Ashbya Genome Database 3.0: a cross-species genome and transcriptome browser for yeast biologists

BACKGROUND: The Ashbya Genome Database (AGD) 3.0 is an innovative cross-species genome and transcriptome browser based on release 40 of the Ensembl developer environment. DESCRIPTION: AGD 3.0 provides information on 4726 protein-encoding loci and 293 non-coding RNA genes present in the genome of the filamentous fungus Ashbya gossypii. A synteny viewer depicts the chromosomal location and orientation of orthologous genes in the budding yeast Saccharomyces cerevisiae. Genome-wide expression profiling data obtained with high-density oligonucleotide microarrays (GeneChips) are available for nearly all currently annotated protein-coding loci in A. gossypii and S. cerevisiae. CONCLUSION: AGD 3.0 hence provides yeast- and genome biologists with comprehensive report pages including reliable DNA annotation, Gene Ontology terms associated with S. cerevisiae orthologues and RNA expression data as well as numerous links to external sources of information. The database is accessible at

Transcriptional profiling of the model organism "A. gossypii" : comparison of life cycle stages and transcription factor deletions

Experiments described in this PhD thesis used for the � rst time custom-made oligonucleotide chips to substantially increase our knowledge and understanding of the model organism A. gossypii. Four sets of experiments of different scales were performed: (1) a transcriptome analysis of two strains at different developmental stages (2) a comparison of the cell wall proteome and transcriptome (3) a bioinformatics search for conserved promoter elements in target genes of the presumptive mating and � lamentation signaling network (4) a transcriptome analysis in strains lacking the transcriptional repressor AgDig1/2 or the transcriptional activators AgSte12 and AgTec1. Chapters 1 to 4 and Appendix 1 and 2 describe in detail the steps of duplicate target preparations and data quality checks (chapter 1) followed by the presentation and discussion of transcriptional pro� les at eight developmental stages: spores and four time points of germination up to bipolar germlings (chapter 2), two stages of hyphal high speed growth, one in liquid medium, the other on agar medium (chapter 3 and appendix 1), and � nally sporulation at limiting nutrient conditions (chapter 4). For these experiments mRNA was isolated from a double auxotrophic derivative (Agleu2�thr4� called �l�t) of the sequenced A. gossypii strain. Transcription pro� les were also determined for germination and high speed hyphae of strain FDAG, a novel natural isolate (Appendix 2). Key results are: (1) very high expression of cell wall genes and genes of unknown function (2) very different expression pro� les of a novel cell wall gene family with similarity to the single copy S. cerevisiae CWP1 gene (3) sporulation-speci� c expression of a novel gene family with similarity to the single gene copy S. cerevisiae HSP26 gene (4) a high percentage (10/20) of homologs to S. cerevisiae twin genes among the most abundant transcripts in spores (5) lack of ribosomal protein transcripts among the top 100 most abundant mRNAs in spores followed by coordinated up-regulation in young germlings (6) an apparently relaxed glucose catabolite repression, because high speed hyphae on agar employ both pathways, glycolysis and gluconeogenesis including a highly active glyoxylate cycle (7) a 150-fold up-regulation of an A. gossypii speci� c gene (NOHBY712) in high speed hyphae on agar medium (8) an up-regulation of a phosphatidyl-inositol-P phosphatase (INP54) in fast speed hyphae indicating substantial changes in membrane composition (9) one third unknown genes among the 15 most down-regulated genes in high-speed hyphae (10) two up-regulated A. gossypii speci� c genes of unknown function at sporulation (11) one highly expressed histone 3 gene during sporulation (also highly abundant mRNA in spores) pointing to substantial gene silencing during germination. Chapter 5 describes results from a collaboration with Frans M. Klis and Piet de Groot (University of Amsterdam) to characterize the cell wall proteome of A. gossypii. Two highly expressed but so far unknown genes were identi� ed as cell wall genes and a complete correlation between 14 cell wall proteins and high expression was established except for AgCCW12, which based on its predicted amino acid sequence, could not yield a tryptic peptide for detection in mass spectrometry. Chapter 6 concerns the comparison of genes controlling the mating and � lamentation pathways in S. cerevisiae with orthologous genes in A. gossypii. To shed light on the functional conservation of the two pathways including transcriptional regulation, a bioinformatics analysis was carried out. This analysis looked into conservation of transcription factor binding sites in promoters of target genes of the two pathways. To experimentally investigate regulatory network conservation, the two transcription factors AgTec1 and AgSte12 and their repressor AgDig1/2 were deleted. The key results are: (1) Components of the mating and � lamentation pathway are highly conserved as concluded from sequence comparison between A. gossypii and S. cerevisiae. However the transcription factors and the repressor seem to have evolved faster during the 100 million years since the separation of the A. gossypii and the S. cerevisiae lineage. (2) The Ste12 binding sites were conserved in 13 out of 18 promoters, which suggest that AgSte12 regulates a similar set of genes as ScSte12. (3) The Tec1 binding sites were conserved in only 7 out of 20 promoters which might suggest that AgTec1 has a (partially) different role and the regulatory network has been rewired during evolution. (4) Mycelia deleted for AgDIG1/2 showed a reduced maximal growth speed of 121 �m/h, which accounts for 67% of wild type growth speed. In addition the colony surface of Agdig1/2� was altered, and no spores were formed in this mutant. (5) Mycelia deleted for AgTec1 grew invasively after 10 days on full medium plates. The average nuclear distance in Agtec1� had nearly doubled compared to wild type. Similar phenotypes were observed in wild type mycelia in response to glucose limitation. (6) Mycelia deleted for AgSTE12 did not show a changed phenotype under conditions where the mating cascade was not induced by external stimuli. Chapter 7 describes the differential gene expression in strains lacking the transcriptional repressor AgDig1/2 or the transcriptional activators AgSte12 and AgTec1. The � fteen top upand down-regulated genes are discussed. To support and/or extent the single gene analysis the activity of prede� ned groups of genes was scored with t-pro� ler (Boorsma et al, 2005). The key results for DIG1/2 are: (1) AgDIG1/2 has a similar role as ScDIG1/2 which is to repress mating genes and � lamentation genes. It also represses a set of genes with S. cerevisiae homologs of unknown function. (2) AgPRY1/PRY2, a gene with homology to the plant PR-1 class of pathogen related proteins, was among the top � fteen up-regulated genes in Agdig1/2� and among the top15 down-regulated genes in Agste12�. This expression pattern suggest a possible role of this gene in mating. (3) Glyoxylate cycle genes were signi� cantly down-regulated in Agdig1/2�. Either the deletion of AgDIG1/2 down-regulates these genes preventing hyphae to reach fast growth speeds on glucose plates. Or, the deletion of AgDIG1/2 leads to an induction of mating genes thus causing a “cell cycle arrest”. (4) Meiosis and sporulation genes were down-regulated in Agdig1/2� which was consistent with the observed sporulation defect. (5) Ribosomal protein genes were down-regulated in Agdig1/2�. The key results for TEC1 are: (1) Thiamine biosynthesis genes and genes involved in utilization of alternative carbon sources were up-regulated in Agtec1�. (2) Ribosome biosynthesis and assembly genes, in particular genes involved in snoRNA binding, processing and maturation of pre-rRNA, were down-regulated in Agtec1�. This included the genes AgNOG1, AgNOP7 and AgRLP24 whose S. cerevisiae homologs code for proteins that form a complex which is tethered to the nucleolus by nutrient depletion causing cessation of late stages of ribosome biogenesis (Honma et al., 2006). (3) TOR (target of rapamycin) broadly controls ribosome biogenesis and in S. cerevisiae the ScNOG1/NOP7/RLP24 complex. Therefore we hypothesize that components of the TOR pathway are upstream of AgTec1 and that AgTec1 is a transcription factor that contributes to control of ribosome biogenesis via AgNOG1, possibly through the putative AgNog1/Nop7/Rlp24 complex. (4) In well fed polarly growing germlings, AgTec1 has a dual role as it acts as a repressor on the expression of genes involved in utilization of alternative carbon sources and thiamine biosynthesis and as an activator on the expression of ribosome biosynthesis genes. The key results for STE12 are: (1) Even under conditions where the mating cascade was not induced, AgSTE12 played a role in the cell. Genes whose expression was positively affected by deletion of AgSTE12 were mainly genes involved in amino acid biosynthesis