BIOLOGY

9 a b s t r a c t 30 The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish 31 here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate 32 oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline 33 carboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolase lyase (UglA), and the fungal-34 specific a-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, 35 while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate 36 pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in 37 some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the 38 urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We 39

Phytopathogenic Cercospora species produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers which absorb light energy and produce extremely cytotoxic, reactive oxygen species. In this work, we used Saccharomyces cerevisiae as a model system for the identi®cation and cloning of genes whose products mediate cercosporin detoxi®cation. Two genesexpressed in high-copy number vectors conferred cercosporin resistance to an otherwise sensitive strain. One gene codes for Snq2p, a well-characterized multidrug, ABCtype, eux protein. The other, designated CPD1 (Cercosporin Photosensitizer Detoxi®cation), encodes a novel protein with signi®cant similarity to the FAD-dependent pyridine nucleotide reductases. We showed that over-expression of either of these proteins can also mediate resistance to other singlet oxygen-generating compounds. The involvement of Snq2p and Cpd1p in photosensitizer detoxi®cation reinforces previous observations which suggested that singlet oxygen acts on membrane lipids and that cellular resistance to cercosporin is mediated by a mechanism involving toxin eux and/or toxin reduction.

A small family of at least four genes encodingmelon ascorbateoxidase (AO) has been identified and three members of it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymes homologousto ascorbate oxidasesfromotherplantsand similar to othermulticopperoxidases. We describedetailed 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.

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 full- length 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 plants. 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. Simi- lar 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.

Background: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.

In Saccharomyces cerevisiae the general amino acid (GAP1) permease catalyses active transport of apparently all amino acids across the plasma membrane. GAP1 activity is regulated by control of synthesis and control of activity in response to the nitrogen source supplied; ammonia and giutamine Inactivate GAP1 function while proline and urea allow its maximum expression. We have isolated and characterized a gene, AUAI, involved in ammonia regulation of GAP1 activity. AUAI is not essential for growth but overexpression of the AUAI transcript in a high-copy vector or due to a regulatory mutation, aua1-1, present -10 bp upstream from the start of >1(y4Mranscription, releases GAP1 activity from ammonia-inactivation without affecting GAP1 transcription. The aua1-1 mutation has no phenotype when ammonia is replaced by proline or glutamate as the nitrogen source or when it is present in a gapi background. AUAI expression is itself ammonia repressible in a wild-type strain but not in the aua1-1 mutant. The AUAI gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11714 Da. This polypeptide is highly hydrophilic and extremely basic. The AUAI product shows no significant similarity with any previousty known protein sequence. Interestingly, a 10-amino acid segment of AUA1 is directly repeated in the most basic segment of the protein. Possible roles of AUAI are discussed.

In this article we study the cellular expression of UapA and AzgA, the two major purine transporters of Aspergillus nidulans, by constructing strains expressing, from their native promoters, fully functional fluorescent (UapA-sGFP, AzgA-sGFP) or immunological (UapA-His) chimeric transporters. Epifluorescence microscopy and immunodetection showed that under different physiological conditions and during Aspergillus development: (i) UapA and AzgA expression in the plasma membrane becomes evident early during germination and remains at a significant basal level in mycelium, (ii) Neither of the two transporters is expressed in the stalk, the vesicle, the phialides and the conidiospores, but surprisingly, UapA is specifically and strongly expressed in the periphery of metulae, (iii) Both transporters are expressed in ascogenous hyphae and in hü lle cells but not in cleistothecia or ascospores, (iv) Purine induction leads to $ 4-fold increase in UapA and AzgA protein content in mycelium, compatible with an analogous increase at the transcriptional level, (v) Ammonium leads to removal of UapA, but not of AzgA, from the plasma membrane by sorting of the protein to the vacuole.

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways. Ó

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. D