Agnès Thierry

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant.

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.