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.     

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.     

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

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.     

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.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

Nitrogen regulation of acid phosphatase in Neurospora crassa.

Neurospora crassa possesses a repressible acid phosphatase with phosphodiesterase activity which appears to permit it to utilize ribonucleic acid as a phosphorus and as a nitrogen source. This acid phosphatase, which is specified by the pho-3 locus, is derepressed approximately eightfold during nitrogen limitation and to an even greater extent during phosphorus limitation, but is unaffected by sulfur limitation. Derepression of the enzyme did not occur when adenosine 5'-monophosphate was the sole phosphorus or nitrogen source. Synthesis of the acid phosphatase is not under the control of the nit-2 locus, which regulates the expression of a large number of other nitrogen catabolic enzymes. The structural gene of the acid phosphatase appears to be a member of both the phosphorus and nitrogen regulatory circuits.     

Properties of Two Nuclease Genes in NEUROSPORA CRASSA

Two genes, nuc-1 and nuc-2, of Neurospora crassa which were responsible for the nucleic acid digestion, were located on linkage group 1 and 2, respectively. A temperature-sensitive mutant (B1ts-2) was obtained from a nuc-2 mutant. Nuclease mutants showed a reduced activity of nuclease N(3) which was found to be a complex consisting of nuclease N(3) (') and inhibitor molecule. Nuclease N(3), nuclease N(3) (') and inhibitor were partially purified and estimated to have the approximate molecular weights of 38,000, 12,500 and 25,000 respectively. A nuc-1 mutant produced the nuclease N(3) (') altered in thermolability. A nuc-2 mutant and B1ts-2 produced the inhibitor altered in the capacity to inhibit nuclease N(3) (') activity. The inhibitor prepared from B1ts-2 was more thermostable than that from other strains. From these results, it was suggested that the nuc-1 gene is the structural gene for nuclease N(3) (') and the nuc-2 gene that for the inhibitor. A possible involvement of this enzyme-inhibitor complex in the regulation of nuclease activity and synthesis of other proteins was suggested.     

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.     

Transcriptional control and patterning of the pho-1 gene, an essential acid phosphatase expressed in the C. elegans intestine

We have previously described an acid phosphatase enzyme, PHO-1, present at the lumenal surface of all but the anterior six cells of the Caenorhabditis elegans intestine. In the present paper, we identify the pho-1 structural gene, which encodes a histidine acid phosphatase showing highest similarity to human prostatic acid phosphatase. The pho-1 5'-flanking DNA is capable of directing reporter gene expression that is both gut specific, correctly timed and correctly "patterned", that is, not expressed in the gut anterior. Furthermore, this anterior-posterior patterning of pho-1 expression responds to the C. elegans Wnt pathway as if pho-1 is repressed (directly or indirectly) by high levels of the HMG effector protein POP-1. Transgenic analysis of the pho-1 promoter shows that gut expression is critically dependent on a single WGATAR site. The gut-specific GATA factor ELT-2 binds to this site in vitro and removal of ELT-2 from the embryo destroys expression of the pho-1 reporter. Thus, all our results indicate that pho-1 is a direct downstream target of ELT-2. Finally, the pho-1 loss-of-function mutation shows an interesting and unexpected phenotype for a somatically-expressed hydrolytic enzyme: loss of pho-1 causes arrest of the majority of embryos but this lethality is a maternal effect. We suggest that pho-1 is required by the maternal intestine to assimilate some nutrient or cleavage product that is subsequently provided to the next generation of embryos.     

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.     

Genetic and regulatory aspects of methionine biosynthesis in Saccharomyces cerevisiae

Methionine biosynthesis and regulation of four enzymatic steps involved in this pathway were studied in Saccharomyces cerevisiae, in relation to genes concerned with resistance to ethionine (eth(1) and eth(2)). Data presented in this paper and others favor a scheme which excludes cystathionine as an obligatory intermediate. Kinetic data are presented for homocysteine synthetase [K(m)(O-acetyl-l-homoserine) = 7 x 10(-3)m; K(i) (l-methionine) = 1.9 x 10(-3)m]. Enzymes catalyzing steps 3, 4, 5, and 9 were repressible by methionine. Enzyme 4 (homoserine-O-transacetylase) and enzyme 9 (homocysteine synthetase) were simultaneously derepressed in strains carrying the mutant allele eth(2) (r). Studies on diploid strains confirmed the dominance of the eth(2) (s) allele over eth(2) (r). Regulation of enzyme 3 (homoserine dehydrogenase) and enzyme 5 (adenosine triphosphate sulfurylase) is not modified by the allele eth(2) (r). The other gene eth(1) did not appear to participate in regulation of these four steps. Gene enzyme relationship was determined for three of the four steps studied (steps 3, 4, and 9). The structural genes concerned with the steps which are under the control of eth(2) (met(8): enzyme 9 and met(a): enzyme 4) segregate independently, and are unlinked to eth(2). These results are compatible with the idea that the gene eth(2) is responsible for the synthesis of a pleiotropic methionine repressor and suggest the existence of at least two different methionine repressors in S. cerevisiae. Implications of these findings in general regulatory mechanisms have been discussed.     

Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae.

Two lines of evidence showed that the PHO8 gene encodes the structure of repressible, nonspecific alkaline phosphatase in Saccharomyces cerevisiae: (i) the enzyme produced by a temperature-sensitive pho8 mutant at the permissive temperature (25 degrees C) was more thermolabile than that of the wild-type strain, and (ii) the PHO8 gene showed a gene dosage effect on the enzyme activity. The pho8 locus has been mapped on chromosome IV, 8 centimorgans distal to rna3. A new mutant carrying the pho9 gene was isolated which lacks repressible alkaline phosphatase, but has the normal phenotype for the synthesis of repressible acid phosphatase. The pho9 gene segregated independently of all known pho-regulatory genes and did not show the gene dosage effect on repressible alkaline phosphatase activity. The pho9/pho9 diploid hardly sporulated and showed no commitment to intragenic recombination when it was inoculated on sporulation medium. Hence the pho9 mutant has a phenotype similar to the pep4 mutant, which was isolated as a pleiotropic mutant with reduced levels of proteinases A and B and carboxypeptidase Y. An allelism test indicated that pho9 and pep4 are allelic.     

Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase.

The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+.     

An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans.

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.     

Control of the production and partial characterization of repressible extracellular 5'-nucleotidase and alkaline phosphatase in Neurospora crass.

A new species of orthophosphate repressible extracellular 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was found to be released into mycelial culture media when a wild type strain of Neurospora crassa was grown on limiting amounts of phosphate. The production of 5'-nucleotidase and extracellular acid and alkaline phosphatase was inhibited by the addition of rifampicin when it was added at the later stage of mycelial growth, but not when it was added at a very early stage. The 5'-nucleotidase and extracellular alkaline phosphatase were partially purified and characterized. pH optimum of the former was 6.8 and that of the latter was higher than 10.0. The 5'-nucleotidase activity was inhibited by ethylenediaminetetraacetate (EDTA) and ZnCl2 at pH 6.8 and stimulated by MnCl2 and CoCl2 at pH 4.0. Alkaline phosphatase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase hydrolyzed various 5'-nucletides but not 3'-nucleotides or other various phosphomono- and diester compounds. Alkaline phosphatase hydrolyzed all the phosphomonoester compounds tested. Mutants, nuc-1 and nuc-2, which were originally isolated by the inability to utilize RNA or DNA as a sole source of phosphate, were unable to produce 5'-nucleotidase or six other repressible enzymes reported previously. These mutants showed no or significantly reduced growth on orthophosphate-free nucleotide media depending on the number of conidia inoculated, mainly because of loss of ability to produce these repressible extracellular phosphatases.     

Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b

The oxidation of methane to methanol in methanotrophic bacteria is catalysed by the enzyme methane monooxygenase (MM0). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the alpha, beta and gamma subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.     

Genetic Control of Phosphate-Metabolizing Enzymes in NEUROSPORA CRASSA: Relationships among Regulatory Mutations

In Neurospora crassa, the phosphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, preg and nuc-1, pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pcon-c (constitutive) mutations reside in the same cistron. preg-c (constitutive) mutations are epistatic to nuc-2 mutations. nuc-1 (null) mutations are epistatic to all others.     

害虫遗传不育技术中致死基因的研究与应用

基于遗传修饰手段的昆虫不育技术(SIT)作为一类物种特异、环境友好、科学高效的新兴策略,在害虫防治中具有广阔的应用前景。释放携带显性致死基因昆虫的技术(RIDL)是改进传统SIT的重要手段之一,主要包括四环素调控系统、特异性启动子、性别特异剪接系统和特异性致死基因等重要元件,其中根据不同昆虫的特点选择合适的特异性致死基因对于构建遗传不育品系至关重要。这些致死基因或受到阻遏调控系统的控制、或特异的在雌虫中表达、亦或直接作用于X染色体,导致后代在特定发育阶段或特定性别中条件致死。本文综述了RHG家族(reapr、hid、grim、michelob_x)细胞凋亡基因、转录激活因子t TA及Nipp1Dm、归巢内切酶基因等在害虫遗传不育技术中的研究和应用,讨论了特定致死基因的效应机理和应用特点,并对其可能的发展方向进行了展望。由于不同效应基因的致死作用和调控机理尚未完全明晰,因此深入研究特异致死基因的凋亡机制和在不同物种中的兼容作用,将为害虫遗传防控提供更多的研究思路和手段。