Characterization of inl + transformants of Neurospora crassa obtained with a recombinant cosmid-pool

Summary We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl ⁺ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl ⁺ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F₁ progeny of the transformants. We were able to recover eleven different plasmids from the F₁ progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl ⁺ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.     

Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia

Genetic and molecular analyses of the phenomenon of senescence—i.e., irreversible loss of growth and reproductive potential upon subculturing—in Neurospora intermedia strain M1991-60A, collected from Maddur in southern India, showed the presence of plasmid pMaddur1, which is homologous to the senescence-inducing circular mitochondrial plasmid, pVarkud. Maternal inheritance of senescence in M1991-60A correlated to the formation of variant pMaddur1, its subsequent insertion into mitochondrial (mt)DNA and the accumulation of defective mtDNA with the pMaddur1insert. PCR-based analyses for similar plasmids in 147 natural isolates of Neurospora from Maddur showed that nearly 40% of the strains had pMaddur1 or pMaddur2 that shared 97–98% sequence homology with pVarkud and pMauriceville. Nearly 50% of the strains that harbored either pMaddur1 or pMaddur2, also contained a circular Varkud satellite plasmid (pVS). Size polymorphism maps to the cluster of PstI sites in the non-coding region. Whereas senescence of nearly 40% of N. intermedia strains may be due to pMaddur, the presence in seven strains of pVS but not pMaddur and the absence of either of these two plasmids in other senescence-prone isolates suggests yet undiscovered mechanisms of senescence in the Maddur strains.     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

A new,rapid and efficient transformation procedure for Neurospora

Summary A new, rapid, efficient and reliable method for transforming Neurospora crassa is described. In this procedure, germinated conidia are treated with lithium acetate, then incubated with DNA, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. Optimal conditions to achieve a high transformation rate are reported. Transformation can be obtained with both circular and linear plasmid DNA and also with genomic DNA. Although the rate is substantially decreased, transformation was also obtained with relatively impure DNA preparations, such as that made via rapid miniprep procedures. This transformation technique is simple and reliable and provides a considerable savings in time and materials.     

Unstable cytoplasms in Hawaiian strains of Neurospora intermedia

Summary By subjecting a large sample of natural isolates of N. intermedia to prolonged serial subculturing, 26 cytoplasmic variants have been identified. These variants show senescence, and finally death at some strain-specific point in the subculture series. All senescent strains are from the Hawaiian archipelago, where their incidence in natural populations is high. Senescent cultures can be female-fertile. Random ascospore analyses show that (i) senescence is maternally inherited; (ii) different stages of senescence give different proportions of senescent progeny; and (iii) ascospores from one cross show different degrees of senescence. These results indicate that senescence is determined by a genetic factor which re sides in the cytoplasm. This factor promotes instability of the cytoplasm, resulting initially in cytoplasmic heterogeneity shown by ascus and conidium sampling, and finally in death. Molecular studies to be published elsewhere show that the progression through senescence to death is correlated with the occurence of abnormalities in cytochrome content and mitochondrial DNA. The Hawaiian word kalilo (dying), symbolised [kal], is proposed to denote these cytoplasms.     

A regulatory phenotype associated with the en-am 1 mutant of Neurospora crassa

Summary We have investigated the nature of the en-am1 mutant of Neurospora crassa and have found that it affects the regulation of proline oxidase and utilisation of other nitrogen sources. This mutant is closely linked to the gln gene but not allelic with it. Data from crosses suggest that the two genes he on opposite sides of the in1 gene on linkage group VR.     

A “Stopper” mutant of Neurospora crassa containing two populations of aberrant mitochondrial DNA

Summary [E35], an extranuclear mutant of Neurospora crassa has all the phenotypic characteristics of the stopper mutants (De Vries et al. 1980). In the present work, the mitochondrial DNA as well as the mitochondrial translation products are characterized further. The primary mutational event appears to have been the deletion of about 4 kbp from the wild-type genome. Moreover, after prolonged vegetative growth the mutant accumulates an 8-m circular mtDNA, which was demonstrated both by electronmicroscopy and by restriction enzyme analysis. Hence, the mutant contains two populations of aberrant mitochondrial DNA, the smaller of which is an amplification of the rRNA-tRNA part of the larger. We propose that the primary deletion has generated a signal in the larger DNA which can cause premature termination of replication at the deletion site, and subsequent circularization of the unfinished daughter molecule. Finally, the deleted part may contain a determinant for synthesis of a protein of 11 kDal. The function of this protein, which is not a subunit of the F₀ ATPase, is not yet known.Abbreviations (k)bp (kilo)basepairs - kDal kilodalton - mt mitochondrial     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

A calcium binding protein from cell wall of Neurospora crassa

Isolated cell wall preparations of N. crassa bind significant levels of Ca, Mg and other divalent cations. Enzymatic treatment of the cell wall with β‐(1,3)‐glucanase, but not with chitinase, resulted in solubilization of only the calcium‐binding protein fraction. A calcium‐binding protein (CaBP) was purified by metal‐chelate affinity chromatography and reversed phase HPLC. CaBP has an Mr of around 6 kDa on SDS‐PAGE and mass spectrometry showed that it has a molecular mass of 5744 Da. One mole of CaBP binds 2 moles of calcium and is partially inhibited (15–50%) by other divalent cations (Mg, Ni and Cu). Quenching of tryptophan fluorescence was observed upon copper binding but not calcium binding. This is a first report of a calcium binding protein from the cell wall of fungi. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Methylation of the foreign transposon Restless in vegetative mycelia of Neurospora crassa

Methylation of foreign and/or repeated sequences in the filamentous fungus Neurospora crassa is believed to be directed against invading transposable elements. To test this hypothesis, the fate of a transposon in N. crassa was investigated. Vectors were constructed which carried the transposon Restless, an active class-II element isolated from the fungus Tolypocladium inflatum. These vectors were introduced into N. crassa strains by protoplast transformation. Two strategies were employed: (1) ectopic multi-copy integration, and (2) site-specific single-copy integration at the his-3 locus. All ectopic transformants exhibited strong methylation as confirmed by Southern hybridization of genomic DNA digested with the methylation-sensitive endonuclease Sau3AI and the methylation-insensitive endonuclease NdeII. Single copies of Restless integrated at the his-3 locus were not methylated. These results are discussed with respect to non-RIP methylation and potential consequences for gene-tagging strategies based on the use of Restless. Received: 27 September / 3 November 1999     

Inactivation of the Neurospora crassa mitochondrial outer membrane protein TOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology

Mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. Recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane). The largest component of this complex is TOM70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP). Mutant strains were identified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70. Strains that lack TOM70 grow more slowly than wild-type strains, conidiate poorly, and contain enlarged mitochondria. In vitro preprotein import studies using TOM70-deficient mitochondria revealed a defect in the uptake of the ADP/ATP carrier. Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondrial-processing peptidase (MPP) which was imported more efficiently by TOM70-deficient mitochondria. These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein import. The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial morphology. Received: 25 March 1999 / 18 May 1999     

Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene

Summary In the further analysis of a cross in which the mis-sense allele, am ³, of the Neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the RIP process whereas the am ³ allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. The am ³ allele had also acquired a new HindIII restriction site. It had no detectable methylation. The mutations responsible respectively for the new restriction site and the modified enzyme properties were separated from each other, and from the original am ³ mutation, by selecting for intragenic recombination on either side of the am ³ site. In this way two new effectively wild-type alleles were generated, one characterised by its heat-sensitive and kinetically modified enzyme product and the other by a new HindIII site. These results demonstrate that the RIP phenomenon can be a source of new functional alleles.     

Heterokaryotic transmission of senescence plasmid DNA in Neurospora

Summary Heterokaryotic transmission is one of the major techniques for the study of cytoplasmic inheritance and here we have applied it to the senescence-determining plasmids kalilo (Hawaiian) and maranhar (Indian). We have shown that kalilo-based senescence is effectively transmitted by cytoplasmic contact, both in N. crassa and in N. intermedia. In the first place, the heterokaryons themselves are senescent, confirming the suppressivity of the senescence phenotype in mixtures of normal and senescent cytoplasms. Second, senescence is found in new nuclear associations, as shown by analysis of conidial isolates and meiocytes stemming from the heterokaryons. In addition, the free plasmid AR-kalDNA, and its form that is inserted into mtDNA, (mtIS-kalDNA), are both transmitted to new nuclear associations. In a transient fusion between senescent N. intermedia and nonsenescent N. crassa cells, AR-kalDNA was transmitted to N. crassa and mtIS-kalDNA was transmitted to N. crassa mtDNA. A cryptic mitochondrial plasmid, not associated with senescence, was also transmitted very efficiently to N. crassa mitochondria. In mixed kalilo/maranhar fusions, both plasmids coexisted, approximately equally, in the heterokaryons themselves, and in conidial isolates. However, in sexual derivatives, AR-marDNA was in an excess and AR-kalDNA was sometimes absent. The efficient heterokaryotic transmission of these elements suggests that this is one of their natural modes of spread in populations.