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In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer × sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer × sensitive crosses. We suggest that the region contains a suppressor of MSUD.

Complexity in and around the A and a mating type idiomorphs in Neurospora crassa was examined. Six sets of transcripts surrounding the idiomorphs were identified by Northern analysis. Several different patterns of regulation were observed. The two pairs of transcripts closest to the centromere-proximal idiomorph flanks (variable regions) exhibited mating type-specific size differences. DNA sequence was obtained for the region surrounding the four transcripts which showed mating type-specific size and expression. One of these pairs encoded amino acid sequences highly similar to a domain present in the plasma membrane ATPase. A mutant allele of one of these genes was induced by repeat-induced point mutation (RIP), which resulted in altered perithecial development and the elimination of ascospore production. These transcripts comprise a cluster of genes which may be involved in the control of mating and sexual development in N. crassa.

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Ψ63, was identified. We characterized the Ψ63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Ψ63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Ψ63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Ψ63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Ψ63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.

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.

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.

Previously we have shown that the nucleolus organizer region (NOR) of Neurospora crassa changes size frequently during the premeiotic portion of the sexual phase. Here, we have investigated whether these changes in size originate only in specific regions of the NOR, or are distributed throughout the NOR. In two special strains of Neurospora, the NOR was divided into proximal and distal segments. In the first, the NOR was divided by a translocation breakpoint and, in the second, the NOR was divided by a meiotic crossover point. The two strains were crossed individually to normal sequence tester strains and the sizes of the proximal and distal segments were followed by pulsed-field gel electrophoresis. The analysis of progeny from both crosses indicates that the events affecting NOR size are not limited to a specific region of the NOR. Additionally, we have obtained evidence that the rDNA of N. crassa can undergo unequal sister chromatid exchange.

A gene unpaired during the meiotic homolog pairing stage in Neurospora generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process, sad-2 . Mutated Sad-2 RIP alleles, like those of Sad-1 , are dominant and suppress MSUD. Crosses homozygous for Sad-2 are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional sad-2 + gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.

AS a new graduate student of Herschel Mitchell at Caltech in 1951, I soon had occasion to visit Horowitz's office. I immediately noticed a newspaper clipping taped to the door. “It is a Privilege to be Living in the Same Century as Horowitz,” gushed the title. The article was, as it turned out, about a recent concert by Vladimir Horowitz, but it prepared me for the wry Horowitz sense of humor, always delivered deadpan. Later, there was Thanksgiving dinner at his house with his wife, Pearl, his son and daughter, and his mother. Norm sat down to the piano and we all sang old chestnuts. Vladimir he was not. But full of food and drink as we were and enthralled with our own wine-enhanced voices, it was a perfect evening. I left knowing Norm Horowitz and I had begun a lifelong friendship.

A putative pheromone precursor gene of Neurospora crassa , mfa-1 (which encodes mating factor a -1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high mfa-1 expression in all mating type a tissues and suggested low expression levels in mat A tissues. The mfa-1 gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3′ untranslated region (3′ UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of Sordaria macrospora and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were mfa-1 mutants with intact ORFs but multiple mutations in the 3′ noncoding region (CAAX mutants). The 3′ UTR is required for the full range of mfa-1 gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 μM] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous mfa-1 crosses. As males, YAAX mat a mutants were unable to attract wild-type mat A trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX mat a mutants were able to mate and produce sexual progeny despite their inability to attract mat A trichogynes. In the mat A background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the mfa-1 gene appears to have multiple roles in N. crassa development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by mat a to attract trichogynes of mat A ; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.

Self-incompatible het-6 OR/het-6 PA partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL → IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that “escape” from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6 OR and het-6 PA) were constructed and used to determine that 80 of 96 escape strains tested were het-6 PA, retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6 OR, retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans.

The transcriptional activator NUC-1 controls the transcription of the genes for phosphorus acquisition enzymes, and its activity is regulated by the negative regulatory factors, PREG and PGOV In this report, we describe the cloning and molecular analysis of the preg+ gene. In Neurospora crassa, as in higher eukaryotes, transformation frequently results in nonhomologous integration of transforming DNA. Insertion of transforming DNA into host genes mutates the gene and provides a molecular tag for cloning it. We obtained two mutants that have an insertion in the preg+ and pgov+ genes, respectively, among 2 x 10(5) transformants. The preg+ gene was cloned by screening a Neurospora genomic DNA library with DNA sequences flanking the transforming DNA of the rescued plasmid. Northern analysis showed that the transcription of the preg+ gene is not regulated by phosphate. The carboxy-terminal half of PREG shows strong homology with Saccharomyces cerevisiae PHO80 whose function is analogous to that of PREG. The pregc mutations are located in the well conserved residues which may directly interact with the residues in the regulatory domain of NUC-1.

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 field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

The organization of the ribosomal DNA (rDNA) repeat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, . tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11 + 0.21 kb; standard error = 0.038; coefficient of variation (C.V.) = 2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5' end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.

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.

About 100 genes coding for 5S RNA in Neurospora crassa are dispersed throughout the genome (Selker et al., Cell 24:815-818, 1981; R. L. Metzenberg, J. N. Stevens, E. U. Selker, and E. Morzycka-Wroblewska, manuscript in preparation). The majority of them correspond to the most abundant species (alpha) of 5S RNA found in the cell. Gene conversion, gene transposition, or both may be responsible for the maintenance of sequence homogeneity (concerted evolution) of alpha-type 5S genes. To explore these possibilities, we isolated and characterized separate 5S regions from two distantly related laboratory strains of N. crassa. Restriction and sequence analyses revealed no differences in molecular location of allelic 5S genes between the two strains. However, the DNA sequences around the 5S genes are ca. 10% divergent. We concluded that transposition is not frequent enough to account for the concerted evolution of N. crassa alpha-5S genes. In contrast to sequence divergence in the flanking regions between the two strains, the 5S transcribed regions are identical (with one exception), suggesting that these genes are being corrected. We have found that flanking sequences of various N. crassa 5S genes within each strain are largely different. Thus, if the correction mechanism is based on gene conversion, it is limited to the transcribed regions of the genes. However, we did find a short region of consensus including the sequence TATA located 25 to 30 nucleotides preceding the position of transcription initiation. This region may be involved in the transcription of N. crassa 5S genes.

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.

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered.

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.

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.

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.

The 5S RNA genes (5S genes) in N. crassa are not tandemly arranged or tightly clustered as in other eucaryotes that have been examined. 55 RNA or cloned 5S DNA hybridizes to at least 30 different restriction fragments of Neurospora DNA. Of 34 5S DNA clones examined, each contains a single 5S gene. Saturation hybridization analyses indicate that there are about 100 copies of 5S genes in the genome of this organism. We have partially or completely sequenced the 5S region of 15 clones. Both identical and highly divergent 5S coding regions were found. Nine are of one type (alpha). The other six include four different types (beta, beta', gamma and delta) which differ from each other and from the alpha genes to various degrees. Eleven of 15 genes have distinct flanking regions. Analysis of Neurospora 5S RNA showed that it consists of one principal species which matches the alpha-type gene sequence. Additional 5S species corresponding to the less abundant 5S gene types were also detected. The pattern of nucleotide substitutions between the predicted Neurospora 5S RNAs and between these and S. cerevisiae 5S RNA suggests that a particular 5S RNA secondary structure occurs in vivo and is conserved.

The DNA sequences upstream of the 5S RNA genes in Neurospora crassa are largely different from one another, but share a short consensus sequence located in the segment 29 to 26 nucleotides preceding the transcribed region. Differences among flanking sequences do not appear to affect transcription. Deletion analysis indicates, however, that a DNA segment including the conserved "TATA box" is required for in vitro transcription of Neurospora 5S RNA genes.

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.

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.