: Gene Expression and Genome Structure I

The facB88 translocation creates a fusion of two regulatory genes which mediates superactivation of the amdS gene of Aspergillus nidulans

Rachael L. Murphy, Meryl A. Davis and Michael J. Hynes, Department of Genetics, University of Melbourne, Parkville 3052, Victoria, Australia.

The acetamidase gene (amdS) of Aspergillus nidulans is subject to complex transcriptional regulation. The facB gene product, which contains a Zn(II)2Cys6 DNA binding motif, mediates acetate induction of amdS and genes required for acetate metabolism via binding to the 5' regions of these genes. The facB88 reciprocal translocation results in very high level, constitutive expression (superactivation) of amdS. The translocation breakpoints lie within facB on chromosome VIII and a previously unidentified gene on chromosome IV, designated amdX. Only one of the hybrid genes created by the translocation, 5'facB-3'amdX, functions to superactivate amdS. The sequence of this hybrid gene revealed that the DNA binding domain of facB is fused to another DNA binding motif, consisting of two C2H2 zinc fingers, encoded by amdX. AmdX binds in vitro to sequences 5' of amdS that are known binding sites for the CreA and AmdA C2H2 zinc finger proteins. Both DNA binding domains are essential for maximal function of 5'facB-3'amdX, as are both the FacB and AmdX binding sites 5' of amdS. Thus the AmdX DNA binding domain contributes significantly to amdS regulation in the facB88 mutant. amdX has been cloned and sequenced. Gene disruption indicated that amdX is a non-essential gene. The phenotype of this loss of function mutant is slightly but consistently weaker growth on acetamide than wild type strains. Our results are consistent with a minor positive role for native amdX in the regulation of amdS expression.

The Aspergillus pacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH

H.N. Arst, Royal Postgraduate Medical School, London, UK

Many microbes encounter large ambient pH variations in their natural environments. Microorganisms capable of growing over a wide pH range require efficient pH homeostasis and a means of ensuring that activities undertaken beyond pH homeostasis boundaries are appropriate to ambient pH. Aspergillus nidulans is able to grow over a range of approximately eight pH units. The formal genetics and physiology of its regulatory system for controlling syntheses of secreted enzymes, permeases and exported metabolites in response to ambient pH have been described. This system enables, inter alia, the secretion of alkaline phosphatase in alkaline environments and acid phosphatase in acidic environments, as well as a considerable elevation in penicillin biosynthesis at alkaline pH. pH regulation of gene expression in A. nidulans is mediated by pacC whose 678 residue derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic-glutamine repeat. Nine pacCc mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth conidiation. A PacC fusion protein binds a hexanucleotide core consensus sequence. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction.

Initiation of conidiophore development in Aspergillus nidulans

Thomas H. Adams, Bee Na Lee, and Jenny Wieser, Texas A&M University, College Station, TX.

Aspergillus nidulans conidiophore formation can initiate as a programmed part of the lifecycle or in response to nutrient deprivation. In both cases, activation of the conidiation pathway ultimately leads to the expression of the complex brlA developmental regulatory locus which in turn results in activation of other genes required for conidiophore formation. Many developmental mutants blocked prior to activation of brlA expression have morphological abnormalities described as "fluffy". These mutants are characterized by an unrestricted proliferation of aerial hyphae resulting in formation of large cotton-like colonies that, unlike wild type, are able to grow into other colonies. One typical fluffy mutant results from mutation in the fluG gene. Strains containing a deletion of fluG are completely aconidial and no brlA expression is detectable during growth on rich medium. However, fluG mutants conidiate when grown on minimal medium suggesting that fluG null mutants maintain the ability to develop in response to nutrient limitation but have lost programmed developmental initiation. fluG mutant strains also conidiate when grown in contact with wild type colonies or with strains carrying different developmental mutations (e.g. brlA) suggesting that fluG mutants are deficient in production of a conidiation signal. We have identified five additional genetic loci, designated flbA, flbB, flbC, flbD, and flbE, that when mutated result in decreased brlA expression and a fluffy phenotype. We have shown that the predicted flbD product is related to mammalian myb proteins and that flbC encodes a protein with two putative C2H2 zinc fingers suggesting that these genes likely encode transcription factors. The flbA gene is predicted to encode a protein with significant similarity to the Saccharomyces cerevisiae SST2 product which is required for adaptation to mating pheromones. We have shown that overexpression of flbA, flbC, flbD, or fluG in vegetative hyphae can activate conidiophore development. These results have led us to propose that each of these genes functions in the programmed initiation of development and that flbA functions in mediating the response to the fluG-conidiation signal through a mechanism that requires the activity of the flbC and flbD DNA binding proteins.

The nitrogen regulatory circuit of Neurospora

George A. Marzluf, The Ohio State University, Columbus, OH

The expression of many genes which encode nitrogen catabolic enzymes is highly regulated by nitrogen repression and pathway-specific induction. Transcription of nit-3, which encodes nitrate reductase, is controlled by NIT2, a positive-acting global nitrogen regulatory protein, and by NIT4, a pathway specific factor which mediates nitrate induction. Three NIT2 and two NIT4 DNA binding sites occur in the nit-3 promoter. NIT4 binds DNA as a homodimeric protein, and recognizes an 8 bp palindromic sequence 5'TCCGCGGA and closely related sequences. NMR is a putative negative- acting regulatory protein that may act in nitrogen repression; nmr mutants are largely insensitive to repression by glutamine or ammonium ion. A specific NIT2-NMR protein-protein interaction has been detected with the yeast 2-hybrid system in vivo and with GST-fusion studies in vitro. NMR binds to two regions of the NIT2 protein, one of which is a short a-helical motif within the DNA binding domain.

Isolation of white collar-1, a central regulator of the blue light response in Neurospora

Paola Ballario and Giuseppe Macino, University of Rome "La Sapienza", Rome, Italy

The filamentous fungus, Neurospora crassa, is responsive to blue light irradiation, through the activation of specific genetic programs like: induction of mycelial carotenogenesis, promotion of conidia and protoperitecia development, phototropism of peritecial beaks and inhibition or shift of the circadian rhythm of conidiation. Two blind mutants, called white collar 1 and 2 (wc-1 and wc-2), defective in all the blue light-induced phenomena are known. The pleiotropic phenotype of these white collar mutants strongly suggests that they are involved in signal transduction. The blue light transduction pathway has never been dissected at molecular level, only recently a putative blue light photoreceptor has been described in Arabidopsis thaliana. We report here the isolation by chromosome walking and mutant complementation of the white collar-1 gene, a further key piece of the blue light puzzle. The wc-1 deduced product is a protein with a predicted molecular weight of 125 kDa, characterized by a single class 4 Zn finger and a polyglutamine stretch. The wc-1 Zn finger is highly homologous to the finger domains of vertebrate GATA factors, of fungal regulator proteins like Nit- 2, AreA, GLN3, DAL80, and of plant NTL1. The wc-1 Zn finger domain expressed in E. coli is able to bind in vitro to the promoter region of the blue light regulated Neurospora albino-3 gene, which contains two GATA sequences. Wc-1 gene expression is self-regulated and induced, at the transcription level, by blue light irradiation.

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