The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5'-end of the mRNA, with a major start at -106 relative to the initiation codon. This indicates that the actual length of the non-transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying beta-galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis-acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis-acting elements is involved in sucrose repression. Strikingly, it is located outside the non-transcribed 525bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5'-deletion up to -56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total-protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA-binding activity that specifically shifts a promoter fragment located between -654 and -455 (relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.

Urogenital system development in mammals requires the coordinated differentiation of two distinct tissues, the ductal epithelium and the nephrogenic mesenchyme, both derived from the intermediate mesoderm of the early embryo. The former give rise to the genital tracts, ureters and kidney collecting duct system, whereas mesenchymal components undergo epithelial transformation to form nephrons in both the mesonephric (embryonic) and metanephric (definitive) kidney. Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the development of both ductal and mesenchymal components of the urogenital system. We report here that Pax-2 homozygous mutant newborn mice lack kidneys, ureters and genital tracts. We attribute these defects to dysgenesis of both ductal and mesenchymal components of the developing urogenital system. The Wolffian and Mullerian ducts, precursors of male and female genital tracts, respectively, develop only partially and degenerate during embryogenesis. The ureters, inducers of the metanephros are absent and therefore kidney development does not take place. Mesenchyme of the nephrogenic cord fails to undergo epithelial transformation and is not able to form tubules in the mesonephros. In addition, we show that the expression of specific markers for each of these components is de-regulated in Pax-2 mutants. These data show that Pax-2 is required for multiple steps during the differentiation of intermediate mesoderm. In addition, Pax-2 mouse mutants provide an animal model for human hereditary kidney diseases.

During gestation, the paired box-containing gene Pax2 is expressed in the mid-hindbrain area, developing eye and inner ear. We generated Pax2 null mutant mice, which show the requirement of Pax2 for the establishment of axonal pathways along the optic stalks and ventral diencephalon. In mutant brains, the optic tracts remain totally ipsilateral due to agenesis of the optic chiasma. Furthermore, Pax2 mutants show extension of the pigmented retina into the optic stalks and failure of the optic fissure to close resulting in coloboma. In the inner ear, Pax2 mutants show agenesis of the cochlea and the spiral ganglion, i.e., the parts of the organ responsible for auditory function and in whose primordium Pax2 is expressed. Our results identify Pax2 as a major regulator of patterning during organogenesis of the eye and inner ear and indicate its function in morphogenetic events required for closure of the optic fissure and neural tube.

Secondary metabolism, usually superfluous under laboratory conditions, is intrinsically elusive to genetic analysis of its regulation. We describe here a method of analyzing regulatory mutations affecting expression of secondary metabolic genes, with an Aspergillus nidulans penicillin structural gene (ipnA [encoding isopenicillin N-synthase]) as a model. The method is based on a targeted double integration of a lacZ fusion reporter gene in a chromosome different from that containing the penicillin gene cluster. The trans-acting regulatory mutations simultaneously affect lacZ expression and penicillin biosynthesis. One of these mutations (npeE1) has been analyzed in detail. This mutation is recessive, prevents penicillin production and ipnA::lacZ expression, and results in very low levels of the ipnA message at certain times of growth. This indicates that npeE positively controls ipnA transcription. We also show that this tandem reporter fusion allows genetic analysis of npeE1 by using the sexual and parasexual cycles and that lacZ expression is an easily scorable phenotype. Haploidization analysis established that npeE is located in chromosome IV, but npeE1 does not show meiotic linkage to a number of known chromosome IV markers. This method might be of general applicability to genetic analysis of regulation of other fungal secondary metabolic pathways.Secondary metabolism in microbes is often elusive to genetic analysis, because most, if not all, pathways classified in this category are dispensable under laboratory conditions. The penicillin biosynthetic pathway (22) is a prototype of such pathways in filamentous ascomycetes and has been extensively used as a model for at least four reasons. (i) It is a rather simple pathway, and only three enzymes are required to convert primary metabolites (three amino acids) into penicillin; (ii) the corresponding structural genes, which are clustered, have been cloned and characterized from several species; (iii) the end product can be sensitively detected with a bioassay; and (iv) it is of obvious biotechnological interest. As a consequence, a wealth of information about this pathway has been accumulated. In contrast, regulation of penicillin biosynthesis is largely unelucidated, possibly because the absence of a sexual cycle in Penicillium chrysogenum has hindered formal genetic analysis of the putative regulatory mechanisms. This problem has been circumvented by using Aspergillus nidulans (26), a closely related plectomycete amenable to formal genetic studies (9) and for which sophisticated molecular biology techniques are available (31).By using molecular techniques to analyze transcription of the A. nidulans ipnA gene (encoding isopenicillin N-synthase, a key enzyme catalyzing the central step in the pathway), we have described two modes of transcriptional regulation of a penicillin structural gene. Carbon regulation in response to the availability of a preferred carbon source modulates ipnA expression through the action of a yet undefined negative-acting regulatory ge...