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H Bertrand

Publications and source records attributed to H Bertrand.

At least 73 records · Page 4Linked to original sources
PubMed1982

RNA splicing in Neurospora mitochondria. Characterization of new nuclear mutants with defects in splicing the mitochondrial large rRNA.

In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal RNA contains an intervening sequence of 2.3 kb. We have identified eight nuclear mutants that are defective in splicing the mitochondrial large ribosomal RNA and that accumulate unspliced precursor RNA. These mutants identify three different nuclear genes required for the same mitochondrial RNA splicing reaction. Some of the mutants have unique phenotypic characteristics (for example, accumulation of an unusual intron RNA) that may provide insight into specific aspects of mitochondrial RNA splicing. Mutations at one locus, cyt4, are subject to partial phenotypic suppression by the electron-transport inhibitor antimycin. This phenomenon suggests that at least one component required for mitochondrial RNA splicing is regulated such that its synthesis or activity is increased in response to impairment of electron transport.

Antimycin A↗
PubMed1982

Mitochondrial variants of Neurospora intermedia from nature.

From a sample of 122 natural isolates of Neurospora intermedia collected recently from around the world, five variants had erratic stop-start growth patterns reminiscent of the phenotype of "stopper" laboratory extranuclear mutants of Neurospora crassa. Like laboratory isolated mutants, the natural "stopper" variants were sterile as protoperithecial parents and transmitted the variant growth phenotypes very inefficiently, if at all, as male parents. Heterokaryon tests could not be made because of strain incompatibilities. Four of the variants have mitochondrial cytochrome aa3 and b deficiencies. These four variants are all defective in mitochondrial ribosome assembly and have abnormal ratios of large to small subunits. Restriction enzyme analyses revealed some similarity of N. intermedia to N. crassa mtDNA. One normal and four variant strains had additional DNA in comparison to a standard normal strain. Cumulatively, the results indicate that the genetic alterations which cause stopper phenotypes of these natural isolates of N. intermedia are of mitochondrial rather than nuclear origin.

Crosses, Genetic↗
PubMed1981

A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase.

[C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25 degrees, but which is deficient in cytochromes b and aa3 when grown at 37 degrees (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37 degrees, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37 degrees-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rate of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37 degrees-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.

Adenosine Triphosphatases↗
PubMed1980

Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates.

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5'-monophosphates. The purine nucleoside 5'-monophosphates are more effective than the pyrimidine homologues. 5'-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825--835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309--316].

Deoxycholic Acid↗
PubMed1980

Substrate kinetics of the alternative oxidase of Neurospora crassa.

The kinetics of the succinate oxidation by cyanide-sensitive and cyanide-insensitive submitochondrial particles of Neurospora crassa cells suggest that both respiratory pathways use the same complex II. This is confirmed by comparing the kinetics of the reductase activities of the isolated succinate-ubiquinone oxidoreductase (complex II) of cyanide-sensitive and cyanide-insensitive cells respectively. No alternative-oxidase activity was found to be associated with the isolated complex II of cyanide-insensitive cells.

Chloramphenicol↗
PubMed1980

Deletion mutants of Neurospora crassa mitochondrial DNA and their relationship to the "stop-start" growth phenotype.

"Stoppers" are a class of Neurospora crassa extranuclear mutants characterized by gross deficiencies of cytochromes b and aa3 and an unusual growth phenotype which involves irregular periods of growth andnongrowth. In the present work, mtDNAs from all four stopper mutants were found to contain deletions or insertions detectable by restriction enzyme analysis. [stp] mtDNA consists predominantly of defective molecules which retain a 16-megadalton segment (EcoRI-1, -4, and -6) of wild-type mtDNA (40 megadaltons). The other stopper mutants show smaller alterations: [stp A18t]-618, a 0.35-kilobase deletion in EcoRI-7b; [stp B2]-651, a 4-kilobase insertion in EcoRI-2; and [stp A]-574, a 5-kilobase deletion in EcoRI-2 and -10. Based on these results, we propose that "stop-start" growth results from competition between certain defective mtDNAs which have a tendency to predominate and low concentrations of less defective mtDNA species which must be retained to sustain growth. Three additional extranuclear mutants ("nonstoppers") have also been found to contain deletions in mtDNA. Remarkably, the defective mtDNA species in two of these mutants ([poky]H1-10 and [SG-3]-551) retain different sizes (18 and 13 megadlatons, respectively) of the same region retained in [stp] mtDNA (i.e., EcoRI-1, -4, and -6). The findings suggest that production of defective mtDNAs in Neurospora is nonrandom with a preferred mechanism leading to retention of this segment. It may be significant that the retained segment contains both mitochondrial rRNA genes and most mitochondrial tRNA genes. These deletion mutants may provide a tool for genetic mapping of Neurospora mtDNA.

DNA, Mitochondrial↗
PubMed1979

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.

Electron Transport Complex IV↗
PubMed1979

Cytochrome c oxidase subunits in nuclear and extranuclear cytochrome-aa3-deficient mutants of Neurospora crassa.

The mitochondria of cytochrome-aa3-deficient Neurospora crassa mutants were screened for the seven polypeptide constiuents of cytochrome c oxidase. The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits. With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. The results were as follows. 1. The mitochondria of the cya-2-8 and cya-3-16 nuclear mutants and the [exn-5] cytoplasmic mutant contained a protein complex immunoprecipitated by anti-holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides. Only the oxidase subunits 5 and 6 were immunoprecipitated by anti-holoenzyme antibody from the mitochondria of the cyt-2-1 and 299-1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation. 2. A 'subunit 1' polypeptide larger than the authentic subunit-1 polypeptide of wild-type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt-2-1, and 299-1 and the [mi-3] cytoplasmic mutant. This larger polypeptide may be an unprocessed precursor of the 'mature' subunit 1 protein of the holoenzyme. No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the [exn-5] cytoplasmic mutant and the cya-2-8 and cya-4-23 nuclear mutants. 3. A nuclear mutant, 299-1, lacks the mitochondrially synthesized subunit-2 polypeptide of cytochrome oxidase. When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants [exn-5], cya-2-8, cya-3-16 and cya-4-23 was lower than in mitochondria from wild-type. This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants. The cya-4-23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6. On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N. crassa cytochrome-aa3-deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme.

Cytochromes↗
PubMed1979

Conversion of a mitochondrial precursor polypeptide into subunit 1 of cytochrome oxidase in the mi-3 mutant of Neurospora crassa.

1. The cytochrome-alpha alpha 3-deficient mi-3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41 000-Mr subunit 1 of the wild-type enzyme. An apparent molecular weight of about 45 000 was estimated for the mutant product. 2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase. 3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45 000-Mr polypeptide is converted to a 41 000-Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse-chase labelling kinetics reveal a typical precursor product relationship. 4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase. A possible physiological role of the post-translational processing of the mitochondrially synthesized component is discussed.

Cross Reactions↗
PubMed1979

Analysis and exploration of age-related changes in mammalian structure and function.

Although there is a large body of data showing an age-associated decline in the physiological systems of mammals, there is a great need for more descriptive information on the morphological and functional changes that occur with age. A powerful approach for ascertaining the extent to which age-related physiological changes limit the functional capacity of mammals is to study the effects of age on the response to challenges. Food restriction that markedly increases the mean life span of rodents also provides a potentially effective experimental probe of the aging process. Results are reported from our current research on morphologic and functional changes during the total life span of rats and on the influence of food restriction on these age-related changes. The data from our study of the lean body mass, adipose tissue mass, and adipose tissue structure indicate that current dogmas need to be reexamined; moreover, they clearly show the need for and the power of life-long descriptive studies. Our research on the influence of food restriction on age-related changes in functional activity supports the hypothesis that one mechanism by which food restriction increases the life span of rats relates to the delay in time of onset and a change in chronologic course of physiologic decline.

Adipose Tissue↗
PubMed1978

A regulatory system controlling the production of cytochrome aa3 in Neurospora crassa.

The mitochondria of the cyt-2-1, cya-3-16, cya-4-23 and 299-1 nuclear mutants and the [mi-3] and [exn-5] cytoplasmic mutants of Neurospora crassa are deficient in cytochrome aa3, while the cyb-1-1 and cyb-2-1 mutants have mitochondrial b-cytochrome deficiencies. However, the mitochondria from cyb-1-1 cyt-2-1, cyb-1-1 [mi-3] and cyb-2-1 [mi-3] double mutants contain 30% to 50% of the amount of cytochrome aa3 that is present in mitochondria from wild-type; i.e. cyb-1-1 and cyb-2-2 act as suppressors of the cytochrome aa3 deficiency phenotypes that are associated with cyt-2-1 and [mi-3] mutations. The production of cytochrome aa3 can be induced in cyt-2-1 and [mi-3] by growing cells in medium containing antimycin A, an inhibitor of electron transport in the cytochrome bc1 segment of the mitochondrial electrontransport chain. Moreover, the growth of the [mi-3] mutant is strongly stimulated by low concentrations of antimycin A. The induction of cytochrome aa3 by antimycin treatments does not occur in [exn-5], cya-4-23 and 299-1 cells; but does take place in cya-3-16 cells. Although some of the seven constituent polypeptides of cytochrome aa3 are present in mitochondria of [mi-3], the holoenzyme complex is not formed in the mutant. In contrast, the mitochondria of cyb-1-1 [mi-3] and cyb-2-2 [mi-3] double mutants contain a fully assembled cytochrome oxidase complex as well as some unassembled subunit polypeptides. The observations are indicative of the existence of at least two regulatory systems controlling the production of cytochrome aa3. One of the circuits appears to control the basal or "constitutive" production of cytochrome oxidase, the other seems to coordinate the level of cytochrome aa3 with some function of the mitochondrial cytochrome bc1 complex, possibly electron transport.

Antimycin A↗
PubMed1978

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa3 have been isolated. When grown at 41 degrees C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa3 and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23 degrees C, they are virtually indistinguishable from wild-type strains. The mutants were selected on the basis of their slow growth at 41 degrees C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41 degrees c. The selecttion technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa3 may be altered in the mutants at the restrictive temperature.

Cold Temperature↗
PubMed1978

Nuclear suppressors of the [poky] cytoplasmic mutant in Neurospora crassa. III. Effects on other cytoplasmic mutants and on mitochondrial ribosome assembly in [poky].

We have previously isolated six non-allelic, nuclear mutations (sui loci) that partially suppress the growth, respiratory and cytochrome abnormalities of the extranuclear [poky] mutant. A comparison of the mitochondrial ribosome profiles of suppressed and unsuppressed [poky] strains revealed that five of the six suppressors alleviate at least partially the deficiency of mitochondrial small ribosomal subunits that is associated with the [poky] genotype. Six independently isolated Group 1 extranuclear mutants, namely [exn-1], [exn-2], [exn-4-a1, [stp-b1], [SG-1] and [SG-3-A1, which have growth and cytochrome phenotypes similar to [poky] also were found to be deficient in small subunits of mitochondrial ribosomes. Using cytochrome aa3 and b production as a criterion for mitochondrial protein synthesis, it could be shown that the nuclear su I suppressors of [poky] also suppress the other six Group I extranuclear mutants. However, differences in the efficiencies of suppression by suI suppressors suggest that at least some of Group I extrachromosomal mutants are not simply re-isolates of [poky], but represent distinct extranuclear mutations.

DNA, Mitochondrial↗