Talks: Gene Expression and Genome Structure II

Physical maps of fungal genomes provide new research strategies for a wide range of fundamental biological problems, for engineering the production of new pharmaceuticals, and for understanding the cause of fungal diseases in plants and animals. The physical maps reported here represent the in vitro reconstruction of entire chromosomes from recombinant DNA libraries and provide useful tools to clone genes, determine genomic structure, and study genome evolution. We report a 29 kb resolution physical map of the entire 31 Mb genome of Aspergillus nidulans reconstructed from a 5134 clone cosmid library using 648 probes. The maps are the result of a novel two-way ordering process in which overlapping clones (redundant) and non-overlapping probes (tiles) are ordered to span the entire genome. The physical map is composed of eight matrices with clones down the rows and probes across the columns, one for each chromosome. The redundant order of clones contains 4550 anchored clones (89% of the cosmid library) into 132 contigs with an average of 17 contigs per chromosome. The compressed map (providing a minimum tiling of all 8 chromosomes) is reduced 5-fold in redundancy and contains 1085 clones. By integrating the physical and genetic maps with chromosome and clone hybridization data, we found that repeated DNA sequences are non-randomly distributed along chromosomes in a way reminiscent of heterochromatic banding patterns on cytological maps in other eukaryotes.

Interaction between specific induction and carbon catabolite repression in the ethanol regulon in A. nidulans

A. nidulans is able to use a wide variety of compounds as sole carbon sources. These pathways have in common the involvement of two transcriptiona1 regulatory circuits. The first one is specific induction mediated by trans-activators which usually belong to the zinc binuclear cluster family (C6 class). The second is carbon catabolite repression, controlled by thw wide domain repressoor CreA, which contains two zinc fingers of the C2H2 class. In the ethanol utilization paathway, specific induction proceeds by the binding of the AlcR activator to a number of specific targets localized to the alcR promoter itself, the alcR gene being positively autoregulated, and to the promoters of structural genes under its control e.g. alcA. Two types of targets, direct and invert repeats containing the same consensus core, are both necessary for full transcriptional induction. The high strength of the alcA promoter results from the synergistic transciptional activation by AlcR mediated by the multiple binding sites The carbon catabolite repressor, CreA, exerts a double transcriptional repression, on the trans-acting gene alcR and independently on structural genes. A great number of CreA binding sites have been identified in the cis-acting regions of the alcR and alcA genes. The disruption of different functional CreA targets all result in superinduced and derepressed alcR and alcA expression. Those which overlap or are in very close proximity to AlcR targets account for a direct competition between AlcR and CreA for the same cis-acting region. The interplay between these two regulatory circuits regulates the expression of the ethanol regulon genes under all physiological conditions.

Promoter analysis of the Neurospora crassa circadian clock-controlled ccg-2 (eas) gene

The N. crassa ccg-2 gene encoding a fungal hydrophobin is transcriptionally regulated by the circadian clock. In addition, ccg-2 is positively regulated by light, and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analysis of the ccg-2 promoter was carried out to localize the cis-acting elements mediating clock, light, and developmental control. A distinct positive clock element was localized to within a 45 nt region, just upstream of the TATA box. Using an unregulated promoter/reporter system we show that this element is necessary and sufficient for conferring clock regulation on the ccg-2 gene. We are currently using this element as a probe in gel-mobility shift assays to identify trans-acting clock factors.

The relationship between DNA methylation and transcription in Neurospora crassa

A correlation between DNA methylation and lack of gene activity has been observed in animal, plant, and fungal systems. Our understanding of the relationship between methylation and transcription remains limited, however. Exploration of this relationship in Neurospora is facilitated by RIP (repeat induced point mutation), which frequently results in methylation of the mutated sequences and by a mutation (dim-2) that appears to prevent all DNA methylation. Using dim-2 and the methylation inhibitor 5-azacytidine, we found that the methylation associated with RIPed genes, and their surrounding sequences, results in a reduction (or abolishment) of stable transcripts. Results of our study also revealed that mutations by RIP can affect the length of transcripts produced from an affected gene. In addition, we determined that the methylation associated with the tandemly repeated rDNA in Neurospora does not affect rRNA levels, probably because the methylation is confined to the non-transcribed spacer regions. We are currently exploring the mechanism by which DNA methylation reduces transcript accumulation. In separate experiments to examine the possible effect of transcription on methylation, we found that placement of an intact promoter in or next to a methylated region does not prevent methylation. Methylation of a region was reduced by placing it upstream of the am gene, however. We are dissecting the upstream am region to determine the cause of this effect.

Substrate specificity and transcriptional regulation of purine permeases in Aspergillus nidulans

There are three purine permeases in Aspergillus nidulans. One, encoded by the uapA gene, is specific for uric acid and xanthine uptake, a second, encoded by the azgA gene, is specific for hypoxanthine, adenine and guanine, while a third, encoded by the uapC gene, is a broad specificity permease able to incorporate all purines into the cell. Early genetical data established that uapA and uapC are under the control of the specific transcription factor UaY, mediating uric acid induction, and of the GATA-like factor AreA, mediating nitrogen metabolite repression. The azgA gene is neither under UaY or AreA control. UaY mediates uric acid induction of at least nine genes of the purine assimilation pathway. There are striking similarities between UaY and the Saccharomyces cerevisiae protein PPR1, involved in the regulation of pyrimidine biosynthesis. Both proteins bind to 5'CGG-6X-CCG sequences. uapA and uapC have been cloned and sequenced. The binding sites for UaY and AreA on the uapA and uapC promoters have been established by gel retardation and footprinting techniques. An allele of areA, which changes a conserved leucine to a valine residue in the DNA binding domain, reduces drastically and specifically the expression of uapA and uapC. Suppressors specific for either gene have been isolated, characterised and sequenced. These provide a quite detailed picture of the structure of the uapA and uapC promoters. The uapA and uapC open reading frames reveal typical membrane proteins of superimposable hydropathicity profiles and show a 58% identity. The construction of hybrid proteins and mutagenesis of selected hydrophilic regions is been used to determine the residues involved in substrate recognition.

Mechanism of coordinate regulation of ribosomal genes by nutrition and growth in Neurospora

In Neurospora crassa, transcription of the large (40S) ribsomal RNA genes by RNA polymerase I and of the ribosomal protein genes by RNA polymerase II are coordinately regulated by the carbon supply. Transcription of the 5S rRNA genes by RNA polymerase III however is largely insensitive to regulation by carbon or growth. We have characterized in detail sequences required for transcription and carbon regulation of the ribosomal protein gene crp-2. The crp-2 promoter contains at least six specific elements within 250 bp of the transcription startpoint. None are similar to UASrpg sequences required for transcription of yeast ribosomal protein genes. Two elements (CGrepeat and Dde box) occur together as a pair twice in the crp-2 promoter. Variations of these elements occur in all 5 ribosomal protein gene promoters examined, and in most cases, the two elements occur as a pair close to -70 with conserved spacing. This pair of elements also occurs with the same spacing within a functional domain of the 40S rRNA promoter, suggesting that the pair is repsonsible for coordinating rRNA and r protein gene expression. The CGrepeat/Dde box pair also corresponds to the region responsible for carbon regulation of crp-2 The CG-repeat binds the Aspergillus carbon catabolite repressor creA. Therefore we have isolated cre-1, the Neurospora homolog of creA, and are investigating whether it is responsible for coordinate regulation of the r-protein and rRNA genes by carbon, mediated via the CGrepeat/Dde box pair.

Regulation of sulfur metabolism in Neurospora crassa

The sulfur regulatory system of N. crassa is composed of a group of highly regulated structural genes (e.g., arylsulfatase, ars-1+) which are under coordinate control of the cys-3+ positive and scon+ (sulfur controller) negative regulatory genes. The CYS3 regulator is a bZIP DNA-binding protein that is necessary and sufficient to induce sulfur structural gene expression. Experiments modifying the dimerization specificity of CYS3, through leucine zipper alterations, demonstrate that CYS3 functions in vivo as a homodimer. The cys-3+ gene is also subject to autoregulation. In vitro studies have defined CYS3 binding sites on the structural and regulatory genes in the system. In vivo studies with the ars-1+ promoter have confirmed the importance of the putative CYS3 binding sites. The ars-1+ promoter constructs included deletions and site-directed alterations to control elements which were assayed following integration at a defined chromosomal site. In addition, a heterologous promoter construct has been used to demonstrate the minimum sequence necessary for CYS3 controlled expression. The scon-2+ negative regulatory gene has been characterized and encodes a beta-transducin homolog with six WD-40 repeats. SCON2 potentially defines a new subclass of WD proteins. Using band shift and Northern blot analysis a control loop has been identified between cys-3+ and scon-2+.

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