Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III2/IV in yeast or I/III2/IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes.

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina.

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.

Recombinant mitochondrial DNA molecules suggest a template switching ability for group-II-intron reverse transcriptase.

Degenerative processes in the filamentous fungus Podospora anserina are strongly correlated with the instability of the mitochondrial genome. Among the sources of instability is the mobile group-II intron COX1-i1, also called intron alpha, which encodes a protein with a reverse transcriptase activity. In this paper we characterize, through PCR experiments, mitochondrial recombinant DNA molecules joining the 5' end of intron alpha to the 3' end of tRNA sequences including the CCA motif. The structure of these junctions led us to propose that they were most probably initiated by a RNA template switching of the reverse transcriptase encoded in COX1-i1. This activity might be involved in a number of mitochondrial rearrangements occurring in degenerative syndromes and in some long-lived mutants.

Intron open reading frames as mobile elements and evolution of a group I intron.

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF-containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.

Plasticity of the mitochondrial genome in Podospora. Polymorphism for 15 optional sequences: group-I, group-II introns, intronic ORFs and an intergenic region.

The mitochondrial chromosome of 15 Podospora anserina and one Podospora comata wild-type strains have been extensively examined for the presence of optional elements and for sequence divergence. Among the P. anserina strains, nine optional sequences were found. By comparing P. anserina with the closely related and weakly interfertile P. comata species, six additional optional sequences were detected. These optional elements correspond to mitochondrial introns belonging to different groups and subgroups (11 cases), intronic open reading frames (two cases), a complex insert and an intergenic region. Although difficult to explain, the distribution of optional mitochondrial sequences among the 15 wild-type isolates of P. anserina is far from random.

Mitochondrial intronic open reading frames in Podospora: mobility and consecutive exonic sequence variations.

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group 1 intronic ORFs are mobile elements and that their transfer, and concomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.

The S12 ribosomal protein of Podospora anserina belongs to the S19 bacterial family and controls the mitochondrial genome integrity through cytoplasmic translation.

In the filamentous fungus Podospora anserina, two nuclear genes are involved in the premature death syndrome associated with a site-specific deletion of the mitochondrial DNA: a mutant allele of the AS1 gene, encoding the cytoplasmic ribosomal protein S12, and an uncharacterized gene closely linked to the mating-type locus. We describe here the cloning and the sequencing of the wild-type and two mutant alleles of the AS1 gene. The P. anserina S12 protein belongs to the bacterial S19 ribosomal protein family and shows 72% identity with the S15 human ribosomal protein. Transformation experiments have shown that the AS1-4 mutation itself is responsible for the premature death phenotype and that it corresponds to a Gly to Asp change in the highly conserved COOH-terminal part of the protein. Use of antibodies directed against S12 did not permit detection of the mutant ribosomal protein inside the mitochondria. However, cross-reactions were observed with at least one mitochondrial ribosomal protein displaying a higher molecular weight than S12. The mitochondrial protein does not seem to be a by-product of the AS1 gene but is more likely the mitochondrial homologue of S12. These results strongly suggest that the mutant S12 protein acts indirectly to promote the mitochondrial deletion, via the cytoplasmic translation.

The in vivo use of alternate 3'-splice sites in group I introns.

Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used.

Transposition of a group II intron.

Among mobile genetic elements, self-splicing introns are of particular interest. They belong to either group I or group II depending on their three-dimensional structure. Homing, the systematic intron invasion of an intronless gene when it encounters its homologous intron-bearing allele, is the only means for intron mobility so far demonstrated. It depends on the activity of the intron-encoded protein and is very specific for the acceptor site. Intron transposition, the transfer of an intron to a novel site, predicted on the basis of phylogenetic studies and in vitro reverse-splicing experiments, has been proposed to be responsible for evolutionary intron spreading. Here we present results from polymerase chain reaction experiments consistent with transposition of a group II intron. This event is proposed to account for the site-specific deletion in the mitochondrial chromosome of the fungus Podospora anserina that is associated with the premature death syndrome and might also be involved in the senescence process affecting this species.

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina.

In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron alpha) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron alpha, was detected in five senescent subclones which all showed strong amplification of the intronic alpha sequence (Sen DNA alpha).

Conditions required for activation of the mouse albumin or alpha-fetoprotein gene in hybrids between mouse lymphoblastoma and rat hepatoma cells.

Activation of two previously silent mouse hepatic genes has been investigated in hybrid cells between pseudodiploid mouse lymphoblastoma cells and hyperdiploid or hypertetraploid rat hepatoma cells. In this material, activation of the mouse albumin gene is a frequent event, whereas activation of mouse alpha-fetoprotein (AFP) occurs only in those cells that produce large amounts of albumin. Quantitative tests of hybrid populations for the activated proteins and their mRNAs revealed the expected sizes and structures: moreover, as in hepatoma cells, the amount of both rat and mouse albumin produced was directly proportional to the intracellular concentration of the corresponding mRNA. The cellular environment required for activation of the liver-specific genes was investigated by cell-by-cell analysis of each hybrid clone. Immunostaining for the presence of rat and mouse albumin and mouse AFP revealed unexpected heterogeneity in the phenotypes of the hybrid populations, which were found to contain cells that: (a) failed to express either of the proteins; (b) produced all three; (c) produced both rat and mouse albumin; or (d) produced rat albumin only. Karyotypic analysis indicated that the hybrid-cell phenotype depended on parental chromosome ratios rather than absolute numbers of chromosomes. It was found for albumin and mouse AFP that the fraction of immunostained cells was equal to the fraction of metaphases that contained a minimal rat-to-mouse chromosome ratio of 2.5 and 9, respectively. It is concluded that in those hybrids, expression of liver-specific genes is regulated by extinguishers, but in a dose-dependent fashion, suggesting the intervention of antagonistic activators from the rat hepatoma chromosomes.

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.

Activation of a silent gene is accompanied by its demethylation.

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat.

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions.

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.

Coordinate secretion of rat and mouse albumin by mouse hepatoma x rat hepatoma hybrid cells directly reflects the intracellular concentration of the corresponding mRNAs.

The ratio of mouse to rat albumin secreted by mouse hepatoma x rat hepatoma hybrid cells is constant (of the order of 5.0) irrespective of the total amounts produced. The present results establish for seven independent hybrid clones that the coordination in the ratio of mouse to rat product applies also at the level of accumulation of albumin mRNAs of the two species. The interpretation that coordinate synthesis reflects coordinate transcription of the relevant genes is thus reinforced.