Cells of the dimorphic yeast Candida albicans are easily induced to germinate in synchrony. Using germinating cells of strain FC18, we examined the effects of several drugs that are known to affect the cytoskeleton on growth and School o f Medicine, Showaku, Nagoya 466, Japan cytoskeletal organization. Cytochalasin A (CA), an inhibitor of actin function, inhibited the germination of the yeast cells and changed the cylindrical expansion of the apex of the germ tube to swelling growth. Effects of CA on the organization of actin were examined with rhodamine-phalloidin (Rh-Ph), which specifically stains F-actin. In CA-untreated cells, Rh-Ph staining resulted in condensed dot-like fluorescence a t the growing tip, as well as filamentous fluorescence (actin cables) that ran from the apex t o the basal region. In CA-treated cells, condensed dot-like fluorescence was still observed a t the swelling tip, but actin cables had disappeared completely. This result indicates that CA does not affect the asymmetrical distribution of actin, and suggests that the actin cables are not required for maintenance of the polarized localization of actin. Benomyl, an anti-microtubule drug, inhibited the germination of yeast cells and the apical growth of germinated cells. Benomyl not only disrupted microtubules (MTs), but also affected the distribution of actin. In benomyl-treated cells, actin dots were randomly dispersed all over the cell. This result indicates that benomyl destroyed the mechanism that maintains the asymmetrical distribution of actin, and suggests that MTs are involved in such a mechanism. The polarized localization of organelles is one of the most important factors associated with dimorphism. Our data suggest that the cytoskeleton, composed of actin and MTs, is involved in the control of polarity in the hyphal growth of C. ahicans, and that actin and MTs are interrelated in the establishment of polarity.

In vitro experiments show that ␥ -tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta , and microtubules can be repolymerized from the isolated plastids. ␥ -Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that ␥ -tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.

Summary Fungal secondary metabolites (SMs) are extremely important in medicine and agriculture, but regulation of their biosynthesis is incompletely understood. We have developed a genetic screen in Aspergillus nidulans for negative regulators of fungal SM gene clusters and we have used this screen to isolate mutations that upregulate transcription of the non-ribosomal peptide synthetase gene required for nidulanin A biosynthesis. Several of these mutations are allelic and we have identified the mutant gene by genome sequencing. The gene, which we designate mcrA, is conserved but uncharacterized, and it encodes a putative transcription factor. Metabolite profiles of mcrA deletant, mcrA overexpressing, and parental strains reveal that mcrA regulates at least ten SM gene clusters. Deletion of mcrA stimulates SM production even in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of these organisms. Deleting mcrA in a genetic dereplication strain has allowed us to discover two novel compounds as well as an antibiotic not known to be produced by A. nidulans. Deletion of mcrA upregulates transcription of hundreds of genes including many that are involved in secondary metabolism, while downregulating a smaller number of genes.

Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus.Chromatin is one of the hallmarks of eukaryotes. The eukaryotic genome DNA is complexed with chromosomal proteins to form the chromatin structure in the nucleus. Nuclear reactions such as DNA replication, transcription, DNA repair, and recombination take place on the chromatin and are regulated by the disruption and assembly of chromatin, namely chromatin remodeling. Recently, a variety of chromatin remodeling factors has been identified, including histone modification enzymes and ATP-dependent factors (reviewed in references 1, 2, 22, and 42). The histone chaperone family is one of the chromatin remodeling factors that binds to core histones and facilitates assembly and remodeling of the chromatin in an ATP-independent manner. This family includes various kinds of proteins, such as nucleoplasmin (31) and N1/N2 (28) in Xenopus, nucleosome assembly protein 1 (designated Nap1 in this paper) and Nap2 (13, 48), template-activating factor I␣

Microtubule plus-end tracking proteins (؉TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how ؉TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G 1 phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of ؉TIPs in cell cycle transition.Microtubule dynamics are essential in many cellular processes, including cell motility, intracellular transport, accurate mitosis, and cytokinesis in all eukaryotes. The regulatory factors for microtubule dynamics can be classified into two main types as follows: microtubule-destabilizing proteins, such as stathmin/Op18 (1) and the Kinesin-13 family (also known as MCAK/KIF2 family) (2), and microtubule-stabilizing proteins, the classic superfamily of microtubule-associated proteins (3). Additionally, the plus-end tracking proteins (ϩTIPs) 3 have recently been identified; this family specifically accumulates at the ends of growing microtubules and regulates the microtubule plus-end targeting to the cell cortex or mitotic kinetochores (4, 5). The EB1 family is a member of the ϩTIPs family and consists of three homologs in mammals: EB1, EB2/RP1 (henceforth, EB2), and EB3 (6). As EB1 was originally identified as a protein that interacts with the well characterized tumor suppressor adenomatous polyposis coli (APC) protein (7), the function of EB1 has been investigated extensively. EB1 interacts with other ϩTIPs, including APC, p150 glued , CLIPs, and CLASP1/2, and the interaction network controls microtubule orientation and microtubule-cortex interaction during cell migration (5, 8, 9). EB1 functions not only in the regulation of interphase microtubule dynamics but also in mitotic spindle regulation. For accurate chromosomal segregation, sister chromatids become aligned to the metaphase plate during metaphase, and the alignment requires spindle-kinetochore attachment. Two models have been proposed; in the first, termed the "search-and-capture" model, EB1 localized at the growing microtubule plusends searches for binding partners located on kinetochores (10, 11). In the second model proposed recently, EB1 makes kinetochore fibers and centrosomal microtubules connect, and it is essential for the formation of a functional bipolar spindle (12). Thus, EB1 is thought to be a master controller of microtubule plus-ends; however, little is known about other EB1 family members. Given that EB3 is localized ...

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Fungi are a major source of valuable bioactive secondary metabolites (SMs). These compounds are synthesized by enzymes encoded by genes that are clustered in the genome. The vast majority of SM biosynthetic gene clusters are not expressed under normal growth conditions, and their products are unknown. Developing methods for activation of these silent gene clusters offers the potential for discovering many valuable new fungal SMs. While a number of useful approaches have been developed, they each have limitations and additional tools are needed. One approach, upregulation of SM gene cluster-specific transcription factors that are associated with many SM gene clusters, has worked extremely well in some cases, but it has failed more often than it has succeeded. Taking advantage of transcription factor domain modularity, we have developed a new approach. We have fused the DNA-binding domain of a transcription factor associated with a silent SM gene cluster with the activation domain of a robust SM transcription factor, AfoA. Expression of this hybrid transcription factor activated transcription of the genes in the target cluster and production of the antibiotic (+)-asperlin. Deletion of cluster genes confirmed that the cluster is responsible for (+)-asperlin production, and we designate it the aln cluster. Separately, co-induction of expression of two aln cluster genes revealed the pathway intermediate (2Z,4Z,6E)-octatrienoic acid, a compound with photoprotectant properties. Our findings demonstrate the potential of our novel synthetic hybrid transcription factor strategy to discover the products of other silent fungal SM gene clusters.

We determined the complete nucleotide sequence of a 2.1-kb HindIII-EcoRI fragment that was cloned from a resident large plasmid of Klebsiella pneumoniae Chedid, a highly virulent and mucoviscous strain of the O1:K2 serotype. This fragment encoded an ability to enhance K2 capsular polysaccharide synthesis in K. pneumoniae, and a 636-bp open reading frame (rmpA2) was found. The 411-bp rmpA reported to be involved in the virulence and mucoid phenotypes of K. pneumoniae by Nassif et al. (Mol. Microbiol. 3:1349-1359, 1989) was a part of rmpA2. Eighty percent homology in nucleotide sequence was found between rmpA2 and rmpA in the corresponding regions. The central domain of the deduced amino acid sequence of RmpA2 showed considerable homology to the central domains of NtrC of K. pneumoniae and Escherichia coli, to which the sigma factor of RNA polymerase binds. The C-terminal domain of RmpA2 also demonstrated considerable homology with the putative helix-turn-helix motifs of LuxR of Vibrio fischeri and FixJ of Rhizobium meliloti. Moreover, RmpA2 also showed some homology in its N- and C-terminal regions to those of RcsA, a transcriptional activator for colanic acid synthesis in E. coli. On the other hand, a sequence upstream of rmpA2 was found to be highly homologous to insertion sequence 3 of members of the family Enterobacteriaceae. Southern hybridization analysis suggested that rmpA2 exists on the large plasmids of all mucoviscous virulent K2 strains but not on those of the slightly mucoviscous avirulent strains. Freeze substitution electron microscopy and fluorescent-antibody staining with anti-K2 serum revealed that K. pneumoniae Chedid has a dense and thick capsule (180 nm) with dense extracapsular substance, whereas K. pneumoniae K2-215, one of the slightly mucoviscous and avirulent strains, has a capsule which is looser and thinner (120 nm) than that of strain Chedid and no extracapsular substance. Introduction of rmpA2 into K2-215 as well as reference strains K. pneumoniae K9 and K72 resulted in a change of the colony phenotype to highly mucoviscous through abundant production of extracapsular substance which reacted with anti-K2, -K9, or -K72, respectively, as did their parental strains. From these results, it is suggested that RmpA2 belongs to the family of transcriptional regulators and confers a highly mucoviscous phenotype on cells of various serotypes of K. pneumoniae by enhancing extracapsular polysaccharide synthesis.