RNA silencing gene truncation in the filamentous fungus Aspergillus nidulans.

The genus Aspergillus is ideally suited for the investigation of RNA silencing evolution because it includes species that have experienced a variety of RNA silencing gene changes. Our work on this subject begins here with the model species Aspergillus nidulans. Filamentous ascomycete fungi generally each encode two of the core RNA silencing proteins, Dicer and Argonaute, but A. nidulans appears to have lost one of each to gene truncation events. Although a role in growth, development, or RNA silencing was not detected for the truncated genes, they do produce spliced and poly(A)-tailed transcripts, suggesting that they may have an undetermined biological function. Population analysis demonstrates that the truncated genes are fixed at the species level and that their full-length orthologs in a closely related species are also unstable. With these gene truncation events, A. nidulans encodes only a single intact Dicer and Argonaute. Their deletion results in morphologically and reproductively normal strains that are incapable of experimental RNA silencing. Thus, our results suggest that the remaining A. nidulans RNA silencing genes have a "nonhousekeeping" function, such as defense against viruses and transposons.

Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans.

We have cloned an 11-kbp segment of the genomic DNA of Aspergillus nidulans which complements mutations in nirA, the pathway-specific regulatory gene of the nitrate assimilation pathway. Gene disruption in the corresponding region of the nuclear DNA leads to a phenotype and a gene complementation pattern indistinguishable from that observed in known noninducible nirA mutants. Transformation studies with subclones of the 11-kbp genomic segment showed that a nonreverting null mutation nirA87, maps to a 1.5-kbp stretch within that segment. These data confirm that the cloned segment contains the nirA gene. The gene is completely encompassed in the 11-kbp genomic segment, as a plasmid carrying the corresponding insert gives rise to multicopy transformants exhibiting better growth than wild type on nitrate or nitrite as the sole nitrogen source. Southern and genetic analyses of transformants obtained with various plasmid subclones established a gene size of at most 5.9 kbp. Northern (RNA) hybridization experiments revealed a 4-kb nirA transcript which is barely visible in the wild type but clearly seen in a transformant carrying about 10 gene copies. In both strains, nirA mRNA is synthesized constitutively. Upstream of nirA, a neighboring transcript about 2.8 kbp in length which is transcribed from the opposite strand with respect to nirA was localized. The transcript levels of niaD and niiA, encoding the nitrate and nitrite reductase core proteins, respectively, were investigated in nirA mutants and a nirA multicopy transformant. The results show that the nirA product regulates the transcript steady-state level of these structural genes and that it is a limiting factor for their expression.

The regulator of nitrate assimilation in ascomycetes is a dimer which binds a nonrepeated, asymmetrical sequence.

The regulation of nitrate assimilation seems to follow the same pattern in all ascomycetes where this process has been studied. We show here by in vitro binding studies and a number of protection and interference techniques that the transcription factor mediating nitrate induction in Aspergillus nidulans, a protein containing a binuclear zinc cluster DNA binding domain, recognizes an asymmetrical sequence of the form CTCC GHGG. We further show that the protein binds to its consensus site as a dimer. We establish the role of the putative dimerization element by its ability to replace the analogous element of the cI protein of phage lambda. Mutagenesis of crucial leucines of the dimerization element affect both the binding ability of the dimer and the conformation of the resulting protein-DNA complex. This is the first case to be described where a dimer recognizes such an asymmetrical nonrepeated sequence, presumably by each monomeric subunit making different contacts with different DNA half-sites.

nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions.

The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

The fungal GATA factors.

The DNA-binding domains of eucaryotic GATA factors comprise a four-cysteine Zn finger and an adjacent basic region. Fungal GATA factors regulate nitrogen metabolism, light induction, siderophore biosynthesis and mating-type switching. Hydrophobic interactions determine binding-site specificity. Interactions with other factors may determine promoter specificity. One GATA factor has recently been shown to determine a drastic chromatin rearrangement.

A single amino acid change in a pathway-specific transcription factor results in differing degrees of constitutivity, hyperinducibility and derepression of several structural genes.

unYc462 is a gain-of-function mutation in the purine catabolism positive regulatory gene of Aspergillus nidulans. This allele leads to a constitutive, hyperinducible and derepressed expression of a least three genes controlled by uaY, and this occurs at different levels depending on the target gene. The uaYc462 allele was mapped physically in relation to known loss-of-function alleles and sequenced. uaYc462 is a one-base change in codon 222, resulting in a serine to leucine change. We propose that this mutation maps in a functional domain involved, directly or indirectly, in the interaction of UaY with other components of the transcriptional apparatus. A sequence similar to the motif surrounding serine 222 may play similar roles in the PPR1 and ADR1 proteins of Saccharomyces cerevisiae.

The analysis of the transcriptional activator PrnA reveals a tripartite nuclear localisation sequence.

Nuclear localisation signals (NLSs) have been classified as either mono- or bipartite. Genetic analysis and GFP fusions show that the NLS of a Zn-binuclear cluster transcriptional activator of Aspergillus nidulans (PrnA) is tripartite. This NLS comprises two amino-terminal basic sequences and the first basic sequence of the Zn-cluster. Neither the two amino-terminal basic sequences nor the paradigmatic nucleoplasmin bipartite NLS drive our construction to the nucleus. Cryosensitive mutations in the second basic sequence are suppressed by mutations that restore the basicity of the domain. The integrity of the Zn-cluster is not necessary for nuclear localisation. A tandem repetition of the two basic amino-terminal sequences results in a strong NLS. Complete nuclear localisation is observed when the whole DNA-binding domain, including the putative dimerisation element, is included in the construction. At variance with what is seen with tandem NLSs, all fluorescence here is intra-nuclear. This suggests that retention and nuclear entry are functionally different. With the whole PrnA protein, we observe localisation, retention and also a striking sub-localisation within the nucleus. Nuclear localisation and sub-localisation are constitutive (not dependent on proline induction). In contrast with what has been observed by others in A. nidulans, none of our constructions are delocalised during mitosis. This is the first analysis of the NLS of a Zn-binuclear cluster protein and the first characterisation of a tripartite NLS.

Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase.

We describe the sequence changes of a number of mutations of the Aspergillus nidulans xanthine dehydrogenase (XDH). We have located the amino acids affected by these changes in the three-dimensional (3D) structure of aldehyde oxido-reductase (MOP) from Desulfovibrio gigas, related to eukaryotic XDHs. Of these, two are loss of function mutations, mapping, respectively, in the molybdenum-pterin co-factor (MoCo) domain and in the domain involved in substrate recognition. Changes in two amino acids result in resistance to the irreversible inhibitor allopurinol. In Arg911 two different changes, conserved among all XDHs and MOP but not in other aldehyde oxidases (AO), change the position of hydroxylation of the analogue 2-hydroxypurine from C-8 to C-6. A number of changes affect residues adjacent to the molybdenum or its ligands. Arg911 is positioned in the substrate pocket in a way that it can account for the positioning of purine substrates in relation to the MoCo reactive center, together with a glutamate residue, universally conserved among the XDHs (Glu833).

Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing.

We present the first indication of a direct relationship between a nuclear and a mitochondrial splicing system. The intron in the precursor of the large, nuclearly coded ribosomal RNA of two species of Tetrahymena possesses all the features of a class of fungal mitochondrial introns. Sequences conserved in mitochondrial introns of different fungal species are also found in the same order in these Tetrahymena nuclear introns, and the intron RNA can be folded to form a secondary structure similar to that proposed for mitochondrial introns by Davies et al. (1982). This "core" secondary structure brings the ends of the intron together. Furthermore, the first intron in the precursor of the large, nuclearly coded rRNA of Physarum polycephalum also has the characteristic conserved sequences and core RNA secondary structure. The limited sequence data available suggest that the intron in the large rRNA of chloroplasts in Chlamydomonas reinhardtii also resembles the mitochondrial introns. Tetrahymena large nuclear rRNA introns also have an internal sequence that can act as an adaptor by pairing with upstream and downstream exon sequences adjacent to the splice junctions to precisely align the splice junctions. These nuclear introns therefore fit the model of the role of intron RNA in the splicing process that was proposed by Davies et al. (1982), suggesting that the mechanisms of splicing may be very similar in these apparently diverse systems. It is therefore probable that the RNA secondary structures for which there is good evidence in the case of mitochondrial introns will be found to form the basis of active site structure and precise alignment in splicing and cyclization of the Tetrahymena intron "ribozyme".

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

A gene coding for the uric acid-xanthine permease of Aspergillus nidulans: inactivational cloning, characterization, and sequence of a cis-acting mutation.

In Aspergillus nidulans, integration of transforming sequences can proceed through recombination with homologous sequences or at heterologous sites in the genome. In a strain with a large deletion in the gene coding for acetamidase (amdS), a plasmid carrying this gene integrates into and inactivates uapA, the putative structural gene for uric acid-xanthine permease, with a frequency of 0.3%. The integration event occurs 3' to the open reading frame of amdS. A 10-nucleotide sequence which occurs in this region is also found within the open reading frame of uapA. We have taken advantage of this integration event to clone the permease gene and to characterize a cis-acting mutation, uap-100, as a duplication of 139 bp located in the upstream region of uapA. Northern and dot blot analyses confirmed earlier results measuring the uptake of uric acid: the transcription of the uapA gene is inducible and the uap-100 mutation results in a bypass of the need for induction while having an 8-fold up-promoter effect under inducing conditions.

Insertional inactivation and cloning of the wA gene of Aspergillus nidulans.

We describe examples of wA gene inactivation (resulting in white conidiospores) obtained during transformation of Aspergillus nidulans. One wA- transformant was obtained by transformation with a prn+ plasmid of a strain with green conidia (wA+) which was unable to catabolize L-proline (prn-). This transformant contains a very large number of plasmid copies integrated at a single site inseparable from the wA locus. Passage of this transformant through the sexual cycle generated a variety of novel phenotypes for L-proline utilization, the number and frequency of which depended upon the cleistothecium from which the progeny were obtained, suggesting that the altered phenotypes were due to premeiotic events. The most extreme phenotype was severe hypersensitivity to L-proline. Hypersensitive progeny had a much reduced number of integrated plasmid copies enabling us to identify and clone putative prn-wA fusion sequences and subsequently retrieve wA sequences from a wild-type gene library. One of the wild-type clones overlapped the different sites of the insertional mutations in two wA- transformants and complemented the wA3 allele. Sequences within this clone hybridized to a transcript that was developmentally regulated in the wild type and absent in a number of mutants defective in conidiospore development. A reiterated sequence was also found in the region of the wA gene.

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans: I. Characterization of alleles, dominance and complementation studies, and a fine structure map of the uaY--oxpA cluster.

In this paper we characterize genetically a positive eukaryotic regulatory gene: the uaY gene of the ascomycete Aspergillus nidulans. Several steps in the uptake and degradation of purines are under the control of the uaY gene (summarized in Scazzocchio and Gorton 1977). In the present paper 12 uaY-mutations are characterized with respect to their inducibility for adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase and by the absence of the uric acid-xanthine permease scored in vivo by resistance to 2-thiouric acid. While 10 mutations are uniformly unleaky, two others are almost wild type for the induction of urate oxidase. A fine structure map of the uaY gene shows that the two "leaky" mutations are not clustered. The fine structure mapping unambiguously positions six uaY alleles and provides preliminary but interesting trends regarding the pattern of gene conversion in the uaY gene. The enzyme levels in all uaY-/uaY+ heterozygous diploids are intermediate between the corresponding uaY-/uaY- and uaY+/uaY+ homozygous diploids, suggesting that one functional copy of the uaY gene is able to mediate the complete induction of only one set of structural genes. No complementation was found between any two uaY- alleles. This establishes that the mutations showing either of the phenotypes are alleles in the same gene; it fails to provide evidence for intracistronic complementation. A mutation, oxpA5, causes resistance to the xanthine analogue oxypurinol (4,6-dihydroxypyrazolo-(3, 4-d)-pyrimidine) and partial constitutivity of adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase. The constitutive phenotype is suppressed by mutations, blocking the synthesis of intracellular inducers. The mutation is recessive and complements fully with the 11 uaY- mutations tested. It maps to the left of all 12 uaY mutations to which it has been crossed. The data indicate that both the resistance and constitutivity arise from one mutational event in a gene, oxpA, different from uaY and possibly adjacent to it. We propose that the oxpA gene codes for a protein involved in limiting the flow of inducers into the cell nucleus. Thus oxpA and uaY constitute a regulatory gene cluster, indicating that uaY is the regulatory gene.

Mitochondrial Four-Point Crosses in ASPERGILLUS NIDULANS : Mapping of a Suppressor of a Mitochondrially Inherited Cold-Sensitive Mutation.

Four-point mitochondrial crosses were conducted in heterokaryons of Aspergillus nidulans. The mutations used were (oliA1), conferring resistance to oligomycin, (camA112), conferring resistance to chloramphenicol; (cs-67), conferring cold-sensitivity, and ( sumD16), a suppressor of (cs-67). Initially, the crosses were conducted by observing the segregation of extranuclear markers in heterokaryotic sectors emerging from the original point of heterokaryosis. This showed that (camA112), (cs-67) and (sumD16) were linked but were probably all unlinked to (oliA1). Second, four-point crosses were conducted using a double marker selection technique, in which (camA112 ) and (oliA1) were always set in repulsion and the frequency of the phenotypes produced by the segregation of the mutant and wild-type alleles of (cs-67) and (sumD) were observed in (camA112 oliA1) recombinants. From these results we concluded that (camA112 ), (cs-67) and (sumD16) were linked and probably mapped in the order given. It was observed that the two nuclear types of conidia from a heterokaryon often had a dissimilar frequency distribution of the segregants of a mitochondrial cross.

Characterisation, cloning and integrative properties of the gene encoding urate oxidase in Aspergillus nidulans.

A number of mutations have been obtained which define the structural gene (uaZ) coding for urate oxidase in linkage group I of Aspergillus nidulans. This gene has been cloned by transformation of a uaZ- null mutant. A chromosome I/VIII translocation which splits the gene has been defined both genetically and physically. All known mutations are contained in a 1-kb fragment, itself contained in the probe which recognizes a 1.2-kb inducible message. Plasmids carrying uaZ show a strict bias towards homologous recombination in transformation experiments.

Cloning and characterization of the ethanol utilization regulon in Aspergillus nidulans.

In Aspergillus nidulans alcohol dehydrogenase (ADH) I and aldehyde dehydrogenase (AldDH) are co-inducible by acetaldehyde (Pateman et al., 1983; Sealy-Lewis and Lockington, 1984) and subject to carbon catabolite repression. The structural genes alcA and aldA are unlinked, but alcA is closely linked to the positive control gene alcR. We have obtained cDNA clones of alcA and aldA and genomic clones comprising alcA and alcR. The location of these genes in a genomic clone carrying a 13-kb insert was determined by subcloning and subsequent transformation of previously characterised point mutants. We have characterised at the physical level some large deletions encompassing both linked genes. We have shown that induction affects the level of RNA hybridisible with alcA and aldA probes. Mutations in the regulatory gene alcR, result in non-inducibility of RNA hybridisible with either probe. Thus the induction process is possibly at the level of transcription. Analogous experiments suggest that carbon catabolite repression of alcohol dehydrogenase I is equally at the level of transcription.

Heterologous expression of the Aspergillus nidulans regulatory gene nirA in Fusarium oxysporum.

We have isolated strains of Fusarium oxysporum carrying mutations conferring a phenotype characteristic of a loss of function in the regulatory gene of nitrate assimilation (nirA in Aspergillus nidulans, nit-4 in Neurospora crassa). One of these nir- mutants was successfully transformed with a plasmid containing the nirA gene of A. nidulans. The nitrate reductase of the transformants is still inducible, although the maximum activity is lower than in the wild type. Single and multiple integration events were found, as well as a strict correlation between the presence of the nirA gene and the Nir+ phenotype of the F. oxysporum transformants. We also investigated how the A. nidulans structural gene (niaD) is regulated in F. oxysporum. Enzyme assays and Northern experiments show that the niaD gene is subject to nitrate induction and that it responds to nitrogen metabolite repression in a F. oxysporum genetic background. This indicates that both the mechanisms of specific induction, mediated by a gene product isofunctional to nirA, and nitrogen metabolite repression, presumably mediated by a gene product isofunctional to the homologous gene of A. nidulans, are operative in F. oxysporum.

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum.

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.

A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans.

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.