The stunted (stuA) gene product is required for the orderly differentiation and spatial organization of cell types of the Aspergillus nidulans conidiophore. Expression of the stuA gene is complex. Two transcripts, stuA~ and stuA~, are initiated from separate promoters. Transcription of both RNAs increases -50-fold during the establishment of developmental competence. Induction-dependent transcriptional and post-transcriptional regulatory mechanisms further enhance expression 15-fold. Consistent with the latter observation, both transcripts have structural features characteristic of RNAs subject to translational control. Conidiophore morphogenesis requires regulatory interactions between the products of the stuA, bristle (brlA), and abacus (abaA) genes. Enhanced stunted expression is cell type specific and dependent on a functional BrlA protein.StuA affects the spatial localization of AbaA by acting directly, or indirectly, to repress abaA expression.

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell‐specific or morphogenesis‐specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63–75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12αp. SteAp and Ste12αp lack the pheromone induction domain found in budding yeast Ste12p, but have C‐terminal C2/H2‐Zn+2 finger domains not present in the other Ste12 proteins. A ΔsteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle‐specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH‐like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. (i) A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC-strain to tipC'. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpCq allele in a minority of the strains. (ii) An A. nidulans tipC' gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC-argB+ strain to tipC'.Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. (iii) The A. nidulans SpoCl Cl-C gene was modified by removal of an internal restriction fragment and introduced into a trpC-strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the Cl-C region separated by plasmid DNA was self-fertilized, and trpC-progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified Cl-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one-or two-step procedures similar to those developed for Saccharomyces cerevisiae.The original demonstration of DNA-mediated transformation of the unicellular yeast Saccharomyces cerevisiae (13) was followed rapidly by the construction of numerous transformation vectors with different properties and the development of a variety of experimental strategies that now make it possible to manipulate the yeast genome with great precision (reviewed in references 7, 20, and 29). The availability of this technology has resulted in many important discoveries about the molecular and genetic mechanisms regulating the activities of eucaryotic cells.One of the most prominent and important properties of the yeast transformation system is that cloned DNA sequences can be introduced into chromosomes by homologous recombination (13, 22). This property allowed the development of one-step (24) and two-step (25) procedures for precise replacement of chromosomal regions with DNA sequences that have been altered in vitro by using recombinant DNA methods. These techniques make it possible to examine the biochemical and biological conse-

The functions of at least four potential regulatory genes are known to overlap temporally during elaboration of the multicellular asexual reproductive apparatus (conidiophore) of Aspergillus nidulans. One of these, the stuA (stunted) gene, has been previously classified as a morphological modifier essential for correct spatial organization of the conidiophore. The gene was cloned by complementation of a strain carrying the stuA1 mutation and has been localized to a 5.0 kb KpnI fragment that encodes a 3.3 kb mRNA. The stuA mRNA was detected at very low levels in mature conidia and in somatic hyphae that had not established developmental competence. A dramatic increase in the abundance of this mRNA occurred coincidentally with the establishment of competence, but prior to the induction of conidiation. RNA abundance remained at this elevated level during conidiophore morphogenesis. These results are consistent with genetic data suggesting that stuA gene function is required from the very earliest events of asexual reproduction until completion of conidiophore development, but is not specifically required for differentiation of conidia. The expression of the stuA transcript was not affected by any of the other characterized developmental mutations. These latter results suggest that transcriptional activation at the onset of competence is mediated by an as yet unidentified genetic locus or loci.

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-β (PLC-β), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.