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  • 1
    ISSN: 1432-0983
    Keywords: Mitochondrial rRNA ; Nuclear/mitochondrial interactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have isolated two non-allelic nuclear mutants of Neurospora crassa that are temperature-sensitive for the production of cytochromes aa 3 and b. When grown at 23 °C the mutants are virtually indistinguishable from the parent wild-type strains. When grown at 41 °C the mutants have large amounts of KCN-insensitive respiration and lack cytochromes aa 3 and b. Further examination of the mutants revealed that they were extremely deficient in their capacity for mitochondrial protein synthesis when grown at 41 °C. This protein synthesis deficiency appears to be related to a virtual absence of both small and large mitochondrial ribosomal subunits following growth at 41 °C. Examination of the mitochondrial RNAs of the mutants suggests that mitochondrial rRNAs are synthesized in greatly reduced amounts or that they are misprocessed when these mutants are grown at the non-permissive temperature.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0983
    Keywords: Cytochrome c ; Cytochrome aa 3 ; Mitochondria ; Neurospora crassa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa 3 and c. Using a sib-selection procedure we have isolated the cyt-12 + allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 + allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa 3 .
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-0983
    Keywords: Key wordsNeurospora ; TOM70 ; Mitochondria ; Protein import
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0983
    Keywords: Neurospora ; Cytochrome c oxidase ; COXII ; Mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa 3 relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 [mi-3] allele, which suppresses both mutations.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0983
    Keywords: Neurospora ; Complex I ; RIP ; Mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have used the procedure of sheltered RIP to generate mutants of the 78-kDa protein of the peripheral arm of Neurospora crassa complex I. The nuclei containing the mutations were initially isolated as one component of a heterokaryon but subsequent analysis showed that nuclei containing null alleles of the gene could be propagated as homokaryons. This demonstrates that the gene does not serve an essential function. Sequence analysis of one allele shows that 61 transition mutations were created resulting in 39 amino-acid changes including the introduction of four stop codons. Mutant strains grow at a slower rate than wild-type and exhibit a decrease in the production of conidia. Electron paramagnetic spectroscopy of mutant mitochondria suggest that they are deficient in Fe−S clusters N-1, N-3, and N-4.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have isolated twenty-six nuclear, singlegene cytochrome-deficient mutants of Neurospora crassa as an initial step toward the study of the structural components and regulatory mechanisms involved in the biogenesis of the mitochondrial cytochrome system. These mutants, together with two previously described mutants, cyt-1 and cyt-2, have been classified into six distinct groups on the basis of cytochrome phenotype: a) cytochrome aa 3 deficiency (due to mutations affecting loci designated cya); b) cytochrome b deficiency (cyb-1 locus); c) cytochrome b deficiency with a partial deficiency of cytochrome aa 3 (cyb-2 locus); d) deficiency of both cytochromes aa 3 and b (cyt loci); e) deficiency of both cytochromes aa 3 and c (cyt-2 locus); and f) partial deficiency of cytochromes aa 3 and c (cyt-12 locus). Four of seven mutations affecting cya loci have been mapped and are located on linkage groups I, II, V, and VI. It is not yet known whether these genes code for structural components of cytochrome oxidase or have a regulatory function that affects synthesis or assembly of the enzyme. The cyb-1 and cyb-2 genes are located on linkage groups V and VI, respectively, and appear to code for regulatory elements that control the biogenesis of cytochromes b and aa 3 . The positions of the cyt mutations that cause a simultaneous deficiency of cytochromes aa 3 and b are dispersed throughout the genome, except for two gene clusters on the left arm of linkage group I. Some of these mutants may be deficient in mitochondrial protein synthesis. Two mutations, cyt-2 and cyt-12, are located on linkage groups VI and II, respectively, and appear to affect genes that code for components of a regulatory system that controls the biogenesis of cytochromes aa 3 and c.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1617-4623
    Keywords: Key wordsNeurospora crassa ; Mitochondria ; Complex I ; Assembly ; Gene disruption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nuclear gene coding for the 20.8k-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) from Neurospora crassa, nuo-20.8 was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain of N. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functional nuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.
    Type of Medium: Electronic Resource
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