Revisiting the Neurospora crassa mitochondrial genome.

The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64 839 bp revealed 21 protein-coding genes and several hypothetical open reading frames with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related to the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in noncoding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.

Quantification of Neurospora crassa mitochondrial DNA using quantitative real-time PCR.

The filamentous fungus Neurospora crassa is a popular model organism used in a wide range of biochemical and genetic studies and vastly used in mitochondrial research. Despite the relevance of mitochondria in N. crassa biology, no method for quantification of mitochondrial DNA (mtDNA) is currently available. Quantitative real-time PCR (qPCR) is a powerful tool, with a wide range of applications, and has been used for the quantification of nucleic acids in humans and a few other species. Here we present a new qPCR assay for relative quantification of N. crassa mtDNA. Three sets of qPCR primers targeting different regions of the mitochondrial genome were tested for mtDNA quantification. The qPCR was successfully validated in N. crassa strains from different geographical locations, representing the vast genetic diversity of this species, and knockout mutant strains. Moreover the assay proved to be efficient in templates with varied amounts of mitochondria, obtained through different DNA extraction methods. The qPCR performed well in all tested samples revealing a higher amount of mtDNA than nuclear DNA in all cases. This technique will facilitate the characterization of mtDNA of N. crassa in future studies and can be used as a tool to validate methods of mitochondria isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: The standardization of quantitative real-time PCR (qPCR) techniques is essential to enable and facilitate future comparisons. Neurospora crassa is a model organism with a lot of potential in different fields of study. Here we use N. crassa to develop and establish an assay to quantify mitochondrial DNA using qPCR. We tested strains with different geographical background and our data demonstrated the usefulness of this assay to quantify mitochondrial DNA in N. crassa. This technique can be useful in a wide variety of applications and in different types of studies.

Complex I from the fungus Neurospora crassa.

Respiratory chain complex I is a complicated enzyme of mitochondria, that couples electron transfer from NADH to ubiquinone to the proton translocation across the inner membrane of the organelle. The fungus Neurospora crassa has been used as one of the main model organisms to study this enzyme. Complex I is composed of multiple polypeptide subunits of dual genetic origin and contains several prosthetic groups involved in its activity. Most subunits have been cloned and those binding redox centres have been identified. Yet, the functional role of certain complex I proteins remains unknown. Insight into the possible origin and the mechanisms of complex I assembly has been gained. Several mutant strains of N. crassa, in which specific subunits of complex I were disrupted, have been isolated and characterised. This review concerns many aspects of the structure, function and biogenesis of complex I that are being elucidated.

Identification of the TYKY homologous subunit of complex I from Neurospora crassa.

A polypeptide subunit of complex I from Neurospora crassa, homologous to bovine TYKY, was expressed in Escherichia coli, purified and used for the production of rabbit antiserum. The mature mitochondrial protein displays a molecular mass of 21280 Da and results from cleavage of a presequence consisting of the first 34 N-terminal amino acids of the precursor. This protein was found closely associated with the peripheral arm of complex I.

Primary structure of a ferredoxin-like iron-sulfur subunit of complex I from Neurospora crassa.

We have isolated cDNA clones encoding an iron-sulfur polypeptide subunit of the mitochondrial complex I of Neurospora crassa. The fungal cDNA library was screened by hybridisation with an heterologous probe from Paracoccus denitrificans. The DNA sequence of relevant isolates was determined and revealed an open reading frame encoding a precursor protein of 219 amino acid residues. The gene product is a ferredoxin-like protein that contains two cysteine-rich motives that may each bind a tetranuclear iron-sulfur cluster. The primary structure of the protein is highly homologous to the 23 kDa iron-sulfur subunit of complex I from bovine and P. denitrificans. Interestingly, an alanine residue within the second cluster-binding motif, which is conserved in complex I but replaced by tyrosine in similar chloroplast genes, is substituted for serine in N. crassa.

Primary structure of the nuclear-encoded 10.5 kDa subunit of complex I from Neurospora crassa.

We have isolated a cDNA clone for the nuclear encoded 10.5 kDa subunit of complex I from N. crassa. DNA sequencing revealed an open reading frame corresponding to a polypeptide with 94 amino acids and a calculated molecular mass of 10531 Da. The protein is synthesized without a cleavable mitochondrial targeting sequence. The N. crassa polypeptide is the fungal equivalent of subunit B8 of bovine complex I.

NADH dehydrogenase in Neurospora crassa contains myristic acid covalently linked to the ND5 subunit peptide.

The mitochondrial, proton-pumping NADH:ubiquinone oxidoreductase consists of at least 35 subunits whose synthesis is divided between the cytosol and mitochondria; this complex I catalyzes the first steps of mitochondrial electron transfer and proton translocation. Radiolabel from [(3)H]myristic acid was incorporated by Neurospora crassa into the mitochondrial-encoded, approximately 70 kDa ND5 subunit of NADH dehydrogenase, as shown by immunoprecipitation. This myristate apparently was linked to the peptide through an amide linkage at an invariant lysine residue (Lys546), based upon analyses of proteolysis products. The myristoylated lysine residue occurs in the predicted transmembrane helix 17 (residues 539-563) of ND5. A consensus amino acid sequence around this conserved residue exists in homologous subunits of NADH dehydrogenase. Cytochrome c oxidase subunit 1, in all prokaryotes and eukaryotes, contains this same consensus sequence surrounding the lysine which is myristoylated in N. crassa.

Primary structure and characterisation of a 64 kDa NADH dehydrogenase from the inner membrane of Neurospora crassa mitochondria.

A cDNA clone encoding a mitochondrial NADH dehydrogenase from Neurospora crassa was sequenced. The total DNA sequence encompasses 2570 base pairs and contains an open reading frame of 2019 base pairs coding for a precursor polypeptide of 673 amino acid residues. The protein is encoded by a single-copy gene located to the right side of the centromere in linkage group IV of the fungal genome. The N-terminus of the precursor protein has characteristics of a mitochondrial targeting pre-sequence. The protein displays homology with mitochondrial NADH dehydrogenases from yeast. In contrast to these polypeptides, however, analysis of its primary structure revealed that it contains a well-conserved calcium-binding domain. Rabbit antiserum against the protein expressed in an heterologous system recognises a mitochondrial protein of N. crassa with an apparent molecular mass of 64 kDa. Analysis of the fungal mitochondria by swelling, digitonin fractionation and alkaline treatment indicate that the protein is located in the inner membrane of the organelles, possibly facing the matrix side.

Complementary DNA sequences of the 24 kDa and 21 kDa subunits of complex I from Neurospora.

We have cloned and sequenced cDNAs coding for two subunits of the peripheral arm of Neurospora crassa complex I. The two polypeptides are synthesized as precursor proteins which are processed to mature forms with predicted molecular masses of 24331 and 20982 Da.

Characterisation of the last Fe-S cluster-binding subunit of Neurospora crassa complex I.

We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.

Respiratory chain complex I is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly.

We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21. 3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.

Inactivation of genes encoding subunits of the peripheral and membrane arms of neurospora mitochondrial complex I and effects on enzyme assembly.

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly.

On the mechanical properties of PLC-bioactive glass scaffolds fabricated via BioExtrusion.

This paper addresses the mechanical characterization of polycaprolactone (PCL)-bioglass (FastOs®BG) composites and scaffolds intended for use in tissue engineering. Tissue engineering scaffolds support the self-healing mechanism of the human body and promote the regrowth of damaged tissue. These implants can dissolve after successful tissue regeneration minimising the immune reaction and the need for revision surgery. However, their mechanical properties should match surrounding tissue in order to avoid strain concentration and possible separation at the interface. Therefore, an extensive experimental testing programme of this advanced material using uni-axial compressive testing was conducted. Tests were performed at low strain rates corresponding to quasi-static loading conditions. The initial elastic gradient, plateau stress and densification strain were obtained. Tested specimens varied according to their average density and material composition. In total, four groups of solid and robocast porous PCL samples containing 0, 20, 30, and 35% bioglass, respectively were tested. The addition of bioglass was found to slightly decrease the initial elastic gradient and the plateau stress of the biomaterial scaffolds.

Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa.

Complex I from Neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. Several subunits were isolated. Polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo. N. crassa cells were pulse-labelled with radioactive amino acids. The time course of incorporation of radioactivity into complex-I polypeptides was studied by immunoprecipitation. The labelling kinetics of whole complex I was found to be similar to that of cytochrome oxidase, displaying a half-maximal labelling time of 10 min. Newly synthesized individual polypeptide subunits (about 23 species) assembled into the holoenzyme at markedly different rates. Two mitochondrially synthesized proteins, a 29-kDa polypeptide (the ND-1 gene product) and a 12-kDa polypeptide were the fastest components to appear in the enzyme. We estimate that the precursor pool sizes of all components range between 1-25% of the amounts present in the final complex. Precursors of polypeptides of complex I were synthesized in an heterologous cell-free system and immunoprecipitated with subunit specific antibodies. Six isolated precursors were compared with the corresponding mature proteins. It appears that four subunits (apparent molecular masses of 22, 25, 31 and 33 kDa) are initially synthesized as larger-molecular-mass precursors. Two subunits (apparent molecular masses of 12.5 and 14 kDa) are made with the same size as their mature forms.

Characterization of a membrane fragment of respiratory chain complex I from Neurospora crassa. Insights on the topology of the ubiquinone-binding site.

1. A membrane fragment of complex I from the fungus Neurospora crassa was isolated by immunoprecipitation from alkaline-extracted mitochondrial membranes. 2. Analysis of the polypeptide composition of this hydrophobic domain of complex I has brought insights on the topology of two subunits of the enzyme, namely the 20.8 and 9.3 kDa components. 3. Our results indicate that the ubiquinone-binding site of complex I resides in the interface of the peripheral and membrane arms of the enzymes. The significance of these findings are discussed.

Two nuclear-coded subunits of mitochondrial complex I are similar to different domains of a bacterial formate hydrogenlyase subunit.

A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.

The membrane domain of complex I is not assembled in the stopper mutant E35 of Neurospora.

The assembly of mitochondrial NADH: ubiquinone oxidoreductase (complex I) was studied in the E35 stopper mutant of Neurospora crassa at different times during growth in liquid media. Assembly of complex I as well as of its membrane domain is impaired in this strain throughout the growth period. Nevertheless, a structure that resembles the peripheral aim of the enzyme is still formed in the mitochondria of this mutant. The absence of the membrane domain of complex I in E35 can be attributed to the specific deletion of the mitochondrial ND2 and ND3 subunits of the enzyme.

Primary structure, in vitro expression and import into mitochondria of a 29/21-kDa subunit of complex I from Neurospora crassa.

A full-length cDNA clone coding for a cytoplasmically-synthesized subunit of complex I from Neurospora crassa (apparent molecular mass of 29 kDa) was isolated. DNA sequencing revealed an open reading frame coding for a protein containing 201 amino acids. A molecular mass of 21323 Da was calculated. The precursor polypeptide was efficiently expressed in vitro and imported into isolated mitochondria. It is synthesized without a cleavable signal sequence and needs a membrane potential in order to bind to the mitochondrial membranes.

Primary structure and expression of a nuclear-coded subunit of complex I homologous to proteins specified by the chloroplast genome.

A 31-kDa subunit of complex I from Neurospora crassa, of nuclear origin, was cloned. The precursor polypeptide (33 kDa) could be efficiently expressed in an in vitro system for transcription and translation. The processing of the precursor to the mature protein was also obtained in vitro. An open reading frame coding for a precursor protein of 283 amino acids (32247 Da) was found by DNA sequencing. The predicted primary structure shows significant homology with proteins made in chloroplast. This supports the hypothesis that an enzyme similar to respiratory chain NADH dehydrogenase might exist in these organelles.