Bernard Dujon

Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure

The complete sequences of nine Saccharomyces cerevisiae mitochondrial introns, six of which carry long open reading frames, have already been published. We have recently determined the sequence of an intron in the large ribosomal mitochondrial RNA of Kluyveromyces thermotolerans (Jacquier et al., in preparation), which we found to be closely related to its S. cerevisiae counterpart. This latter result prompted us to undertake a systematic search for possible homologous elements in the other, available sequences with the help of an original computer program. A previously unsuspected wealth of evolutionarily conserved sequences and secondary structures was thus uncovered. Seven at least of the available sequences may be folded up into elaborate secondary structure models, the cores of which are nearly identical. These models result in bringing together the exon-intron junctions into relatively close spatial proximity and looping out either all or most of the sequences in open reading frame, when present. These results and their possible implications with respect to the mechanism of splicing are discussed in the light of available genetic and biochemical data.

Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region.

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR–proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans–hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

Genetic and physical characterization of a segment of yeast mitochondrial DNA involved in the control of genetic recombination

Genetic recombination between the 3 RIB (ribosomal) loci of yeast mitochondrial DNA is under the control of a mitochondrial locus named omega (with alleles omega+ and omega-) which is tightly linked to the RIBI locus. We have attempted to elucidate the molecular mechanisms(s) involved by using rho- mutants with similar (RIBI+ RIB2+ RIB3(0) genotype but different recombination properties in rho- x rho+ crosses. These were obtained through pedigree analysis and their mitochondrial DNAs were mapped on a high resolution physical map of the RIB section that had been built by analysis of thermal denaturation profiles and electron microscopy of partially denatured molecules. By comparison of physical and genetic data it can be shown that possession of the omega+ allele by the rho- cell is not sufficient for its expression in crosses, some additional DNA segments(s) in the ribosomal region being needed. This result and several features of the rho+ x rho- crosses are discussed in the light of current concepts in mitochondrial genetics of yeast and the recently discovered fact that omega+ and omega- strains differ by the presence of a 1000 base pairs insertion in the former.

Sequence of a mitochondrial plasmid of sunflower (Helianthus annuus) and its relationship to other mitochondrial plasmids

Abstract A circular plasmid called plT is found in mitochondria of sunflower ( Helianthus annuus ). Its nucleotide sequence has been determined and analyzed in order to understand its possible role and origin. The nucleotide sequence exhibits a 159 base pair region with highly organized repeats. The sequence of plT shows no major homology with other plasmids of higher plants although organized patterns are present in the circular plasmids. We also report the existence of two related mitochondrial plasmids in a line of H. annuus and in the H. petiolaris fallax species. In addition, plT plasmid has been detected in total cellular DNA of male-sterile sunflower with a copy number of a hundred times lower than in the male-fertile one.

[5] In Vitro fragmentation of yeast chromosomes and yeast artificial chromosomes at artificially inserted sites and applications to genome mapping

Publisher Summary Techniques for large-scale genome mapping, cloning, and sequencing have been developed in the bakers yeast, Saccharomyces cerevisiae , a particularly suitable model and tool for such experiments for several reasons. The discovery of endonucleases cutting very rarely (meganucleases) and encoded by mobile group I introns has permitted to develop new tools for genome mapping that are particularly well suited to the case of yeast chromosomes and yeast artificial chromosomes (YACs). The chapter discusses the use of intron-encoded endonucleases for site-directed chromosomal fragmentation and its applications to the physical mapping of intact yeast chromosomes, YACs, or fragments thereof. The methods described in the chapter include the integration of I- Sce I recognition sites in yeast chromosomes and in YACs, the preparation of yeast DNA in agarose plugs for PFGE after I- Sce I and/or I- Ppo I digestion, the purification of chromosome fragments and the labeling of chromosomal DNA probes in agarose plugs, and the hybridization of cosmid, plasmid, or λ clones with chromosome fragments. The examples of the applications of such methods are also summarized in the chapter.

The hierarchical approach to the DNA stability problem: II. — Some applications and speculations with yeast mitochondrial DNA as an example

As discussed in the preceding article [1] hierarchical analysis of DNA sequences should make it possible to treat complex unfolding (and refolding) processes involving both equilibrium and non-equilibrium subtransitions. Hence a variety of actual experimental situations may be analyzed. This is demonstrated with the help of a 1950 bp yeast mitochondrial DNA sequence encompassing part of the 21S ribosomal RNA gene: excellent fit of complex denaturation and renaturation profiles is achieved with only two adjustable parameters. The advantage of dealing with objectively defined stability units is also apparent when stability profiles are compared to known functional maps: striking correlations may be brought out and their possible significance is briefly discussed.