François Michel

Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis

Alignment of the 87 available sequences of group I self-splicing introns reveals numerous instances of covariation between distant sites. Some of these covariations cannot be ascribed to historical coincidences or the known secondary structure of group I introns, and are, therefore, best explained as reflecting tertiary contacts. With the help of stereochemical modelling, we have taken advantage of these novel interactions to derive a three-dimensional model of the conserved core of group I introns. Two noteworthy features of that model are its extreme compactness and the fact that all of the most evolutionarily conserved residues happen to converge around the two helices that constitute the substrate of the core ribozyme and the site that binds the guanosine cofactor necessary for self-splicing. Specific functional implications are discussed, both with regard to the way the substrate helices are recognized by the core and possible rearrangements of the introns during the self-splicing process. Concerning potential long-range interactions, emphasis is put on the possible recognition of two consecutive purines in the minor groove of a helix by a GAAA or related terminal loop.

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

SummaryRecently, the nucleotide sequences for three “mitochondrial plasmids” associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed ε senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian “URF1” gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.

Physical and genetic organization of petite and grande yeast mitochondrial DNA: III. High resolution melting and reassociation studies☆

Mitochondrial DNAs from 13 petite mutants have been analyzed by means of high-resolution melting and reassociation in solution, in an attempt to relate their physical chemical properties to the mitochondrial genotype, which displays various combinations of genetic marker deletions. The kinetic complexities of the petite mtDNAs were found to range from 13 down to 1500 of that of the grande mtDNA; the loss in sequential complexity undergone by petite mtDNAs parallels the loss in mitochondrial genetic complexity. Melting profiles of petite mtDNAs can be resolved into well-defined peaks. Some of them are specific to the marker genes. The genotypic specificity increases as the sequential complexity of mtDNA decreases. The mtDNA region conferring resistance to erythromycin could in this way be shown to be characterized by two melting peaks, at 72 °C and 75 °C. The results are interpreted in terms of selective enrichment in gene-specific sequences.

Incipient mitochondrial evolution in yeasts: II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution☆

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.

Physical and genetic organization of petite and grande yeast mitochondrial DNA: II. DNA-DNA hybridization studies and buoyant density determinations☆☆☆

Abstract Buoyant density of mitochondrial DNA from 14 cytoplasmic petite mutants issued from the same grande yeast Saccharomyces cerevisiae was determined. Mutants that have retained the mitochondrial gene conferring resistance to erythromycin displayed higher buoyant density, while mutants that have retained the mitochondrial gene conferring resistance to chloramphenicol displayed lower buoyant density. It is inferred that the segment which carries the ER gene has a higher G + C content than the segment which carries the CR gene. DNA-DNA filter hybridizations were carried out systematically in different reciprocal pair-wise combinations between mtDNAs purified from various mutants and from the grande. All petites were found to be deleted in 42 to 93% of the grande sequence, depending on the mutant studied. Sequence homology between petite mtDNAs was greatest in mutants retaining common genetic markers and was least when different genetic markers were retained. Practically no hybridization was found between some CREO and COER mutants. Correlations established between the extent of DNA-DNA hybridization, kinetic and genetic complexity show that a selective enrichment of gene specific sequences occurs in mtDNA of petites.

Sequence of the mitochondrial gene encoding subunit I of cytochrome oxidase in Saccharomyces douglasii

We have determined the complete sequence of the mitochondrial (mt) gene (COXI) coding for cytochrome oxidase subunit I of Saccharomyces douglasii. This gene is 7238 bp long and includes four introns. The salient feature of the S. douglasii COXI gene is the presence of two introns, Sd.ai1 and Sd.ai2, which have not been observed in S. cerevisiae genes. Both are group-I introns and are located at novel positions compared with the S. cerevisiae COXI. Interestingly, one of these introns (the second one) is inserted at the same position as intron 2 of COXI of Kluyveromyces lactis and also as intron 8 of the same gene in Podospora anserina. The ORFs contained in these three introns display a high degree of similarity. Comparisons of exonic and intronic sequences of the COXI of two Saccharomyces species reinforces our previous conclusions: the evolution of mt genes in yeast obeys different rules to those found in vertebrates.

Divergence in mtDNA and Effects in Interspecific Combinations of Nuclear and Mitochondrial Genomes in the Yeast Genus Saccharomyces

Yeast is an exceptional material for the study of the cytochromes of the respiratory chain. In the species Saccharomyces cerevisiae both nuclear and mitochondrial genetic maps are well defined with numerous mutants available. As this organism is a facultative aerobe it is possible to obtain mutations interrupting the respiratory chain without any lethal effect. In the First International Symposium on Plant Mitochondria held in 1978 it was reported on the modifications of mitochondrial translation products in cytochrome b deficient mutants of S. cerevisiae, which suggested a mosaic organisation for the cytochrome b structural genel. It has since been demonstrated that as many as 6 exons and 5 introns may comprise this mitochondrial gene named cob-box2, 3, 4. About 200 mutations have been mapped on this mitochondrial DNA segment and span about 7300 base pairs4, i.e. roughly seven times more than needed to specify the cytochrome b apoprotein sequence.