The FFAG R&D and medical application project RACCAM

JACoW web site http://accelconf.web.cern.ch/AccelConf/e06/Pre-Press/WEPCH161.pdf WEPCH161International audienceThe RACCAM project (Recherche en ACCelerateurs et Applications Medicales) has recently obtained fundings, extending over three years (2006-2008), from the French National Research Agency (ANR). RACCAM is a tripartite collaboration, involving (i) the CNRS Laboratory IN2P3/LPSC, (ii) the French magnet industrial SIGMAPHI, and (iii) the nuclear medecine Departement of Grenoble Hospital. The project concerns fixed field alternating gradient accelerator (FFAG) research on the one hand, and on the other hand their application as hadrontherapy and biology research machines. RACCAM's goal is three-fold, (i) participate to the on-going international collaborations in the field of FFAGs and recent concepts of "non-scaling" FFAGs, with frames for instance, the Neutrino Factory (NuFact) and the EMMA project of an electron model of a muon FFAG accelerator, (ii) design, build and experiment a prototype of an FFAG magnet proper to fulfil the requirements of rapid cycling acceleration, (iii) develop the concepts, and show the feasibility, of the application of such FFAG beams to hadrontherapy and to biology research

Evolutionary dynamics of hAT DNA transposon families in Saccharomycetaceae

Transposable elements (TEs) are widespread in eukaryotes but uncommon in yeasts of the Saccharomycotina subphylum, in terms of both host species and genome fraction. The class II elements are especially scarce, but the hAT element Rover is a noteworthy exception that deserves further investigation. Here, we conducted a genome-wide analysis of hAT elements in 40 ascomycota. A novel family, Roamer, was found in three species, whereas Rover was detected in 15 preduplicated species from Kluyveromyces, Eremothecium, and Lachancea genera, with up to 41 copies per genome. Rover acquisition seems to have occurred by horizontal transfer in a common ancestor of these genera. The detection of remote Rover copies in Naumovozyma dairenensis and in the sole Saccharomyces cerevisiae strain AWRI1631, without synteny, suggests that two additional independent horizontal transfers took place toward these genomes. Such patchy distribution of elements prevents any anticipation of TE presence in incoming sequenced genomes, even closely related ones. The presence of both putative autonomous and defective Rover copies, as well as their diversification into five families, indicate particular dynamics of Rover elements in the Lachancea genus. Especially, we discovered the first miniature inverted-repeat transposable elements (MITEs) to be described in yeasts, together with their parental autonomous copies. Evidence of MITE insertion polymorphism among Lachancea waltii strains suggests their recent activity. Moreover, 40% of Rover copies appeared to be involved in chromosome rearrangements, showing the large structural impact of TEs on yeast genome and opening the door to further investigations to understand their functional and evolutionary consequences

Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3′-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment

Whole-Genome Sequencing and Intraspecific Analysis of the Yeast Species Lachancea quebecensis

The gold standard in yeast population genomics has been the model organism Saccharomyces cerevisiae. However, the exploration of yeast species outside the Saccharomyces genus is essential to broaden the understanding of genome evolution. Here, we report the analyses of whole-genome sequences of nineisolates from the recently described yeast species Lachancea quebecensis. The genome of one isolate was assembled and annotated, and the intraspecific variability within L. quebecensis was surveyed by comparing the sequences from the eight other isolates to this reference sequence. Our study revealed that these strains harbor genomes with an average nucleotide diversity of pi = 2 x 10(-3) which is slightly lower, although on the same order of magnitude, as that previously determined for S. cerevisiae (pi = 4 x 10(-3)). Our results show that even though these isolates were all obtained from a relatively isolated geographic location, the same ecological source, and represent a smaller sample size than is available for S. cerevisiae, the levels of divergence are similar to those observed in this model species. This divergence is essentially linked to the presence of two distinct clusters delineated according to geographic location. However, even with relatively similar ranges of genome divergence, L. quebecensis has an extremely low global phenotypic variance of 0.062 compared with 0.59 previously determined in S. cerevisiae

Génolevures: protein families and synteny among complete hemiascomycetous yeast proteomes and genomes

The Génolevures online database (http://cbi.labri.fr/Genolevures/ and http://genolevures.org/) provides exploratory tools and curated data sets relative to nine complete and seven partial genome sequences determined and manually annotated by the Génolevures Consortium, to facilitate comparative genomic studies of Hemiascomycete yeasts. The 2008 update to the Génolevures database provides four new genomes in complete (subtelomere to subtelomere) chromosome sequences, 50 000 protein-coding and tRNA genes, and in silico analyses for each gene element. A key element is a novel classification of conserved multi-species protein families and their use in detecting synteny, gene fusions and other aspects of genome remodeling in evolution. Our purpose is to release high-quality curated data from complete genomes, with a focus on the relations between genes, genomes and proteins

RIPCAL: a tool for alignment-based analysis of repeat-induced point mutations in fungal genomic sequences

Background Repeat-induced point mutation (RIP) is a fungal-specific genome defence mechanism that alters the sequences of repetitive DNA, thereby inactivating coding genes. Repeated DNA sequences align between mating and meiosis and both sequences undergo C:G to T:A transitions. In most fungi these transitions preferentially affect CpA di-nucleotides thus altering the frequency of certain di-nucleotides in the affected sequences. The majority of previously published in silico analyses were limited to the comparison of ratios of pre- and post-RIP di-nucleotides in putatively RIP-affected sequences – so-called RIP indices. The analysis of RIP is significantly more informative when comparing sequence alignments of repeated sequences. There is, however, a dearth of bioinformatics tools available to the fungal research community for alignment-based RIP analysis of repeat families. Results We present RIPCAL http://www.sourceforge.net/projects/ripcal, a software tool for the automated analysis of RIP in fungal genomic DNA repeats, which performs both RIP index and alignment-based analyses. We demonstrate the ability of RIPCAL to detect RIP within known RIP-affected sequences of Neurospora crassa and other fungi. We also predict and delineate the presence of RIP in the genome of Stagonospora nodorum – a Dothideomycete pathogen of wheat. We show that RIP has affected different members of the S. nodorum rDNA tandem repeat to different extents depending on their genomic contexts. Conclusion The RIPCAL alignment-based method has considerable advantages over RIP indices for the analysis of whole genomes. We demonstrate its application to the recently published genome assembly of S. nodorum

Short interspersed nuclear element (SINE) sequences in the genome of the human pathogenic fungus Aspergillus fumigatus Af293.

Copyright: © 2016 The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kanhayuwa L, Coutts RHA (2016) Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293. PLoS ONE 11(10): e0163215. https://doi.org/10.1371/journal.pone.0163215.Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.Peer reviewedFinal Published versio

Competitive Repair by Naturally Dispersed Repetitive DNA during Non-Allelic Homologous Recombination

Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR–dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR–dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer