Genetic Control of Alkaline Phosphatase Synthesis in Neurospora: The Use of Partial Diploids in Dominance Studies

In wild-type Neurospora, alkaline phosphatase is made under conditions of phosphate limitation, but not conditions of phosphate sufficiency. Mutants at two unlinked loci, nuc-1 and nuc-2, do not make alkaline phosphatase under any conditions, while mutants at two quite closely linked loci, pcon and preg, make alkaline phosphatase even under conditions of phosphate sufficiency. pcon is extremely closely linked to nuc-2. nuc-2 and preg(c) (constitutive) mutants are recessive to their wild-type alleles in partial diploids as well as in heterokaryons, while pcon(c) mutants are dominant or co-dominant. nuc-1 is epistatic to both pcon(c) and preg(c) mutants. The implications of these findings for theories of metabolic control in eukaryotes are briefly discussed.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Isolation of Mutants Deficient in the Repressible Alkaline Phosphatase

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-pcon(c), and preg(c). 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.     

Control of the formation of extracellular ribonuclease in Neurospora crassa.

A finding was made that a species of ribonuclease is released into mycelial culture media when a wild-type strain of Neurospora crassa was grown on limiting amounts of phosphate. The ribonuclease activity in the fully derepressed state extends to about 60 to 100 fold of that in the repressed state. The synthesis of the ribonuclease was inhibited by the addition of rifampicin, cycloheximide or orthophosphate. Three molecular species of the ribonuclease were found. Two enzyme fractions showing larger molecular weights were suspected to be aggregates containing the enzyme showing the smallest molecular weight (molecular weight of 10 300). All three fractions showed pH optima of around 7, preferential hydrolysis of polyguanylic acid and poor hydrolysis of guanosine 2',3',-cyclic monophosphate. These characteristics were the same as those of ribonuclease N1, and it was suggested that ribonuclease N1 is a repressible extracellular enzyme. Mutations in the genes nuc-1 and nuc-2 caused loss of ability to derepress this enzyme, but heterokaryon between them partially restored the ability. The nuc-1 mutation was epistatic to the nuc-2 alleles which are partly constitutive in the ribonuclease production.     

Genetic Control of Phosphorus Assimilation in NEUROSPORA CRASSA: Dose-Dependent Dominance and Recessiveness in Constitutive Mutants

Mutants called nuc-1c, constitutive for alkaline phosphatase synthesis, were isolated and mapped very close to nuc-1 mutants in which this enzyme is not expressed. nuc-1 is epistatic to nuc-1c. nuc-1c acts only if it is cis to normal nuc-1 function. The preparation of partial diploids heterozygous for various nuc-1 alleles is described; nuc-1c is dominant to nuc-1+, which in turn is dominant to nuc-1. In heterocaryons with nuc-1+, nuc-1c is dominant when it is present in high proportion, but essentially recessive if it is present in low proportions. In heterocaryons with nuc-1, nuc-1c is again dominant when present in high proportions, but in low proportions it "complements" to give essentially normal repressibility. A model of regulation consistent with these findings is presented.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Identification of the Structural Gene for Repressible Acid Phosphatase

A mutant of Neurospora crassa with an altered repressible acid phosphatase has been isolated. The enzyme is much more thermolabile than that of wild type, and has an increased Michaelis constant. Tests of allelic interactions (in partial diploids) and in vitro mixing experiments were consistent with the mutation being in the structural gene for the enzyme. This gene, pho-3, was found to be located in the right arm of Linkage Group IV (LGIV). Thus, pho-3 and the structural gene for repressible alkaline phosphatase, pho-2 (LG V), map in separate linkage groups and cannot be part of the same operon. Neither of these structural genes is linked to the known regulatory genes, nuc-1 (LG I), nuc-2 (LG II), and preg (LG II).     

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.     

Extragenic suppressors of yeast glucose derepression mutants leading to constitutive synthesis of several glucose-repressible enzymes

Saccharomyces cerevisiae regulatory genes CAT1 and CAT3 constitute a positive control circuit necessary for derepression of gluconeogenic and disaccharide-utilizing enzymes. Mutations within these genes are epistatic to hxk2 and hex2, which cause defects in glucose repression. cat1 and cat3 mutants are unable to grow in the presence of nonfermentable carbon sources or maltose. Stable gene disruptions were constructed inside these genes, and the resulting growth deficiencies were used for selecting epistatic mutations. The revertants obtained were tested for glucose repression, and those showing altered regulatory properties were further investigated. Most revertants belonged to a single complementation group called cat4. This recessive mutation caused a defect in glucose repression of invertase, maltase, and iso-1-cytochrome c. Additionally, hexokinase activity was increased. Gluconeogenic enzymes are still normally repressible in cat4 mutants. The occurrence of recombination of cat1::HIS3 and cat3::LEU2 with some cat4 alleles allowed significant growth in the presence of ethanol, which could be attributed to a partial derepression of gluconeogenic enzymes. The cat4 complementation group was tested for allelism with hxk2, hex2, cat80, cid1, cyc8, and tup1 mutations, which were previously described as affecting glucose repression. Allelism tests and tetrad analysis clearly proved that the cat4 complementation group is a new class of mutant alleles affecting carbon source-dependent gene expression.     

Control of the activity of intracellular nucleases in Neurospora crassa.

Summary At least four species of nucleases (nuclease N₁, N₂, N₃ and N₄) and one ribonuclease (ribonuclease N₃) were detected in extract of wild type mycelia grown in high phosphate media by gel filtration of 0–65% ammonium sulfate precipitate through Sephadex G-100. Nuclease N₄ eluted the first is a latent nuclease, the activity of which is not detectable within a week after preparation of the extract but a significant increase in nuclease activity was observed during additional one or two weeks by standing the fraction at 4°C. Nuclease N₁ eluted the second is very labile and nuclease N₂ eluted the third is stable at the temperature. Nuclease N₃ eluted the last was activated within two or three weeks at 4°C. Although all the four nucleases were detected independent of the concentration of orthophosphate in culture media, significantly large amounts of latent ribonuclease (ribonuclease N₃) and a number of nucleases including at least one latent nuclease were observed in wild type mycelia grown in low phosphate media. Ribonuclease N₃ was determined to be a repressible enzyme. The activities of these constitutive latent nucleases, ribonuclease N₃ and a number of nucleases specifically present in wild type mycelia grown in low phosphate media were not observed or significantly reduced in both nuc-1 and nuc-2 mutants, which were deficient to derepress at least eight orthophosphate repressible enzymes relating to phosphate metabolism. A revertant from nuc-2 restored the ability to show activation of at least one of the constitutive latent nucleases.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Multiple regulatory genes control expression of a gene family during development of Dictyostelium discoideum.

Mutant strains of Dictyostelium discoideum carrying dis mutations fail to transcribe specifically the family of developmentally regulated discoidin lectin genes during morphogenesis. The phenotypes of these mutants strongly suggested that the mutations reside in regulatory genes. Using these mutant strains, we showed that multiple regulatory genes are required for the expression of the lectin structural genes and that these regulatory genes (the dis+ alleles) act in trans to regulate this gene family. These regulatory genes fall into two complementation groups (disA and disB) and map to linkage groups II and III, respectively. A further regulatory locus was defined by the identification of an unlinked supressor gene, drsA (discoidin restoring), which is epistatic to disB, but not disA, and results in the restoration of lectin expression in cells carrying the disB mutation. Mutant cells carrying the drsA allele express the discoidin lectin gene family during growth and development, in contrast to wild-type cells which express it only during development. Therefore, the suppressor activity of the drsA allele appears to function by making the expression of the discoidin lectins constitutive and no longer strictly developmentally regulated. The data indicate that normal expression of the discoidin lectins is dependent on the sequential action of the disB+, drsA+, and disA+ gene products. Thus, we described an interacting network of regulatory genes which in turn controls the developmental expression of a family of genes during the morphogenesis of D. discoideum.     

A complement of ten essential and pleiotropic arabidopsis COP/DET/FUS genes is necessary for repression of photomorphogenesis in darkness.

Two genetic screens, one for mutations resulting in photomorphogenic development in darkness and the other for mutants with fusca phenotype, have thus far identified six pleiotropic Arabidopsis COP/DET/FUS genes. Here, we characterized representative mutants that define four additional pleiotropic photomorphogenic loci and a null mutant allele of the previously defined DET1 locus. Dark-grown seedlings homozygous for these recessive mutations exhibit short hypocotyls and expanded cotyledons and are lethal before reaching reproductive development. Dark-grown mutant seedlings also display characteristic photomorphogenic cellular differentiation and elevated expression of light-inducible genes. In addition, analyses of plastids from dark-grown mutants reveal partial chloroplast differentiation and absence of etioplast development. Root vascular bundle cells of light-grown mutant seedlings develop chloroplasts, suggesting that these FUS gene products are important for suppression of chloroplast differentiation in light-grown roots. Double-mutant analyses indicate that these pleiotropic cop/det/fus mutations are epistatic to mutations in phytochromes, a blue-light photoreceptor, and a downstream regulatory component, HY5. Therefore, there is a complement of at least 10 essential and pleiotropic Arabidopsis genes that are necessary for repression of photomorphogenic development.     

Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes.     

Coordination of the initiation of recombination and the reductional division in meiosis in Saccharomyces cerevisiae

Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.     

A Pleiotropic Phospholipid Biosynthetic Regulatory Mutation in Saccharomyces Cerevisiae Is Allelic to Sin3 (Sdi1, Ume4, Rpd1)

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe(-) (constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OPI3) constitutively. The Cpe(-) mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe(-) phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe(-) mutations isolated in this study.