Berl R. Oakley

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

Abstract We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

γ-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans

We have recently discovered that the mipA gene of A. nidulans encodes gamma-tubulin, a new member of the tubulin superfamily. To determine the function of gamma-tubulin in vivo, we have created a mutation in the mipA gene by integrative transformation, maintained the mutation in a heterokaryon, and determined the phenotype of the mutation in spores produced by the heterokaryon. The mutation is lethal and recessive. It strongly inhibits nuclear division, less strongly inhibits nuclear migration, and, as judged by immunofluorescence microscopy, causes a reduction in the number and length of cytoplasmic microtubules and virtually a complete absence of mitotic apparatus. We conclude that gamma-tubulin is essential for microtubule function in general and nuclear division in particular. Immunofluorescence microscopy of wild-type hyphae with affinity-purified, gamma-tubulin-specific antibodies reveals that gamma-tubulin is a component of interphase and mitotic spindle pole bodies. We propose that gamma-tubulin attaches microtubules to the spindle pole body, nucleates microtubule assembly, and establishes microtubule polarity in vivo.

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuAΔ) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuAΔ and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

Centrosome-independent mitotic spindle formation in vertebrates

BACKGROUND In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.

γ-Tubulin is present in Drosophila melanogaster and homo sapiens and is associated with the centrosome

The mipA gene of A. nidulans encodes a newly discovered member of the tubulin superfamily of proteins, gamma-tubulin. In A. nidulans, gamma-tubulin is essential for nuclear division and microtubule assembly and is associated with the spindle pole body, the fungal microtubule organizing center. By low stringency hybridizations we have cloned cDNAs from D. melanogaster and H. sapiens, the predicted products of which share more than 66% amino acid identity with A. nidulans gamma-tubulin. gamma-Tubulin-specific antibodies stained centrosomes of Drosophila, human, and mouse cell lines. Staining was most intense in prophase through metaphase when microtubule assembly from centrosomes was maximal. These results demonstrate that gamma-tubulin genes are present and expressed in humans and flies; they suggest that gamma-tubulin may be a universal component of microtubule organizing centers; and they are consistent with an earlier hypothesis that gamma-tubulin is a minus-end nucleator of microtubule assembly.

Chromatin-level regulation of biosynthetic gene clusters

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

The Tip Growth Apparatus of Aspergillus nidulans

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 Spitzenkörper (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 mum 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.

Nuclear movement is β-tubulin-dependent in Aspergillus nidulans

Abstract We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case ( ben A15) and super-sensitivity in another ( ben A16). Nuclear movement was resistant to benomyl in the strain carrying ben A15 and super-sensitive in the strain carrying ben Al6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nud A-E, were analyzed genetically and found not to be allelic to the ben A (β-tubulin) tubA α-tubulin genes.

A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans

The genome sequencing of Aspergillus species including A. nidulans reveals that the products of many of the secondary metabolism pathways in these fungi have not been elucidated. Our examination of the 27 polyketide synthases (PKS) in A. nidulans revealed that one highly reduced PKS (HR-PKS, AN1034.3) and one nonreduced PKS (NR-PKS, AN1036.3) are located next to each other in the genome. Since no known A. nidulans secondary metabolites could be produced by two PKS enzymes, we hypothesized that this cryptic gene cluster produces an unknown natural product. Indeed after numerous attempts we found that the products from this cluster could not be detected under normal laboratory culture conditions in wild type strains. Closer examination of the gene cluster revealed a gene with high homology to a citrinin biosynthesis transcriptional activator (CtnR, 32% identity/47% similarity), a fungal transcription activator located next to the two PKSs. We replaced the promoter of the transcription activator with the inducible alcA promoter, which enabled the production of a novel polyketide that we have named asperfuranone. A series of gene deletions has allowed us to confirm that the two PKSs together with five additional genes comprise the asperfuranone biosynthetic pathway and leads us to propose a biosynthetic pathway for asperfuranone. Our results confirm and substantiate the potential to discover novel compounds even from a well-studied fungus by using a genomic mining approach.