George Diallinas

Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA

A change of a universally conserved leucine to valine in the DNA‐binding domain of the GATA factor AreA results in inability to activate some AreA‐dependent promoters, including that of the uapA gene encoding a specific urate–xanthine permease. Some other AreA‐ dependent promoters become able to function more efficiently than in the wild‐type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild‐type and mutant forms of AreA. In vitro binding studies of wild‐type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild‐type and mutant AreA–DNA complexes derived from the atomic coordinates of the GATA‐1–AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA‐binding sites, is sufficient to ensure activation of transcription of the uapA gene.

Nucleobase transporters (review).

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members fromarchea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.

A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid biosynthesis involved in the synthesis of a multiplicity of plant natural products. We have isolated and characterized a nearly fulllength cDNA clone (pmPAL-1) corresponding to a melon fruit (Cucumis melo L. var. reticulatus) gene coding for a protein which is highly similar to PAL from other lants. Melon fruit PAL is transcriptionally induced both in response to fruit ripening and wounding. PAL gene expression follows the kinetics of expression of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes show different induction kinetics compared to PAL expression in response to wounding. Similar results have been found for two other genes coding for enzymes involved in flavonoid biosynthesis (chalcone synthase, CHS; chalcone isomerase, CHI). Our results imply that regulation of defense gene expression in melon is a co-ordinated process in response to both ethylene and an ethylene-independent wound signal.

Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues

In Aspergillus nidulans, purine uptake is mediated by three transporter proteins: UapA, UapC and AzgA. UapA and UapC have partially overlapping functions, are 62% identical and have nearly identical predicted topologies. Their structural similarity is associated with overlapping substrate specificities; UapA is a high‐affinity, high‐capacity specific xanthine/uric acid transporter. UapC is a low/moderate‐capacity general purine transporter. We constructed and characterized UapA/UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutations. The region including residues 378–446 in UapA (336–404 in UapC) has been shown to be critical for purine recognition and transport. Within this region, we identified: (i) one amino acid residue (A404) important for transporter function but probably not for specificity and two residues (E412 and R414) important for UapA function and specificity; and (ii) a sequence, (F/Y/S)X(Q/E/P) NXGXXXXT(K/R/G), which is highly conserved in all homologues of nucleobase transporters from bacteria to man. The UapC/UapA series of chimeras behaves in a linear pattern and leads to an univocal assignment of functional domains while the analysis of the reciprocal and sandwich’ chimeras revealed unexpected inter‐domain interactions. cDNAs coding for transporters including the specificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.

The nucleobase–ascorbate transporter (NAT) family: genomics, evolution, structure–function relationships and physiological role

This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.

Operator derepressed mutations in the proline utilisation gene cluster of Aspergillus nidulans

SummaryThe proline utilisation gene cluster of Aspergillus nidulans can be repressed efficiently only when both repressing nitrogen and repressing carbon sources are present. We show that two cis-acting mutations in this cluster permit the efficient transcription of the prnB gene under repressing conditions, resulting in direct or indirect derepression of two other transcripts of the pathway. These mutations are transitions that define a 5′GAGACCCC3′ sequence. Similar sequences are found upstream of other genes subject to carbon catabolite repression. We propose that this sequence defines the binding site for the negatively-acting CreA protein, which mediates carbon catabolite repression in this fungus.

Functional Characterization of a Maize Purine Transporter by Expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport–deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (Km = 30 ± 2.5 μM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.

Melon ascorbate oxidase: cloning of a multigene family, induction during fruit development and repression by wounding.

A small family of at least four genes encoding melon ascorbate oxidase (AO) has been identified and three members of it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymes homologous to ascorbate oxidases from other plants and similar to other multicopper oxidases. We describe detailed molecular studies addressing melon AO expression during organ specific differentiation, fruit development and ripening, and in response to wounding. In particular, AO transcript accumulation was induced in ovaries and the outer mesocarp of mature preclimacteric melon fruits, before the expression of genes encoding the necessary enzymatic activities for ethylene biosynthesis. On the other hand, AO was not expressed in late stages of fruit ripening and was repressed in wounded fruits. The role of ethylene in transcriptional regulation of AO is discussed.

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.