Synthesis of signals for de novo DNA methylation in Neurospora crassa

Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)(n) or (TTAA)(n) were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.     

Induction and de novo synthesis of uricase, a nitrogen-regulated enzyme in Neurospora crassa.

Two efficient procedures are presented for the purification of the purine catabolic enzyme uricase from Neurospora crassa. A specific antiserum for uricase was prepared and used to examine the regulation of uricase expression. Even when wild-type cells are growing under full nitrogen repression conditions, they possess a considerable basal level of uricase. Induction results in a severalfold increase in the level of this enzyme and reflects de novo enzyme synthesis. Identical forms of uricase were translated in vitro from RNA isolated from control and induced cells, but, unexpectedly, induced cells contained less translatable uricase mRNA than did control cells. Although uricase is localized in peroxisomes, the enzyme subunit appears to be synthesized in mature form without any requirement for processing.     

Signal for DNA methylation associated with tandem duplication in Neurospora crassa.

Most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of Neurospora crassa. The region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5S rRNA gene. The repeated elements have diverged about 15% by the occurrence of numerous CG to TA mutations, which probably resulted from deamination of methylated cytosines. Most but not all common laboratory strains of N. crassa have methylated duplicated DNA at the zeta-eta locus. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA and it is not methylated. A cross of strains differing at the zeta-eta locus produced progeny which all had duplicated, methylated, or unique, unmethylated DNA, like the parental strains. We conclude that a signal causing unprecedented heavy DNA methylation is present in the zeta-eta region.     

DNA methylation associated with repeat-induced point mutation in Neurospora crassa.

Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.     

An inducible gene expression system for Neurospora crassa.

Neurospora crassa acetyl CoA synthetase is highly induced when the growing mycelium is transferred from sucrose- to acetate-based medium. The inducible promoter of this gene has been isolated and used to control the expression of glutamate dehydrogenase. Transformants containing this expression cassette show gdh levels up to 25 times higher than the nontransformed host strain. This expression cassette will form the basis of a system of heterologous gene expression.     

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

A photoaffinity label for the siderophore-mediated iron transport system in Neurospora crassa

A photoaffinity derivative of the coprogen class of siderophores, p-azidobenzoylcoprogen B, has been synthetized. In the dark it is recognized and taken up by the iron transport system in Neurospora crassa (arg-5, ota, aga) in a concentration-dependent manner (K_m = 6 μM, V_max = 0.2 nmol·min⁻¹·mg⁻¹). It is also a competitive inhibitor of coprogen uptake, K_I ≈ 5 μM. Photolyssis of cells with near-ultraviolet light during transport in the presence of the photoaffinity label results in approx. 50% irreversible inhibition of both coprogen and ferrichrysin uptake. Uptake of p-azidobenzoylcoprogen B itself is also inhibited upon illumination. It is proposed that this affinity label be used in isolation of the iron receptor protein(s) in N. crassa.     

An inducible acetate transport system in Neurospora crassa conidia.

Neurospora crassa conidia possess an active transport system for the uptake of acetate. This system was characterized as: (a) energy dependent; (b) taking place against a concentration gradient; (c) saturating at higher substrate concentrations and (d) competitively inhibited by propionate. Activity of the acetate transport system can be further enhanced by preincubating conidia in 1 mM acetate medium for 180 min (the inducible transport system). The conidial system and the inducible system have similar properties. The development of the inducible transport was dependent on RNA and protein synthesis. A genetic control of this system was further confirmed by isolating a mutant acp-i acetate permease, inducible) that fails to develop the inducible transport system.     

Carbamyl phosphate synthetase A of Neurospora crassa.

Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.