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Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid.

The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism's mtDNA.

Base Sequence↗

Electron spin resonance investigations of mitochondrial electron transport in Neurospora crassa. Characterization of paramagnetic intermediates in a standard strain.

1. Submitochondrial particles from Neurospora strain inl-89601 have been analyzed by electron spin resonance spectroscopy (ESR). Numerous signals due to iron-sulfur proteins are observed at low temperatures. Analysis of these ESR signals at various temperatures allows the assignment of resonances to iron-sulfur centers 1-5 that have been described in other organisms. There are no discrepancies between the signals seen in Neurospora and those described in other organisms and it is likely that Neurospora mitochondria contain the same iron-sulfur centers that are observed elsewhere. 2. NADPH and NADH act to reduce the iron-sulfur centers of respiratory complex I. 3. The drug pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorphenyl)pyrrole] is an effective inhibitor of both NADH-supported and succinate-supported electron transport in Neurospora. 4. Analysis of pyrrolnitrin inhibition curves, respiration studies, ESR spectra, and the steady-state level of reduction of cytochrome b in the presence and absence of the drug shows that pyrrolnitrin acts to inhibit electron transport in Neurospora mitochondria at multiple sites in the region between ubiquinone and cytochrome b.

Depression, Chemical↗

DNA photolyase from the fungus Neurospora crassa. Purification, characterization and comparison with other photolyases.

A phr-gene from the filamentous fungus Neurospora crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,10-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurospora photolyase is shifted from ca 380 nm to 391 nm (epsilon = 34,800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiae photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurospora photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.

Deoxyribodipyrimidine Photo-Lyase↗

Cloning of the arg-12 gene of Neurospora crassa and regulation of its transcript via cross-pathway amino acid control.

The arg-12 locus of Neurospora crassa encodes ornithine carbamoyl transferase, which is one of many amino acid synthetic enzymes whose activity is regulated through cross-pathway (or general) amino acid control. We report here the use of probes derived from the functionally equivalent arg-B gene of Aspergillus nidulans to identify and clone a 10 kb Neurospora DNA fragment carrying the arg-12 gene. Short Neurospora DNA probes derived from this fragment were used to identify a 1.5 kb polyA+ transcript of the arg-12 region. Arg-12 transcript levels increased approximately 20 fold under conditions of arginine or histidine limitation in strains having normal cross-pathway regulation (cpc-1+) but showed no such response in a cpc-1 mutant strain impaired in this regulation. Time course studies in cpc-1+ strains revealed a rapid response (within 10 m) of arg-12 transcript levels following inhibition of histidine synthesis by 3 amino 1,2,4 triazole, but a delayed response following arginine deprivation of an arginine requiring strain. In contrast to the behaviour of arg-12 mRNA, the level of the Neurospora am gene transcript (specifying NADP dependent glutamate dehydrogenase) was unaffected either by amino acid limitation or by the cpc-1 mutation. A possible role for the cpc-1+ product as a positive regulator of transcription of genes subject to cross-pathway control is discussed.

Cloning, Molecular↗

Cloning of the structural gene for orotidine 5'-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli.

A Neurospora gene bank in plasmid pRK9 was used to complement pyrimidine auxotrophs in E. coli. Two plasmids were obtained that complement a pyrF mutant of E. coli. These plasmids hybridise to Neurospora DNA and transform a pyr-4 strain of Neurospora. The promoter used in expressing the orotidine 5'-monophosphate carboxylase in E. coli is within the Neurospora sequence.

Carboxy-Lyases↗

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.

Cytochrome Reductases↗

Gamma-ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines.

Well characterized gamma-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Normal human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline sucrose gradient centrifugation, repression of DNA synthesis by low doses of gamma-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression in synthesis of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW DNA synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.

Ataxia Telangiectasia↗

Characterization of telomere DNA from Neurospora crassa.

The nucleotide sequence of the telomere at the right end of linkage group V (VR) in the standard OR23-IV-A strain of the filamentous fungus, Neurospora crassa, reveals the following features. At the chromosome terminus, tandem repeats of the hexanucleotide TTAGGG are present. Immediately centromere-proximal to the simple sequence repeat is a more complex element called Pogo that is reiterated 5-10 times in the genomes of various Neurospora strains. The element possesses several features characteristic of a transposable element: direct repeats of 318 bp flank the element, there is a long internal open reading frame (ORF), and a 3-bp duplication is found at its borders. However, Pogo has other structural features that are more difficult to reconcile with the standard model of a transposable element. A second telomere from Neurospora was also cloned by screening a genomic lambda library with a synthetic oligodeoxyribonucleotide homologous to the simple sequence repeats. This telomere is entirely non-homologous with the VR telomere except for the TTAGGG repeats, has no associated copy of Pogo, and has no nearby ORFs. There are no long stretches of TTAGGG repeats present in the Neurospora genome at non-telomeric sites.

Amino Acid Sequence↗

Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid.

We previously described two plasmids that replicate autonomously in both Neurospora and E. coli (Stohl and Lambowitz, Proc. Natl. Acad. Sci., U.S.A. 80, 1058-1062, 1983). One plasmid, pALS1, consists of the Neurospora ga-2+ gene (3 kb Hind III-fragment), the mitochondrial plasmid from N. intermedia strain P405-Labelle, and E. coli plasmid pBR325. The other, pALS2, is a putative deletion derivative of pALS1 that lacks most or all of the Labelle insert and that was repeatedly recovered from Neurospora transformants. We have now sequenced the region encompassing the deletion in five pALS2 plasmids isolated independently in two different laboratories. All five plasmids are identical in this region and completely lack the Labelle insert. We have also characterized an additional deletion derivative that retains a small (approximately 0.5 kb) segment of the Labelle insert. The results for pALS2 suggest that pBR325 plus the ga-2+ segment constitute a Neurospora replicon.

Base Sequence↗

Structure of a Neurospora RNA polymerase I promoter defined by transcription in vitro with homologous extracts.

A Neurospora in vitro transcription system has been developed which specifically and efficiently initiates transcription of a cloned Neurospora crassa ribosomal RNA gene by RNA polymerase I. The initiation site of transcription (both in vitro and in vivo) appears to be located about 850 bp from the 5' end of mature 17S rRNA. However, the primary rRNA transcripts are normally cleaved very rapidly at a site 120-125 nt from the 5' end in vitro and in vivo. The nucleotide sequence surrounding the initiation site has been determined. The region from -16 to +9 exhibits partial homology to the corresponding sequences from a wide variety of organisms including yeast, but the most striking similarity is to the initiation region from Dictyostelium discoideum which displays 73% homology to the Neurospora sequence from -23 to +47. The Neurospora sequences from -96 to +97 have been shown to be sufficient for transcription. This region contains two sequences displaying 8/9 bp matches to elements of the 5S rDNA promoter.

Base Sequence↗

A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.

The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system.

Amino Acid Sequence↗

Comparison and cross-species expression of the acetyl-CoA synthetase genes of the Ascomycete fungi, Aspergillus nidulans and Neurospora crassa.

The genes encoding the acetate-inducible enzyme acetyl-coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu-5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N-terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position. The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5' coding sequence not homologous with acu-5, and four within the sequence that is largely similar to that of acu-5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu-5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate-induction of acetyl-CoA synthetase, implying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5' ends.

Acetate-CoA Ligase↗

The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation.

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.

Adenosine Triphosphatases↗

A portable signal causing faithful DNA methylation de novo in Neurospora crassa.

Methylation of cytosine residues in eukaryotic DNA is common, but poorly understood. Typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified. Neurospora crassa DNA containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct DNA methylation de novo. DNA stripped of its methylation by propagation in Escherichia coli was reintroduced into Neurospora crassa by transformation. The zeta-eta region reproducibly became "properly" methylated whether inserted at its native chromosomal position or at ectopic sites. Adjacent Neurospora and bacterial sequences in the transforming DNA rarely became methylated. A model is presented that accounts for position-independent faithful methylation as observed in the zeta-eta region, as well as position-dependent methylation, as occasionally observed, especially with sequences not native to Neurospora.

DNA, Fungal↗

Deficient cyclic adenosine 3',5'-monophosphate control in mutants of two genes of Neurospora crassa.

Strains of Neurospora crassa mutant in either of two genes, Crisp-1 (cr1) and Frost (fr), showed no increase of cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels when subjected to several treatments which produce large increases of cyclic AMP in wild-type Neurospora. Evidently, the previously reported deficiencies of adenylate cyclase in these mutants were sufficient to block the normal increases. This fact suggests that both mutants could be used to help determine which control phenomena involve cyclic AMP and to interrupt the control of established cyclic AMP-regulated functions. Earlier studies had suggested an interdependence of the cyclic AMP level and the electric potential difference across the plasma membrane of Neurospora. Present experiments, therefore, employed several strains with the cr1 mutation to test for possible roles of cyclic AMP in recovery and oscillatory behavior of the Neurospora membrane potential. The results showed all such phenomena to be normal in the adenylate cyclase-defective strains, which demonstrates that variations of cyclic AMP are not obligatorily involved in the apparent control processes. Evidence is also presented that the induction of both glucose transport system II and the alternative oxidase do not require elevated cyclic AMP levels.

2,4-Dinitrophenol↗

Presence of abnormal synaptonemal complexes in heterothallic species of Neurospora.

Synaptonemal complex abnormalities are frequent in reconstructed meiotic prophase nuclei of Neurospora crassa and Neurospora intermedia. Three kinds of synaptonemal complex anomalies were seen: lateral component splits, lateral component junctions, and multiple complexes. The anomalies apparently are formed during or after the pairing process, as they were not seen in the largely unpaired early zygotene chromosomes. Their presence at all the other substages from mid-zygotene to late pachytene indicates that they are not eliminated before the synaptonemal complex decomposes at diplotene. Abnormal synaptonemal complexes were seen in all 19 crosses of N. crassa and N. intermedia that were examined, including matings between standard laboratory strains, inversions, Spore killers, and strains collected from nature. The frequency of affected nuclei and degree of abnormality within a nucleus varied in different matings. No abnormalities were present in the homothallic species Neurospora africana and Neurospora terricola. Structural chromosome aberrations, introgression, and heterozygosity have been eliminated as causes for pairing disorder. The abnormal synaptonemal complexes seemingly do not interfere with normal ascus development and ascospore formation. The affected nuclei are not aborted during meiotic prophase, nor are they eliminated by abortion of mature asci. The abnormal meiocytes do not lead to aneuploidy, as judged by the low frequency of white ascospores in crosses between wild type strains that have many abnormalities. Thus, the abnormal synatonemal complexes do not appear to prevent chiasma formation between homologues.

Cell Nucleus↗

Isolation of specific protease inhibitors from Neurospora crassa.

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography pand gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000-24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

Bacterial Proteins↗

An immunochemical study of Neurospora nucleases.

Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.

Antibodies↗