11. "Post-transcriptional control and kinetic characterization of proline transport in germinating conidiospores of Aspergillus nidulans. "
U.H. Tazebay, V. Sophianopoulou, B. Cubero, C. Scazzocchio and G. Diallinas.
FEMS Microbiology Letters, vol. 132, pages 27-37, (1995).   View at publisher's site   Request copy
Abstract: In the filamentous fungus Aspergillus nidulans, L-proline uptake is mediated by the product of the prnB gene which codes for a member of a family of amino acid transporters found both in pro- and eukaryotes. Regulation of prnB gene expression has previously been studied in great detail at the molecular level. However, no studies have addressed possible post-transcriptional controls or the kinetic characterisation of the PrnB transporter. Here we develop a rapid and efficient method for direct uptake measurements of proline in germinating conidiospores of A. nidulans. We make use of this method and Northern blot analyses in parallel to study the regulation of PrnB expression both at the level of prnB message accumulation and at a post-transcriptional level. These studies show that (i) pathway-specific and wide-domain regulatory systems, previously shown to control prnB gene expression in multicellular mycelia, also operate in unicellular conidia committed to germination; and (ii) PrnB activity is regulated in response to the nitrogen source present in the medium and the level of internally accumulated proline or other amino acids. We also characterise kinetically the PrnB transporter and a secondary proline transport system. Our results open new possibilities for studies using unicellular conidiospores of filamentous fungi and constitute a necessary first step for a subsequent structure-function analysis of the PrnB transporter.

10. "Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes."
G. Diallinas, L. Gorfinkiel, H.N.Jr. Arst, G. Cecchetto and C. Scazzocchio.
Journal of Biological Chemistry, vol. 270, pages 8610-8622, (1995).   View at publisher's site   Request copy
Abstract: In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources. The residual growth of the mutant strains is due to the activity of a broad specificity purine permease. We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations. Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively. The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed. uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene. The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments. The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters. Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships.

9. "Amino acid transporters of lower eukaryotes: regulation, structure and topogenesis."
V. Sophianopoulou and G. Diallinas.
FEMS Microbiology Reviews, vol. 16, pages 53-75, (1995).   View at publisher's site   Request copy
Abstract: Lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans possess a multiplicity of amino acid transporters or permeases which exhibit different properties with respect to substrate affinity, specificity, capacity and regulation. Regulation of amino acid uptake in response to physiological conditions of growth is achieved principally by a dual mechanism; control of gene expression, mediated by a complex interplay of pathway-specific and wide-domain transcription regulatory proteins, and control of transport activities, mediated by a series of protein factors, including a kinase, and possibly, by amino acids. All fungal and a number of bacterial amino acid permeases show significant sequence similarities (33-62% identity scores in binary comparisons), revealing a unique transporter family conserved across the prokaryotic-eukaryotic boundary. Prediction of the topology of this transporter family utilizing a multiple sequence alignment strongly suggests the presence of a common structural motif consisting of 12 alpha-helical putative transmembrane segments and cytoplasmically located N- and C-terminal hydrophilic regions. Interestingly, recent genetic and molecular results strongly suggest that yeast amino acid permeases are integrated into the plasma membrane through a specific intracellular translocation system. Finally, speculating on their predicted structure and on amino acid sequence similarities conserved within this family of permeases reveals regions of putative importance in amino acid transporter structure, function, post-translational regulation or biogenesis. .

8. "A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding."
G. Diallinas and A.K. Kanellis.
Plant Molecular Biology, vol. 26, pages 473-479, (1994).   View at publisher's site   Request copy
Abstract: 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. 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. .

7. "Genetic and biochemical evidence for yeast GCN2 protein kinase polymerization."
G. Diallina and G. Thireos.
Gene, vol. 143, pages 21-27, (1994).   View at publisher's site   Request copy
Abstract: The GCN2 (general control kinase 2) protein is an eIF2-alpha (eukaryotic initiation factor alpha) kinase which mediates translational derepression of the yeast general control transcriptional activator, GCN4, upon amino-acid starvation. We isolated and characterized GCN2 mutations differentially affecting GCN2 function. Mutations mapping in, or close to, the ATP-binding site of the kinase moiety result in constitutively activated GCN2 molecules. A C-terminal regulatory mutation dramatically affects translation initiation rates resulting in pleiotropic phenotypes. The effect of mutations in both regions were found to depend on eIF2-alpha phosphorylation. We have demonstrated that GCN2 mutants have altered autophosphorylation activities in vitro, depending on the presence or absence of a wild-type GCN2 gene and that GCN2 elutes in gel-filtration chromatography fractions with high apparent molecular mass. Both these genetic and biochemical findings suggest that GCN2 functioning might involve polymerization to form dimers or tetramers. .

6. "Structure-function analysis of the proline permease (PRNB) of the filamentous fungus Aspergillus nidulans."
U. Tazebay, V. Sophianopoulou, A. Rosa, C. Scazzocchio and G. Diallinas. G.
Folia Microbiologica (Praha), vol. 39, pages 551, (1994).   View at publisher's site   Request copy
Abstract: No abstract available.

5. "Genetic and molecular characterisation of purine permease genes of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes."
G. Diallinas, L. Gorfinkiel, H.N.Jr. Arst, G. Cecchetto and C. Scazzocchio.
Folia Microbiologica (Praha), vol. 39, pages 513–514, (1994).   View at publisher's site   Request copy
Abstract: The ascomycete fungus Aspergillus nidulans can utilize purines (adenine, guanine, hypoxanthine, xanthine, and uric acid) as sole nitrogen sources. The expression of most structural genes involved in the pathway of purine uptake and catabolism is subject to uric acid induction, mediated by the product of the positive regulatory gene uaY, and to nitrogen metabolite repression, mediated by the product of the general, positive-acting, GATA-like transcription factor, encoded by the areA gene.

4. "Sequence and regulation of the uapA gene encoding a uric acid-xanthine permease in the fungus Aspergillus nidulans."
L. Gorfinkiel, G. Diallinas and C. Scazzocchio.
Journal of Biological Chemistry, vol. 268, pages 23376–23381, (1993).   View at publisher's site   Request copy
Abstract: The nucleotide sequence of the uapA gene, coding for the uric acid-xanthine permease of Aspergillus nidulans, has been determined. The predicted uapA gene product comprises 595 amino acids (M(r) 63,365); it is a highly hydrophobic protein with 12-14 putative transmembrane segments and shows no striking similarity to any other membrane protein of either prokaryotes or eukaryotes, except for a short highly hydrophobic amino acid sequence conserved in a number of different permeases. The presence of an acidic, amphipathic region overlapping with the last hydrophobic segment of UAPA could also be of interest. The results presented suggest that the UAPA permease represents a new type of membrane protein, not described previously. The transcription of uapA is inducible by 2-thiouric acid, and it is highly repressible by ammonium. It is almost absolutely dependent on the presence of functional uaY and areA regulatory gene products. A specific mutation in the GATA binding zinc finger of the AREA protein nearly abolishes uapA transcription. The uap100 cis-acting, up-promoter, constitutive mutation is a duplication that comprises two GATA sites and suppresses weakly the AREA zinc finger mutation but does not alleviate the need for functional UAY and AREA proteins.

3. "AUA1, a gene involved in ammonia regulation of amino acid transport in Saccharomyces cerevisiae."
V. Sophianopoulou and G. Diallinas.
Molecular Microbiology, vol. 8, pages 167–178, (1993).   View at publisher's site   Request copy
Abstract: 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 glutamine inactivate GAP1 function while proline and urea allow its maximum expression. We have isolated and characterized a gene, AUA1, involved in ammonia regulation of GAP1 activity. AUA1 is not essential for growth but overexpression of the AUA1 transcript in a high-copy vector or due to a regulatory mutation, aua1-1, present approximately 10 bp upstream from the start of AUA1 transcription, 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 gap1 background. AUA1 expression is itself ammonia repressible in a wild-type strain but not in the aua1-1 mutant. The AUA1 gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11,714 Da. This polypeptide is highly hydrophilic and extremely basic. The AUA1 product shows no significant similarity with any previously 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 AUA1 are discussed. .

2. "Operator derepressed mutations in the proline utilisation gene cluster of Aspergillus nidulans."
V. Sophianopoulou, T. Suárez, G. Diallinas and C. Scazzocchio.
Molecular and General Genetics, vol. 236, pages 209-213, (1993).   View at publisher's site   Request copy
Abstract: The 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.

1. "A gene coding for the uric acid-xanthine permease of Aspergillus nidulans: inactivational cloning, characterization, and sequence of a cis-acting mutation "
G. Diallinas and C. Scazzocchio.
Genetics, vol. 122, pages 341-350, (1989).   View at publisher's site   Request copy
Abstract: 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.