The putative Cryptococcus neoformans pheromone receptor gene CPR␣ was isolated and studied for its role in mating and filamentation. CPR␣ is MAT␣ specific and located adjacent to STE12␣ at the MAT␣ locus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-proteincoupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of the CPR␣ gene exhibited drastic reductions in mating efficiency but were not completely sterile. ⌬cpr␣ cells displayed wild-type mating efficiency when reconstituted with the wild-type CPR␣ gene. Hyphal production on filament agar was not affected in the ⌬cpr␣ strain, indicating no significant role for CPR␣ in sensing environmental cues during haploid fruiting. The wild-type MAT␣ CPR␣ strain produced abundant hyphae in response to synthetic MATa pheromone; however, the hyphal response to pheromone by ⌬cpr␣ cells was significantly reduced. Exposure of wild-type cells to synthetic MATa pheromone for 2 h induced MF␣ pheromone expression, whereas unexposed cells showed only basal levels of the MF␣ transcript. The ⌬cpr␣ cells, however, exhibited only basal levels of MF␣ message with or without pheromone exposure, suggesting that CPR␣ and MF␣ are components of the same signaling pathway.Cryptococcus neoformans is the etiologic agent of cryptococcosis, one of the most serious fungal diseases encountered by immunocompromised patients worldwide (26). The fungus is a bipolar heterothallic basidiomycete in which the meiotic cycle is dependent upon interactions between cells of the MAT␣ and MATa types (24). The initial interaction between cells of the two compatible mating strains is believed to involve pheromone-receptor pairs of both mating types (34). In Saccharomyces cerevisiae, MATa cells secrete an a-factor pheromone and express the ␣-factor receptor Ste2p whereas MAT␣ cells secrete an ␣-factor pheromone and express the a-factor receptor Ste3p. Both receptors belong to a large class of G-proteinlinked seven-transmembrane-domain receptors (reviewed in references 23, 29, and 40) and are expressed mostly at the tips of schmoos, where cell fusions subsequently occur (23). Binding of pheromone to the receptor induces the pheromone response signal transduction pathway via the mitogen-activated protein (MAP) kinase cascade, which leads to activation of genes required for mating (19,23). For C. neoformans, identification of the STE3 homolog has been reported but detailed information is lacking (30).The mating system of C. neoformans has received considerable attention due to the preponderance of MAT␣ strains among both environmental and clinical isolates (25, 44) and its increased virulence compared to the MATa type (28). Recently, a physical map of the MAT␣ locus from the C. neoformans strain B-4500 (JEC21), chosen for the C. neoformans genome project, was constructed (21). In contrast to other fungi, homologs of several S. cerevisiae pheromone response MAP kinase cascade genes such as STE20␣, STE11␣, and STE12␣, as w...

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (K m ‫؍‬ 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/ 4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

In this study we investigated the relationship between the MAT␣ locus of Cryptococcus neoformans and several MAT␣-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12␣, STE11␣, and STE20␣. To resolve the location of the genes, we screened a cosmid library of the MAT␣ strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MAT␣ locus. It was found that STE12␣, STE11␣, and STE20␣ are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MF␣, the mating type ␣-pheromone gene, a MAT␣-specific myosin gene, and a pheromone receptor (CPR␣) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MAT␣ locus. The MAT␣ locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.

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Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hym A mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym = hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44 kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A. nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hym A mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hym A is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.

Asexual fruiting body development in Aspergillus nidulans requires a precise spatial and temporal coordinated expression of many genes. Insertional mutagenesis was used to isolate and characterize a new mutant of A. nidulans in which hyphal growth was slightly reduced and conidiophore development was specifically blocked at the metula stage. In contrast to the uninucleate metulae of the wild-type, in the mutant these structures were elongated, multinucleate and septate. Further differentiation and production of phialides by a budding-like process was not observed. The mutant metulae thus resembled hyphae rather than metulae and the gene was therefore named hypha-like metulae (hymA). The hymA gene was mapped to linkage group VI. The integrated vector was rescued with border sequences from the integration site. The border sequences were used to isolate a cosmid from a wild-type library which was subcloned to a 5 kb fragment able to complement the mutation in trans. This fragment encoded a 1-8 kb transcript expressed in hyphae and throughout development. It is proposed that hymA is involved in budding processes, and is required for the formation of metulae and for their further differentiation.

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