Isolates of Fusarium oxysporum obtained from the roots of these plants were characterized by means of the vegetative-compatibility grouping method, using nitrate non-utilizing (nit) mutants to determine possible relationships among them and with the carnation wilt pathogen F. oxysporum f.sp. dianthi. No relatedness was evident between any of the isolates, except for five out of 26 combinations of isolates originating from the same plant. Wild populations are therefore diverse and do not constitute a distinct carnation-specific vegetative-compatibility group of F. oxysporum. None of 33 isolates originating from root tissues of wild carnation were vegetatively compatible with F. oxysporum f.sp. dianthi.

Nitrate-nonutilizing mutants used to study heterokaryosis and vegetative compatibility in Glomerella graminicola ( Colletotrichum graminicola)

Nitrate-nonutilizing (nit) mutants of Glomerella graminicola were recovered by selecting chlorate-resistant sectors. Heterokaryons were formed by complementation between two different classes of nit mutants. Complementation groups were distinguished in nitrogen feeding tests and segregated as two, unlinked genes among random progeny of sexual crosses. The two genes are comparable to those encoding the nitrate reductase enzyme and one of a series of molybdenum cofactors in Aspergillus nidulans and Neurospora crassa . Heterokaryon tests were reliable indicators of allelic and dominance relationships between mutations with similar phenotypes. Vegetative compatibility (VC) between two strains of G. graminicola appeared to be regulated by approximately five unlinked VC loci, analogous to those described for other fungi.

Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor.

Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis.

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit+ transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit- mutant strains, the motility phenotype cosegregated with the Nit+ phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.

Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging.

Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas.

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii.

Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wild-type strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit- mutants of C. reinhardtii, but failed to produce His+ revertants or Arar mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three hcr loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit+ and nit- chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.

Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii

1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 mumol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105,000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase present in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1.998; g3 = 1.993 and with gav. = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O4(2-) bleached the e.p.r. signals which, on reoxidation after the addition of NO3(2-) to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and gav. = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent Km 998 microM) and reduced Bromophenol Blue (apparent Km 158 microM) were effective. With these donors the apparent Km values for nitrate were 70 microM and 217 microM respectively.

Nitrate-nonutilizing mutants of Gibberella zeae (Fusarium graminearum) and their use in determining vegetative compatibility

Twenty-four single-spore isolates of Fusarium graminearum were obtained from scabby wheat seeds or glumes collected from 23 locations in Kansas in 1990. All isolates were sexually fertile and homothallic. Nitrate-nonutilizing ( nit) mutants of each isolate were generated on a medium amended with 1.5% KCIO 3. Of 378 mutants, 161 were able to utilize nitrite and hypoxanthine ( nit1), 165 utilized hypoxanthine but not nitrite ( nit3), 47 utilized nitrite but not hypoxanthine (NitM), and 5 appeared to be global nitrogen regulatory mutants similar to the previously described nnu mutant. Complementation was tested by pairing nit1 mutants of each isolate with either a NitM or a nit3 mutant from each isolate on media containing nitrate as the sole nitrogen source. Complementation was more pronounced when nit1 and NitM mutants were paired. Mutants were only able to complement with other mutants from the same wild-type isolate. Therefore, each wild-type isolate belonged to a genetically distinct vegetative compatibility group. The genetic diversity suggests that sexual genetic recombination may be important in the field.

Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.

Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population 'A' (Fusarium moniliforme).

We examined 25 strains of Fusarium moniliforme from eight states known to be associated with equine leukoencephalomalacia, a disease caused by the mycotoxin fumonisin B1. We determined the mating population, mating type, and vegetative compatibility group to which each of these strains belonged. All 25 strains were in the 'A' mating population; 12 were A+ and 13 were A-. Seventeen of the 25 strains were female fertile; these strains also averaged higher levels of fumonisin B1 production than did the strains that were female sterile. Nitrate non-utilizing (nit) mutants were generated in all 25 strains and each strain was assigned to a unique vegetative compatibility group based on the inability of the derived nit mutants to form a prototrophic heterokaryon with complementary nit mutants derived from any of the other strains examined. From these data, we concluded that the production of fumonisin B1 is a general characteristic of strains from the 'A' mating population of Gibberella fujikuroi associated with equine leukoencephalomalacia, since all 25 of the isolates that we examined were genetically distinct individuals.