We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkö rper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 m behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position. INTRODUCTIONPolarized cell growth occurs in most eukaryotic phyla, and it includes a plethora of important phenomena, such as neuronal growth cone extension in animals and pollen tube extension in vascular plants. It is particularly important in filamentous fungi where nearly all growth occurs by hyphal tip extension (reviewed by Momany, 2002). Given that some filamentous fungi are important fermentation organisms, the growth of which is of considerable economic importance, whereas others are serious plant, animal, and human pathogens, there is considerable interest in the mechanisms of tip growth in these organisms.A great deal of progress has been made in understanding fungal tip growth (summarized by Harris et al., 2005; Steinberg, 2007a,b;Riquelme et al., 2007), but key questions remain unanswered. There is general agreement that fungal tip growth involves the synthesis of cell wall components in the cell body, the incorporation of these components into vesicles, the transport of these vesicles to the cell tip, the fusion of these vesicles with the plasma membrane in the area of the cell tip (exocytosis) to release their contents, and the cross-linking of the components after release. It is clear that both microtubules and actin microfilaments play important roles in fungal tip growth, but their exact functions are not yet defined. The exact sites of exocytosis and endocytosis also remain to be determined. The positions of the site(s) of exocytosis are particularly important because fungal walls are relatively stiff structures, and once they have formed the shape of the hypha is established. Hyphal shape is thus determined to a very significant extent by where the wall precursors are released from the cytoplasm, i.e., by the positioning of the site(s) of exocyto...

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape.

Background:The hallmark of Parkinson disease is ␣-synuclein aggregation, whose cytotoxic effects are reproducible in yeast. Results: ␣-Synuclein aggregate clearance is impaired by vacuolar protease inhibitor PMSF but not by proteasome inhibitor MG132. Conclusion: Yeast recovers from ␣-synuclein-induced toxicity by clearing aggregates via autophagy and vacuolar pathways. Significance: The data provide insight into the mechanisms used by yeast cells to clear toxic aggregated proteins.

a b s t r a c tThe identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.