Publications by authors named "Peter Philippsen"

33 Publications

Mechanism of nuclear movements in a multinucleated cell.

Mol Biol Cell 2017 Mar 11;28(5):645-660. Epub 2017 Jan 11.

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Multinucleated cells are important in many organisms, but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungus , nuclei move back and forth, occasionally bypassing each other, preventing the formation of nuclear clusters. This is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei that are pulled by dynein motors anchored at the cortex. Using three-dimensional stochastic simulations with parameters constrained by the literature, we predict the cortical anchor density from the characteristics of nuclear movements. The model accounts for the complex nuclear movements seen in vivo, using a minimal set of experimentally determined ingredients. Of interest, these ingredients power the oscillations of the anaphase spindle in budding yeast, but in , this system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.
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http://dx.doi.org/10.1091/mbc.E16-11-0806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328623PMC
March 2017

Organization of organelles within hyphae of Ashbya gossypii revealed by electron tomography.

Eukaryot Cell 2013 Nov 14;12(11):1423-32. Epub 2013 Jun 14.

European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.
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http://dx.doi.org/10.1128/EC.00106-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837942PMC
November 2013

Genomes of Ashbya fungi isolated from insects reveal four mating-type loci, numerous translocations, lack of transposons, and distinct gene duplications.

G3 (Bethesda) 2013 Aug 7;3(8):1225-39. Epub 2013 Aug 7.

Duke Institute of Genome Sciences and Policy, Duke University, Durham, North Carolina 27710, USA.

The filamentous fungus Ashbya gossypii is a cotton pathogen transmitted by insects. It is readily grown and manipulated in the laboratory and is commercially exploited as a natural overproducer of vitamin B2. Our previous genome analysis of A. gossypii isolate ATCC10895, collected in Trinidad nearly 100 years ago, revealed extensive synteny with the Saccharomyces cerevisiae genome, leading us to use it as a model organism to understand the evolution of filamentous growth. To further develop Ashbya as a model system, we have investigated the ecological niche of A. gossypii and isolated additional strains and a sibling species, both useful in comparative analysis. We isolated fungi morphologically similar to A. gossypii from different plant-feeding insects of the suborder Heteroptera, generated a phylogenetic tree based on rDNA-ITS sequences, and performed high coverage short read sequencing with one A. gossypii isolate from Florida, a new species, Ashbya aceri, isolated in North Carolina, and a genetically marked derivative of ATCC10895 intensively used for functional studies. In contrast to S. cerevisiae, all strains carry four not three mating type loci, adding a new puzzle in the evolution of Ashbya species. Another surprise was the genome identity of 99.9% between the Florida strain and ATCC10895, isolated in Trinidad. The A. aceri and A. gossypii genomes show conserved gene orders rearranged by eight translocations, 90% overall sequence identity, and fewer tandem duplications in the A. aceri genome. Both species lack transposable elements. Finally, our work identifies plant-feeding insects of the suborder Heteroptera as the most likely natural reservoir of Ashbya, and that infection of cotton and other plants may be incidental to the growth of the fungus in its insect host.
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http://dx.doi.org/10.1534/g3.112.002881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737163PMC
August 2013

Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii.

J Cell Sci 2012 Dec 26;125(Pt 23):5830-9. Epub 2012 Sep 26.

European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.
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http://dx.doi.org/10.1242/jcs.111005DOI Listing
December 2012

Regulation of exit from mitosis in multinucleate Ashbya gossypii cells relies on a minimal network of genes.

Mol Biol Cell 2011 Sep 7;22(17):3081-93. Epub 2011 Jul 7.

Department of Molecular Microbiology, Biozentrum, University of Basel, 4056 Basel, Switzerland.

In Saccharomyces cerevisiae, mitosis is coupled to cell division by the action of the Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN) regulatory networks, which mediate exit from mitosis by activation of the phosphatase Cdc14. The closely related filamentous ascomycete Ashbya gossypii provides a unique cellular setting to study the evolution of these networks. Within its multinucleate hyphae, nuclei are free to divide without the spatial and temporal constraints described for budding yeast. To investigate how this highly conserved system has adapted to these circumstances, we constructed a series of mutants lacking homologues of core components of MEN and FEAR and monitored phenomena such as progression through mitosis and Cdc14 activation. MEN homologues in A. gossypii were shown to have diverged from their anticipated role in Cdc14 release and exit from mitosis. We observed defects in septation, as well as a partial metaphase arrest, in Agtem1Δ, Agcdc15Δ, Agdbf2/dbf20Δ, and Agmob1Δ. A. gossypii homologues of the FEAR network, on the other hand, have a conserved and more pronounced role in regulation of the M/G1 transition. Agcdc55Δ mutants are unable to sequester AgCdc14 throughout interphase. We propose a reduced model of the networks described in yeast, with a low degree of functional redundancy, convenient for further investigations into these networks.
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http://dx.doi.org/10.1091/mbc.E10-12-1006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164456PMC
September 2011

Clustering of nuclei in multinucleated hyphae is prevented by dynein-driven bidirectional nuclear movements and microtubule growth control in Ashbya gossypii.

Eukaryot Cell 2011 Jul 3;10(7):902-15. Epub 2011 Jun 3.

Department of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, Basel, Switzerland.

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.
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http://dx.doi.org/10.1128/EC.05095-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147418PMC
July 2011

Evolution of multinucleated Ashbya gossypii hyphae from a budding yeast-like ancestor.

Fungal Biol 2011 Jun 26;115(6):557-68. Epub 2011 Feb 26.

Universität Osnabrück, Institut für Genetik, Barbarastr. 11, 49076 Osnabrück, Germany.

In the filamentous ascomycete Ashbya gossypii polarity establishment at sites of germ tube and lateral branch emergence depends on homologues of Saccharomyces cerevisiae factors controlling bud site selection and bud emergence. Maintenance of polar growth involves homologues of well-known polarity factors of budding yeast. To achieve the much higher rates of sustained polar surface expansion of hyphae compared to mainly non-polarly growing yeast buds five important alterations had to evolve. Permanent presence of the polarity machinery at a confined area in the rapidly expanding hyphal tip, increased cytoplasmic space with a much enlarged ER surface for generating secretory vesicles, efficient directed transport of secretory vesicles to and accumulation at the tip, increased capacity of the exocytosis system to process these vesicles, and an efficient endocytosis system for membrane and polarity factor recycling adjacent to the zone of exocytosis. Morphological, cell biological, and molecular aspects of this evolution are discussed based on experiments performed within the past 10 y.
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http://dx.doi.org/10.1016/j.funbio.2011.02.015DOI Listing
June 2011

Formation and stability of eisosomes in the filamentous fungus Ashbya gossypii.

J Cell Sci 2011 May 26;124(Pt 10):1629-34. Epub 2011 Apr 26.

Biozentrum, University of Basel, Klingelbergstraße 50-70, CH 4056 Basel, Switzerland.

One hallmark of the rapid expansion of the polar surface of fungal hyphae is the spatial separation of regions of exocytosis and endocytosis at hyphal tips, as recently shown for Ashbya gossypii and Aspergillus nidulans. To determine where cortex-associated eisosomes form with respect to these two regions, we monitored fluorescently marked eisosomes in A. gossypii. Each minute, 1.6 ± 0.5 eisosomes form within the first 30 μm of each hypha and are exclusively subapical of the endocytosis region. This spatial separation of the processes of eisosome formation and endocytosis, and the much lower frequency of eisosome formation compared with that of endocytic vesicle production do not support a recently proposed role for eisosomes in endocytosis. Levels of mRNA encoding eisosome components are tenfold higher in spores than in hyphae, explaining the observed higher eisosome density at the cortex of germ bubbles. As in Saccharomyces cerevisiae, eisosomes in A. gossypii are very stable. In contrast to S. cerevisiae, however, the A. gossypii homologue of Pil1, one of the main eisosome subunits, is very important for polar growth, whereas the homologue of Nce102, which colocalizes with eisosomes, is not needed for eisosome stability. By testing partial deletions of the A. gossypii homologue of Ymr086w, another component of the eisosome, we identified a novel protein domain essential for eisosome stability. We also compare our results with recent findings about eisosomes in A. nidulans.
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http://dx.doi.org/10.1242/jcs.082487DOI Listing
May 2011

Dynamics of multiple nuclei in Ashbya gossypii hyphae depend on the control of cytoplasmic microtubules length by Bik1, Kip2, Kip3, and not on a capture/shrinkage mechanism.

Mol Biol Cell 2010 Nov 15;21(21):3680-92. Epub 2010 Sep 15.

Department of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.
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http://dx.doi.org/10.1091/mbc.E10-06-0527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965685PMC
November 2010

Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum.

Eukaryot Cell 2010 Aug 11;9(8):1216-24. Epub 2010 Jun 11.

Departamento de Genética, Universidad de Córdoba, Edificio Gregor Mendel, Campus de Rabanales, 14071 Córdoba, Spain.

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.
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http://dx.doi.org/10.1128/EC.00040-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2918926PMC
August 2010

Structural mutants of the spindle pole body cause distinct alteration of cytoplasmic microtubules and nuclear dynamics in multinucleated hyphae.

Mol Biol Cell 2010 Mar 6;21(5):753-66. Epub 2010 Jan 6.

Department of Molecular Microbiology, Biozentrum University of Basel, Basel, Switzerland.

In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Delta, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Delta and Agcnm67Delta lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Delta only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.
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http://dx.doi.org/10.1091/mbc.e09-07-0555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2828962PMC
March 2010

Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii.

Mol Biol Cell 2010 Jan 12;21(1):18-28. Epub 2009 Nov 12.

Department of Molecular Microbiology, Biozentrum University of Basel, 4056 Basel, Switzerland.

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 microm/min), rotations (up to 180 degrees in 30 s), and long-range nuclear bypassing (up to 9 microm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis-oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.
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http://dx.doi.org/10.1091/mbc.e09-01-0063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801712PMC
January 2010

Of bars and rings: Hof1-dependent cytokinesis in multiseptated hyphae of Ashbya gossypii.

Mol Cell Biol 2009 Feb 24;29(3):771-83. Epub 2008 Nov 24.

Biozentrum, Basel, Switzerland.

We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.
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http://dx.doi.org/10.1128/MCB.01150-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630676PMC
February 2009

Growth-speed-correlated localization of exocyst and polarisome components in growth zones of Ashbya gossypii hyphal tips.

J Cell Sci 2008 Dec 4;121(Pt 23):3878-89. Epub 2008 Nov 4.

Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland.

We use the fungus Ashbya gossypii to investigate how its polar growth machinery is organized to achieve sustained hyphal growth. In slowly elongating hyphae exocyst, cell polarity and polarisome proteins permanently localize as cortical cap at hyphal tips, thus defining the zone of secretory vesicle fusion. In tenfold faster growing hyphae, this zone is only slightly enlarged demonstrating a capacity of hyphal growth zones to increase rates of vesicle processing to reach higher speeds. Concomitant with this increase, vesicles accumulate as spheroid associated with the tip cortex, indicating that a Spitzenkörper forms in fast hyphae. We also found spheroid-like accumulations for the exocyst components AgSec3, AgSec5, AgExo70 and the polarisome components AgSpa2, AgBni1 and AgPea2 (but not AgBud6 or cell polarity factors such as AgCdc42 or AgBem1). The localization of AgSpa2, AgPea2 and AgBni1 depend on each other but only marginally on AgBud6, as concluded from a set of deletions. Our data define three conditions to achieve fast growth at hyphal tips: permanent presence of the polarity machinery in a confined cortical area, organized accumulation of vesicles and a subset of polarity components close to this area, and spatial separation of the zones of exocytosis (tip front) and endocytosis (tip rim).
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http://dx.doi.org/10.1242/jcs.033852DOI Listing
December 2008

The Paxillin-like protein AgPxl1 is required for apical branching and maximal hyphal growth in A.gossypii.

Fungal Genet Biol 2008 Jun 25;45(6):829-38. Epub 2008 Mar 25.

Molecular Microbiology, Biozentrum der Universität Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.

The development from young, slowly growing hyphae to fast growing hyphae in filamentous fungi is referred to as hyphal maturation. We have identified the Paxillin-like protein AgPxl1 in Ashbyagossypii as a developmental protein that is specifically required for hyphal maturation. The early development of A.gossypii strains lacking AgPxl1 is indistinguishable from wild-type. However, at later developmental stages the maximal hyphal extension rate is less than half compared to wild-type and apical branching is affected. Apical branching is characterised as the symmetric division of fast growing hyphal tips resulting in two sister hyphae. In Agpxl1Delta strains two thirds of the apical branching events lead to asymmetric sister hyphae where growth of one branch is either completely aborted or slowed down while extension of the other branch is not affected. This suggests that AgPxl1 plays a role in the organisation of growth and efficient division of growth upon apical branching in mature mycelia. The conserved C-terminal LIM domains are necessary for AgPxl1 function and also contribute to tip localisation. AgCLA4, a PAK-like kinase, is epistatic to AgPXL1 and robust localisation of AgPxl1 depends on AgCla4. This suggests that AgCla4 acts upstream of AgPxl1.
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http://dx.doi.org/10.1016/j.fgb.2008.03.010DOI Listing
June 2008

Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex.

Nat Cell Biol 2007 Jul 3;9(7):788-96. Epub 2007 Jun 3.

Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we discovered an unexpected role for yeast dynein light chain (Dyn2) in the NPC. Dyn2 is a previously undescribed nucleoporin that functions as molecular glue to dimerize and stabilize the Nup82-Nsp1-Nup159 complex, a module of the cytoplasmic pore filaments. Biochemical analyses showed that Dyn2 binds to a linear motif (termed DID(Nup159)) inserted between the Phe-Gly repeat and coiled-coil domain of Nup159. Electron microscopy revealed that the reconstituted Dyn2-DID(Nup159) complex forms a rigid rod-like structure, in which five Dyn2 homodimers align like 'pearls on a string' between two extented DID(Nup159) strands. These findings imply that the rigid 20 nm long Dyn2-DID(Nup159) filament projects the Nup159 Phe-Gly repeats from the Nup82 module. Thus, it is possible that dynein light chain plays a role in organizing natively unfolded Phe-Gly repeats within the NPC scaffold to facilitate nucleocytoplasmic transport.
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http://dx.doi.org/10.1038/ncb1604DOI Listing
July 2007

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

BMC Genomics 2007 Jan 9;8. Epub 2007 Jan 9.

Biozentrum & Swiss Institute of Bioinformatics, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.

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 http://agd.vital-it.ch/.
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http://dx.doi.org/10.1186/1471-2164-8-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1779777PMC
January 2007

The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells.

Eukaryot Cell 2007 Feb 8;6(2):182-97. Epub 2006 Dec 8.

University of Basel Biozentrum, Molecular Microbiology, Klingelbergstrasse 0/70, 4056 Basel, Switzerland.

Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.
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http://dx.doi.org/10.1128/EC.00364-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797942PMC
February 2007

Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells.

Eukaryot Cell 2007 Mar 22;6(3):473-86. Epub 2006 Nov 22.

Department of Molecular Microbiology, Biozentrum University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland.

Cyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in A. gossypii, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in A. gossypii to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in A. gossypii are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells.
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http://dx.doi.org/10.1128/EC.00273-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828934PMC
March 2007

The SH3/PH domain protein AgBoi1/2 collaborates with the Rho-type GTPase AgRho3 to prevent nonpolar growth at hyphal tips of Ashbya gossypii.

Eukaryot Cell 2006 Oct 1;5(10):1635-47. Epub 2006 Sep 1.

Molecular Microbiology, Biozentrum der Universität Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.

Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.
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http://dx.doi.org/10.1128/EC.00210-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1595331PMC
October 2006

Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast.

Dev Cell 2006 Apr;10(4):425-39

Institute of Biochemistry, Biology Department, Swiss Federal Institute of Technology, ETH Zürich, 8093 Zürich, Switzerland.

The orientation of the mitotic spindle plays a key role in determining whether a polarized cell will divide symmetrically or asymmetrically. In most cell types, cytoplasmic dynein plays a critical role in spindle orientation. However, how dynein directs opposite spindle poles toward distinct and predetermined cell ends is poorly understood. Here, we show that dynein distributes preferentially to the spindle pole bodies (SPB) and astral microtubules (MTs) proximal to the bud in metaphase yeast cells. Dynein asymmetry depended on the bud neck kinases Elm1, Hsl1, and Gin4, on the spindle pole components Cnm67 and Cdk1, and on the B-type cyclins Clb1 and Clb2. Furthermore, phenotypic and genetic studies both indicated that dynein is unable to orient the spindle when it localizes to both poles and associated microtubules. Together, our data indicate that proper orientation of the spindle requires dynein to act on a single spindle pole.
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http://dx.doi.org/10.1016/j.devcel.2006.02.018DOI Listing
April 2006

Asynchronous nuclear division cycles in multinucleated cells.

J Cell Biol 2006 Jan;172(3):347-62

Department of Molecular Microbiology, Biozentrum University of Basel, 4056 Basel, Switzerland.

Synchronous mitosis is common in multinucleated cells. We analyzed a unique asynchronous nuclear division cycle in a multinucleated filamentous fungus, Ashbya gossypii. Nuclear pedigree analysis and observation of GFP-labeled spindle pole bodies demonstrated that neighboring nuclei in A. gossypii cells are in different cell cycle stages despite close physical proximity. Neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and mitotic cyclins were present regardless of cell cycle stage, suggesting that the complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking NH(2)-terminal destruction box sequences did not block cell cycle progression. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of a nuclear division cycle in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins.
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http://dx.doi.org/10.1083/jcb.200507003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063645PMC
January 2006

From function to shape: a novel role of a formin in morphogenesis of the fungus Ashbya gossypii.

Mol Biol Cell 2006 Jan 19;17(1):130-45. Epub 2005 Oct 19.

Biozentrum der Universität Basel, 4056 Basel, Switzerland.

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.
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http://dx.doi.org/10.1091/mbc.e05-06-0479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1345653PMC
January 2006

Homologues of yeast polarity genes control the development of multinucleated hyphae in Ashbya gossypii.

Curr Opin Microbiol 2005 Aug;8(4):370-7

Applied Microbiology, Biozentrum, University of Basel, Klingelbergstr. 50-70, CH-4056 Basel, Switzerland.

A few years ago, A. gossypii became recognized as an attractive model to study the growth of long and multinucleated fungal cells (hyphae) because of its small genome, haploid nuclei, and efficient gene targeting methods. It is generally assumed that a better understanding of filamentous fungal growth will greatly stimulate the development of novel fungicides. The use of Ashbya gossypii as a model is particularly promising because of the high level of gene order conservation (synteny) between the genomes of A. gossypii and the yeast Saccharomyces cerevisiae. Thus, a similar set of genes seems to control the surprisingly different growth modes of these two organisms, which predicts that orthologous growth control genes might not play identical cellular roles in both systems. Analyzing the phenotypes of A. gossypii mutants lacking factors with known functions in yeast morphogenesis and nuclear dynamics confirm this hypothesis. Comparative genomics of both organisms also reveals rare examples of differences in the gene sets for some cellular processes, which as shown for phosphate homeostasis can be associated with differences in control levels.
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http://dx.doi.org/10.1016/j.mib.2005.06.021DOI Listing
August 2005

Contribution of the endoplasmic reticulum to peroxisome formation.

Cell 2005 Jul;122(1):85-95

Department of Cellular Protein Chemistry, University of Utrecht, Padualaan 8, NL-3548 CH Utrecht, The Netherlands.

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.
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http://dx.doi.org/10.1016/j.cell.2005.04.025DOI Listing
July 2005

The Ashbya Genome Database (AGD)--a tool for the yeast community and genome biologists.

Nucleic Acids Res 2005 Jan;33(Database issue):D348-52

Department of Applied Microbiology, Biozentrum, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.

The Ashbya Genome Database (AGD) is a comprehensive online source of information covering genes from the filamentous fungus Ashbya gossypii. The database content is based upon comparative genome annotation between A.gossypii and the closely related budding yeast Saccharomyces cerevisiae taking both sequence similarity and synteny (conserved order and orientation) into account. Release 2 of AGD contains 4718 protein-encoding loci located across seven chromosomes. Information can be retrieved using systematic or standard locus names from A.gossypii as well as budding and fission yeast. Approximately 90% of the genes in the genome of A.gossypii are homologous and syntenic to loci of budding yeast. Therefore, AGD is a useful tool not only for the various yeast communities in general but also for biologists who are interested in evolutionary aspects of genome research and comparative genome annotation. The database provides scientists with a convenient graphical user interface that includes various locus search and genome browsing options, data download and export functionalities and numerous reciprocal links to external databases including SGD, MIPS, GeneDB, KEGG, GermOnline and Swiss-Prot/TrEMBL. AGD is accessible at http://agd.unibas.ch/.
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http://dx.doi.org/10.1093/nar/gki009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC539963PMC
January 2005

A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii.

Mol Biol Cell 2004 Oct 28;15(10):4622-32. Epub 2004 Jul 28.

Department of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.
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http://dx.doi.org/10.1091/mbc.e04-02-0104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC519154PMC
October 2004

The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome.

Science 2004 Apr 4;304(5668):304-7. Epub 2004 Mar 4.

Biozentrum der Universität Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.

We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.
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http://dx.doi.org/10.1126/science.1095781DOI Listing
April 2004

Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii.

Mol Biol Cell 2003 Oct 22;14(10):4140-54. Epub 2003 Aug 22.

Applied Microbiology, Biozentrum, Universitaet Basel, 4056 Basel, Switzerland.

We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.
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http://dx.doi.org/10.1091/mbc.e03-03-0167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207006PMC
October 2003

Reinvestigation of the Saccharomyces cerevisiae genome annotation by comparison to the genome of a related fungus: Ashbya gossypii.

Genome Biol 2003 25;4(7):R45. Epub 2003 Jun 25.

Institute of Applied Microbiology, Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Background: The recently sequenced genome of the filamentous fungus Ashbya gossypii revealed remarkable similarities to that of the budding yeast Saccharomyces cerevisiae both at the level of homology and synteny (conservation of gene order). Thus, it became possible to reinvestigate the S. cerevisiae genome in the syntenic regions leading to an improved annotation.

Results: We have identified 23 novel S. cerevisiae open reading frames (ORFs) as syntenic homologs of A. gossypii genes; for all but one, homologs are present in other eukaryotes including humans. Other comparisons identified 13 overlooked introns and suggested 69 potential sequence corrections resulting in ORF extensions or ORF fusions with improved homology to the syntenic A. gossypii homologs. Of the proposed corrections, 25 were tested and confirmed by resequencing. In addition, homologs of nearly 1,000 S. cerevisiae ORFs, presently annotated as hypothetical, were found in A. gossypii at syntenic positions and can therefore be considered as authentic genes. Finally, we suggest that over 400 S. cerevisiae ORFs that overlap other ORFs in S. cerevisiae and for which no homolog can be detected in A. gossypii should be regarded as spurious.

Conclusions: Although, the S. cerevisiae genome is rightly considered as one of the most accurately sequenced and annotated eukaryotic genomes, we have shown that it still benefits substantially from comparison to the completed sequence and syntenic gene map of A. gossypii, an evolutionarily related fungus. This type of approach will strongly support the annotation of more complex genomes such as the human and murine genomes.
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http://dx.doi.org/10.1186/gb-2003-4-7-r45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC193632PMC
July 2003