No abstract

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA‐type transcription factor with the type IVb zinc finger DNA‐binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over‐expressed, sexual‐specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).

We isolated the ganB gene encoding the G␣-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPAI (also known as SCG1), recently shown to be highly homologous to genes encoding the a subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.G proteins represent a group of highly homologous proteins involved in receptor-mediated signal transduction (see reference 50 for a review). They apparently serve as informational transducers between a diverse group of cell surface receptors and an equally diverse assortment of second message effectors. In vertebrates, G proteins have been found to be ubiquitous in terms of their tissue and phylogenetic distribution.The a subunits of G proteins are part of a larger family of guanyl nucleotide-binding proteins, all of which show some primary structure homology, particularly in domains thought to bind GTP and to be responsible for GTP hydrolysis (30,51). Other members of this family include the ras proteins (10), discovered originally as the products of viral oncogenes, and the rho proteins (32), which were identified because of homology to members of the ras family. Although a role in signal transduction is suspected, the functions of members of the ras and rho protein families in higher organisms are not known. With the goal of applying genetic approaches to the resolution of this issue, homologs of both the ras (6) and rho (33) families have been identified in Saccharomyces cerevisiae. It has been determined that ras and rho homologs in S. cerevisiae perform essential functions (33,41) and that at least one of the functions of the yeast RAS proteins is to regulate adenylate cyclase (3, 55), consistent with a role for such proteins in signal transduction. Recently a gene encoding a protein highly homologous to the a subunit of mammalian G proteins, for which a role in signal transduction has already been established, was identified in S. cerevisiae (7,37 This study " For strains congenic with 381G. only markers different from the background are given. The same convention has been used with respect to the W303-1B background.b Strains carrying the ste3::LEU2 insertion mutation were constructed by fragment-mediated gene conversion (45) (MATa) was superimposed by replic...

heterotrimeric G protein-coupled cell surface receptor (encoded by STE3 or STE2). Many of the other components of this transduction pathway are known. The induction of mating pheromone-specific genes occurs through the action of the STE12 gene product as a consequence of its binding to a pheromone-specific transcription-activating sequence known as the pheromone response element (11,14). Several of these pheromone-specific genes are known to be involved in the mating process. In contrast, the mechanism by which the same pathway results in G1-specific cell cycle arrest is not understood. Although STE12 function has been implicated in this arrest, the nature of its involvement is not known (10).Cell cycle progression in budding yeasts is known to require the activity of the CDC28 gene product (20,34,35), a serine/threonine protein kinase of the Cdk (cyclin-dependent kinase) family, which includes the Cdc2 protein kinase (reviewed by Pines and Hunter [32]). The function of the CDC28 gene product is essential for passage through the G1/S and G2/M transitions. Its role at each of these transitions is performed in conjunction with those of distinct families of cyclin proteins, the G2/M function requiring B-type cyclins encoded by the CLB genes (16,40) and the role during G1 phase requiring the G1 cyclins encoded by the CLN genes. The CLN gene family consists of three genes, CLN1, CLN2, and CLN3, which perform an overlapping function that is essential for progression through G1 phase (5,18,27,36

No abstract

In examining the production of valuable compounds by marine microorganisms, we isolated a novel yeast strain that produces a large amount of squalene and several polyunsaturated fatty acids. Molecular and phylogenetic analyses of the ribosomal DNA suggest that the isolate belongs to the genus Pseudozyma, which comprises ustilaginomycetous anamorphic yeasts. The nucleotide sequence of an internally transcribed spacer region from isolate Pseudozyma sp. JCC207 showed 98% similarity with those of Pseudozyma rugulosa and Pseudozyma aphidis, which are close relatives of the isolate. In considering use of Pseudozyma sp. JCC207 for squalene production, the efficiency of squalene production was investigated under different conditions. Glucose was the best carbon source for the production of squalene. In the presence of yeast extract, squalene production was activated and an optimum ratio of glucose to yeast extract was 4.5. For the optimal squalene production, the concentration of glucose was 40 g l(-1) and the best nitrogen source was sodium nitrogen. Pseudozyma sp. JCC207 was shown to produce up to 5.20 g/L of biomass and 340.52 mg/L of squalene. In an optimal condition, the content and yield of squalene produced by Pseudozyma sp. JCC207 were much greater than those obtained from microorganisms previously reported as squalene producers. We identified, classified, and characterized Pseudozyma sp. JCC207 as a novel squalene producer. The squalene production rate of Pseudozyma sp. JCC207 makes it an ideal candidate for the commercialization of microbial squalene.

Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hülle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.