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To study, in vivo, potential P i –water oxygen exchange catalyzed by each of two high-affinity P i symporters of Neurospora crassa, we have developed methods for the purification of P i from whole-cell extracts and the subsequent derivatization of P i for analysis by GC–MS. We have also modified a published procedure for the preparation of 18 O-P i . However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of 18 O-P i in cells incubated in the presence of H 2 18 O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular P i –water oxygen exchange is 4.36 nmol 18 O incorporated into P i per second per milligram of cell protein. The 18 O isotopic distribution of the intracellular P i closely resembles that predicted for random exchange of a single P i oxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular P i pool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.

In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

Peleg, Y., Addison, R., Aramayo, R., and Metzenberg, R. L. 1996. Translocation of Neurospora crassa transcription factor NUC-1 into the nucleus is induced by phosphorus limitation. Fungal Genetics and Biology 20, 185–191. NUC-1, a basic helix–loop–helix zipper protein, activates the expression of several genes involved in phosphorus acquisition in Neurospora crassa. In the present study we investigated whether posttranscriptional mechanisms control the activity of NUC-1. The NUC-1 level was higher (up to fivefold) in wild-type cells grown at low external phosphate concentration and in mutant strains expressing the phosphorus acquisition genes constitutively than in a wild-type strain grown at high external phosphate concentration. Using indirect immunofluorescence we demonstrated that NUC-1 is localized at least predominantly in the cytosol when wild-type N. crassa is grown with an adequate supply of phosphate, whereas NUC-1 is largely concentrated in the nucleus upon limitation of external phosphate. In mutant strains expressing the phosphorus acquisition genes constitutively, NUC-1 localization was also primarily in the nucleus. Thus, subcellular compartmentation of regulatory proteins is an important mechanism in regulating gene expression in filamentous fungi.

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

In response to phosphorus starvation, Neurospora crassa makes several enzymes that are undetectable or barely detectable in phosphate-sufficient cultures. The nuc-1+ gene, whose product regulates the synthesis of these enzymes, was cloned and sequenced. The nuc-1+ gene encodes a protein of 824 amino acids with a predicted molecular weight of 87,429. The amino acid sequence shows homology with two yeast proteins whose functions are analogous to that of the NUC-1 protein. Two nuc-1+ transcripts of 3.2 and 3.0 kilobases were detected; they were present in similar amounts during growth at low or high phosphate concentrations. The nuc-2+ gene encodes a product normally required for NUC-1 function, and yet a nuc-2 mutation can be complemented by overexpression of the nuc-1+ gene. This implies physical interactions between NUC-1 protein and the negative regulatory factor(s) PREG and/or PGOV. Analysis of nuc-2 and nuc-1; nuc-2 strains transformed by the nuc-1+ gene suggests that phosphate directly affects the level or activity of the negative regulatory factor(s) controlling phosphorus acquisition.

The Neurospora crassa phosphate-repressible alkaline phosphatase-encoding gene pho -2 + was cloned and its nucleotide sequence was determined. An open reading frame was found that contains four introns and encodes a putative protein of 555 amino acids. ‘Activator-independent expression’ of ectopically integrated pho -2 + was observed, as noted before for ectopically integrated pho -4 + .

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa . Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S -adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes. Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me -1 and me -8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me -1, me -6 (folate polyglutamate synthetase deficient) and me -8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.

When N. crassa is starved for phosphate the rate of synthesis of total RNA, as measured by the incorporation of uridine, rapidly dclines, attaining a value of 2% of the control after 4 h. Synthesis of ribosomal RNA (rRNA), measured by direct hybridization to ribosomal DNA covalently coupled to diazobenzyloxymethyl (DBM) paper, also declines to a value 3%–4% that of the control after 4 h of phosphate starvation. Measurement of rRNA synthesis in regulatory mutant strains expressing “phosphorus-family” enzymes indicates that two of these mutant strains, pgovc12 and nuc-1, respond differently to phosphate starvation from the response in wild-type or the other five mutant strains. The results suggest that the wild-type products of the regulatory loci, pgov+ and nuc-1+ may have a role in regulating rRNA synthesis as well as “phosphorus family” enzymes.

The repressible alkaline phosphatase of Neurospora crassa was purified from both the mycelium of a wild type strain and from the medium in which cultures of the slime mutant (which lacks the normal cell wall) had been grown. The enzyme preparations from the two sources had similar amino acid compositions, immunological properties, specific activities, thermal stabilities, and kinetic constants, but differed in a number of other properties. Both enzyme preparations contained carbohydrate, but the carbohydrate content of the enzyme isolated from slime medium was almost double that of the enzyme from wild type mycelium (24 and 14%, respectively). The molecular weight of the enzyme secreted by slime cells, estimated by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was higher than that of the enzyme from wild type mycelium by an amount consistent with its increased carbohydrate content. Electrophoresis at pH 4.7 and 9.5 indicated that the enzyme isolated from slime medium is more anionic than the enzyme from wild type mycelium. Chemical analysis revealed the presence of approximately 8 phosphate groups per enzyme molecule in the purified slime extracellular enzyme, whereas the wild type enzyme contained less than 0.5 phosphate molecule per enzyme molecule. The presence of phosphate in the slime extracellular enzyme, and the lack of significant amounts of phosphate in the wild type mycelial enzyme, was also demonstrated by determination of 32P associated with the enzymes isolated from the two sources following derepression in the presence of 32PO43-. A significant portion of the repressible alkaline phosphatase produced by derepressed wild type N. crassa was found to be secreted into the growth medium. The electrophoretic mobility of the enzyme isolated from the wild type culture medium resembled that of the enzyme isolated from the slime culture medium rather than that of the enzyme isolated from wild type mycelium.

Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (Gleason and Metzenberg 1974). The five new mutants, as well as three previously isolated by Gleason and Metzenberg (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.

A mutant, eth-1 r , of Neurospora crassa is ethionine-resistant, temperature-conditional lethal, and nonrepressible by methionine for the synthesis of aryl sulfatase. Raising the osmolar concentration with glycerol repairs all of these defects. All of these defects are also corrected by reversion events that occur near or in the region of the original eth-1 r alteration.

Trevithick, John R. (University of Wisconsin Medical School, Madison), Robert L. Metzenberg and Donald F. Costello. Genetic alteration of pore size and other properties of the Neurospora cell wall. J. Bacteriol. 92:1016-1020. 1966.-Several properties of the cell walls of wild type and the osmotic mutant of Neurospora crassa have been examined. The peameability of the isolated cell walls to polyethylene glycol and dextran polymers of different molecular weights was investigated by the volume of distribution technique. The exclusion thresholds were evaluated by a statistical treatment. The molecular weights corresponding to these thresholds for wild type and osmotic were approximately 4,750 and 18,500, respectively; these values are significantly different. The cell walls of osmotic appeared to be thinner, more easily broken, and more easily compressed to ribbonlike shapes, whereas those of wild type were tubular and strong. Chemical analysis showed that osmotic walls had roughly a 30-fold higher galactosamine-glucosamine ratio than did wild type. It is proposed that the osmotic mutant has a cell wall with abnormally large pores, and that this may account for the increased rate of egress of invertase and the decreased fractionation of light from heavy invertase in this strain.

A mutant of Neurospora crassa, called UW-6, differs from wild type in being partially constitutive for synthesis of a species of alkaline phosphatase, and also for a species of phosphate permease that has a high affinity for phosphate at high pH. UW-6 is possibly allelic with a mutant called nuc-2 that was previously isolated by Ishikawa. nuc-2 has the converse phenotype, in that it cannot be derepressed for either of these two activities. UW-6 is co-dominant with its wild-type allele in heterokaryons and in partial diploids. An unlinked mutant, nuc-1, is like nuc-2 in that it fails to make the alkaline phosphatase or the permease referred to above. nuc-1 is epistatic to UW-6 in the double mutant. The control of phosphorus metabolism is discussed, and is compared with some other control systems in filamentous fungi.

By selection on a medium containing both ethionine and p-fluorophenylalanine a mutant strain of Neurospora has been isolated. This strain, nap, shows greatly decreased transport of neutral and acidic amino acids. The mutation also confers resistance to 4-methyltryptophan, aminoprotein and glycine. The physiological defect appears to be specific since the mutant showed a normal doubling time for protein, normal oxygen consumption and normal transport of glucose, sulfate and basic amino acids. This gene represents a third locus which governs neutral and acidic amino acid transport in Neurospora, the two previously described being un-t (55701) and mtr.

The uptake of glucose, fructose, and allose by Neurospora was shown to be energy-dependent, to follow Michaelis-Menten kinetics, and to display competition in certain combinations. Neurospora was shown to be unable to transport sucrose into cells. After hydrolysis of the disaccharide by transmembrane invertase, only the glucose moiety enters the cell at a significant rate. The possibility was tested that invertase releases product (s) in an oriented fashion that favors transport into the cell. The experimental results do not strongly support this possibility. Results roughly similar to those given by sucrose were obtained by use of the artificial substrate allosucrose; in the latter case, a weakly oriented release of products may have been detected.

A particulate fraction isolated from Neurospora mycelia was found to contain aryl sulfatase activity. The association of aryl sulfatase with this fraction was labile to shear and to incubation with Lubrol, but not to short incubation at 37 ° or to nystatin. Mycelia were disrupted by three different techniques and the particulate fraction prepared from each homogenate was analyzed on a sucrose density gradient. A constant feature in these gradients was a peak of aryl sulfatase activity at a density of 1.16 to 1.17 g/ml. These results are consistent with a location for aryl sulfatase in Neurospora analogous to the mammalian lysosome. Alternative possible locations are discussed.

The kinetics of the inhibition of the invertase of Neurospora crassa by the products of the hydrolysis, sorbose, and the unusual inhibitor, aniline, have been investigated. The fructosyl transferase activity with glucose or fructose as acceptors of fructose was negligible. The kinetics of the inhibitions were interpreted in terms of a simple three-step mechanism with the enzyme in three forms: free enzyme (E), enzyme-substrate (EFA), and enzyme-fructose (EF). For fructose inhibition of sucrose hydrolysis reciprocal 1 v against 1 S plots showed that the slopes of the lines could be described as a parabolic function of fructose concentration. This was interpreted as nonspecific binding of fructose at the site which normally binds the afructone moiety. The inhibitions by sorbose and aniline were characterized as linear noncompetitive, while that by glucose could be either competitive or noncompetitive, since the effect on the intercepts ( 1 V ) of the lines was at the borderline of experimental error. By contrast, the effect of aniline was mainly on the intercepts of the lines, and was interpreted as a strong combination of aniline with the enzyme-fructose complex, with a much weaker interaction with the free enzyme form.

Invertase has been purified from Neurospora crassa. Unlike the corresponding enzyme from yeast, it contains little or no mannose, though small amounts of hexosamine have been detected. It is suggested that the latter reflects the in vivo location of invertase in the cell wall of Neurospora, as does the presence of mannose in yeast. Neurospora invertase has an unusually high extinction at 280 mμ. Various physical and kinetic properties of the enzyme are reported.

1. 1. The s-fructofuranosidase (s-d-fructofuranoside fructohydrolase, EC 3.2.1.26) of Neurospora conidia is fully accessible to substrate, to aniline (an inhibitor), and to H+, as judged by the similarity of the kinetic properties of the conidial enzyme to those of highly purified preparations. The conidial and mycelial forms of s-fructofuranosidase are rapidly and completely inactivated under conditions which do not impair the viability of conidia, and fail to destroy alkaline phosphatase, which has been inferred on other grounds to be intracellular. 2. 2. A limited fraction of the conidial s-fructofuranosidase is easily removed by washing. 3. 3. The bound conidial s-fructofuranosidase, but not the crude or purified soluble enzyme, is resistant to pepsin inactivation. 4. 4. The results favor the view that this enzyme is located between the cell wall and the cell membrane.

Growth of Neurospora on media containing mannose results in low activities of invertase and trehalase, whereas very high levels of these enzymes are attained during growth in galactose media. A mutant has been isolated which, under suitable conditions of culture, produces much larger amounts of these two enzymes than does the parental strain. Evidence is presented that the levels of enzymes are affected by a single gene alteration.

A serine auxotroph of Neurospora requires exogenous serine to repress the arylsulfatase, suggesting that this enzyme is repressed by cysteine and not by methionine.

Genetic studies suggest that the so-called “phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and “phosphorus-family” enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pconc, and pregc. Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1r locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1r. They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1r, is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.—It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-metr mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The γ-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.

Mutations which affect the regulation of Neurospora repressible alkaline phosphatase do so by altering the rate of de novo alkaline phosphatase synthesis. In regulatory mutants the rate of alkaline phosphatase polypeptide synthesis can vary over a 1000-fold range. Following transfer to phosphate-free medium, the wild-type cell is capable of increasing the rate of synthesis of alkaline phosphatase molecules within 30–45 min.

THE synthesis of citrulline by preparations from mammalian liver requires the addition of ornithine, ammonia, bicarbonate ion, magnesium ion, adenosine triphosphate, and an appropriate derivative of glutamic acid1. Synthetic compounds which have been shown to be active in the latter capacity are the N-carbamyl, N-formyl, N-acetyl and N-chloroacetyl derivatives of L-glutamic acid1. These compounds have been shown to be catalytically active in the synthesis of carbamyl phosphate, an intermediate precursor of the carbamyl group of citrulline1–3.

1. 1. The basis of ethionine resistance in two temperature-sensitive irreparable mutants of Neurospora crassa (55701 and r-eth -1) has been examined. 2. 2. One of these mutants (55701) appears to be resistant due to an impaired ability to concentrate ethionine from the medium. 3. 3. The strain r-eth -1 has a normal uptake system for ethionine, but fails to incorporate this analog into protein. Evidence is offered that although r-eth -1 overproduces methionine, this fact accounts for only part of the resistance to ethionine, and that this strain has an ability to discriminate against this analog that is not a trivial consequence of methionine overproduction. 4. 4. It is proposed that a single gene alteration in r-eth -1 genes rise to three pleiotropic effects: (a) a component of the protein-synthesizing machinery is able to discriminate between methionine and ethionine, (b) this component is involved in the regulation of methionine synthesis, (c) the change in this component renders it abnormally temperature-labile.

Three enzymes of sulfur metabolism are simultaneously lost in the cys -3 mutant of Neurospora . Revertants of cys -3 have been selected and appear to arise from mutation within the original locus. The revertants can be grouped into at least three classes according to the amounts of the enzymic activities that are restored and by temperature sensitivity. The properties of the revertant strains indicate that the cys -3 mutant is not a large deletion, a super-repressor mutation, or an extreme polar mutant; nor does the cys -3 locus code for a polypeptide chain common to the three enzymes. We suggest that cys -3 is a regulatory gene which exerts positive control over enzyme synthesis.

Trevithick, John R. (University of Wisconsin Medical School, Madison), and Robert L. Metzenberg. Molecular sieving by Neurospora cell walls during secretion of invertase isozymes. J. Bacteriol. 92: 1010–1015. 1966.—The secretion of invertase by young mycelia of Neurospora was studied. The process of secretion was found to be dependent upon growth. The results indicate that fractionation of light invertase, the monomer, from heavy invertase, the aggregated form, occurs at the cell wall. Neurospora strains wild type, crisp, osmotic, and the double mutant crisp osmotic were tested. An inverse relation exists between the fraction of the total invertase activity of the culture which the mold secretes into the medium and the degree of fractionation, defined as the ratio of the fraction of the invertase secreted into the medium that is light invertase to the fraction of the invertase remaining associated with the cells that is light invertase. The hypothesis is offered that the increased secretion of invertase and decreased degree of fractionation seen in osmotic mutants, and to a lesser extent in the other mutants, can be explained by an increased porosity of the cell wall.

1. 1. Neurospora invertase (s- d -fructofuranoside fructohydrolase, EC 3.2.1.26) (s20,W = 10.3) can be dissociated to active subunits (s20,W = 5.2) by heat or by high salt concentrations at low or high pH. The subunits can reassociate to give the polymeric form. Both forms of the enzyme are active per se. 2. 2. Both forms of the enzyme are present in crude extracts of mycelia or washings from conidia. 3. 3. The polymeric form can be separated from the active subunit by gel electrophoresis or Sephadex G-200 filtration. 4. 4. When the polymeric form is subjected to conditions which cause partial dissociation, a transient form (s20,W = 9.8) is present. This probably represents the polymeric enzyme in a pre-clastic conformation. 5. 5. Some observations on the surface activity of invertase are briefly noted.

A group of enzymes of sulfur metabolism (arylsulfatase, cholinesulfatase, and a number of others) are normally repressed in Neurospora crassa by an abundant supply of a “favored” sulfur source such as methionine or inorganic sulfate. A mutant called scon c was isolated in which the formation of each of these enzymes is largely or completely nonrepressible. The structural genes for three of these enzymes have been mapped; scon c is not linked to any of them. It is also not linked to cys-3 , another gene which is involved in control of the same group of enzymes. Two alleles of the structural gene for arylsulfatase [ ars + and ars ( UFC-220 )] produce electrophoretically distinguishable forms of arylsulfatase. Heterokaryons with the constitution scon c ars + + scon + ars ( UFC-220 ) were prepared. These heterokaryons produce both forms of arylsulfatase under conditions of sulfur limitation, but produce only the wild-type ( ars + ) form under conditions of sulfur abundance. When the alleles of ars and scon are in the opposite relationship, only the ars ( UFC-220 ) form of arylsulfatase can be detected under conditions of sulfur abundance. Thus the effect of the scon c mutation seems to be limited to its own nucleus. The implications of these findings are discussed.

A temperature-conditional lethal mutant of Neurospora crassa, un-t ( 55701 ), was resistant to neutral amino acid analogues by virtue of a decreased ability to transport these analogues and their natural congeners across the cell membrane. The uptake of acidic, but not basic, amino acids was also impaired, as was the uptake of potassium ions. After preincubation above the tolerated temperature, the ability to take up a still wider variety of metabolites was greatly reduced. Protoplasts of the mutant were more osmotically fragile than those of wild type. The possibility that the mutant has a generalized membrane defect is discussed.

Aryl sulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was found to have multiple locations in Neurospora conidia. Some enzyme activity remained in the supernatant when a spore suspension was centrifuged or filtered. Part of the cell-bound activity could be detected by adding the assay ingredients to a suspension of intact spores (patent enzyme), and additional activity was only detectable when the spores were first treated to destroy their permeability barriers (cryptic enzyme). Such treatments include: disruption with an X-press, brief rinsing with chloroform or acetone, incubation at 60 C for 5 min, and incubation with phenethyl alcohol, nystatin, or ascosin. Part of the patent aryl sulfatase was inactivated by briefly acid treating the intact spores (no loss of conidial viability). This enzyme was considered to have a cell surface location. Some enzyme was acid-resistant in intact spores, but all of the enzyme was acid-sensitive in spores whose permeability barriers had been disrupted. The p H dependence, kinetic properties, and p -nitrophenyl sulfate uptake were investigated in acid-treated conidia. No aryl sulfatase was detected in ascospores. Young mycelia contained more aryl sulfatase than did conidia, but little, if any, was secreted into the growth medium. Cryptic activity was demonstrated in young mycelia by brief chloroform treatment or by rinsing the cells with 0.1 m acetate buffer. Enzyme activity in young mycelia was completely labile to acid treatment, as was cell viability.

The synthesis of a number of enzymes of sulfur assimilation in Neurospora crassa is controlled by the sulfur source. Mutants in one regulatory gene, cys-3, are unable to make any of the enzymes. This locus is thought to specify a macromolecule that is required for the expression of the structural genes. A mutant, sconc, of another regulatory gene is nonrepressible for the synthesis of the enzymes. We report here the isolation of suppressed sconc strains which are actually the double mutant, sconc,cys-3. These strains are phenotypically indistinguishable from single mutants at the cys-3 locus. Thus cys-3 is epistatic to sconc. Evidence that the expression of the cys-3 gene is itself controlled is also presented.