©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

George Diallinas (1)(§), Lisette Gorfinkiel (1) (3)(¶), Herbert N. ArstJr. (2), Gianna Cecchetto (1) (3)(**), Claudio Scazzocchio (1)

From the (1) Institut de Génétique et Microbiologie, Unité Associé au Centre National de la Recherche Scientifique 1354, Université de Paris-Sud, Batiment 409, Centre d'Orsay, F-91405, France, the (2) Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom, and the (3) Seccion Bioquimica, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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= 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.


INTRODUCTION

The ascomycete Aspergillus nidulans is able to utilize purines as nitrogen sources. The pathway of purine catabolism has been studied genetically and physiologically (Scazzocchio and Darlington, 1968; Scazzocchio et al., 1982). The expression of the genes involved is subject to pathway-specific induction by uric acid (Scazzocchio, 1973; Sealy-Lewis et al., 1978), mediated by the uaY-positive regulatory gene product (Scazzocchio et al., 1982; Suárez et al., 1991a, 1991b) and to nitrogen metabolite repression mediated by the GATA-like, areA-positive regulatory gene product (Arst and Cove, 1973; Kudla et al., 1990).

Early genetic evidence suggested the existence of two specific, active transport systems responsible for the uptake of various purines and their analogues: (i) a system responsible for the uptake of uric acid and xanthine, their thioanalogues 2-thiouric acid and 2-thioxanthine, allopurinol, and oxypurinol (Darlington and Scazzocchio, 1967; Arst and Cove, 1973; Arst and Scazzocchio, 1975; Scazzocchio and Arst, 1978). The uapA gene has been genetically identified as the gene coding for the major permease of this system. Similar to previously isolated uapAloss-of-function mutations (Darlington and Scazzocchio, 1967), uapA insertional inactivation mutations (Diallinas and Scazzocchio, 1989) result in leaky growth on uric acid or xanthine as nitrogen sources and in partial resistance to their thioanalogues, allopurinol and oxypurinol. These observations suggested the existence of, at least, one other permease with similar specificity with the uapA gene product. The uapA gene has been cloned (Diallinas and Scazzocchio, 1989), its sequence determined, and its expression studied at the level of message accumulation (Gorfinkiel et al., 1993). uapA codes for a highly hydrophobic protein with 12-14 putative transmembrane domains. Its expression is inducible by 2-thiouric acid (and by implication by uric acid), repressible by ammonium, and almost absolutely dependent on functional uaY and areA gene products. (ii) A system specific for the uptake of adenine, guanine, hypoxanthine, and the analogues 8-azaguanine and purine; loss-of-function mutations in the corresponding gene, azgA, led to azaguanine resistance and to reduced growth on adenine, guanine, and hypoxanthine as nitrogen sources. In contrast to the highly regulated expression of the uapA gene, azgA expression is probably constitutive (Scazzocchio and Darlington, 1967). Similar to uapAmutants, azgAloss-of-function mutants have leaky phenotypes, suggesting the existence of a ``secondary'' permease transporting the same purines and analogues as the azgA gene product (Darlington and Scazzocchio, 1967).

The mutation areA102 is a leucine to valine change in the zinc finger of the AreA regulatory protein (Kudla et al., 1990) altering specifically the expression of several genes under areA control. It results in enhanced expression of a number of genes, abolishes the expression of others, and does not affect the expression of some others (Hynes and Pateman, 1970; Arst and Cove, 1973; Arst and Scazzocchio, 1975). Although areA102 mutants show normal expression of all genes encoding enzymes involved in purine catabolism, they cannot grow on uric acid or xanthine as nitrogen sources due to reduced uptake of these purines (Arst and Scazzocchio, 1975; Gorton, 1983). Strains carrying this mutation show unimpaired growth on the uric acid precursor, hypoxanthine. We have shown that the areA102 mutation leads to drastically reduced uapA gene expression (Arst and Scazzocchio, 1975; Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993). The inability of the strains carrying areA102 mutation to grow on uric acid or xanthine is absolute, in contrast to strains carrying uapAmutations. Interestingly, the areA102 mutation also eliminates the residual growth of azgAmutants on hypoxanthine (or adenine or guanine) (see below). These observations suggested that this mutation affects similarly the expression of uapA and of the gene(s) coding for the uptake proteins responsible for the residual growth of strains carrying the uapAand azgAmutations. As areA102 single mutants are not affected in their utilization of hypoxanthine it can be concluded that the azgA gene is normally expressed in the presence of the areA102 mutation.

We have previously referred to the gene uapC (Scazzocchio and Gorton, 1977; Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993, Scazzocchio, 1994) as a gene which might code for a secondary uric acid-xanthine permease. Here, we present rigorous genetic and biochemical evidence that the uapC gene product is a wide specificity purine permease responsible for the leaky phenotypes of uapAand azgAmutants on various purines as nitrogen sources. We report the sequence of the uapC gene and analyze its putative product which shows approximately 58% identity to the UapA permease and significant similarities to a number of bacterial pyrimidine trans-porters. Our analyses also suggested that two previously reported open reading frames of unknown function in Escherichia coli and Clostridium perfrigens might encode purine transporters. Finally, we study the regulation of uapC message accumulation under different physiological conditions of growth and in different regulatory mutants.


EXPERIMENTAL PROCEDURES

Media and Growth Conditions

Standard media and growth conditions of A. nidulans were previously described (Cove, 1966; Scazzocchio et al., 1982). Supplements were added when necessary. Non-inducing, inducing, and inducing-repressing conditions have been previously established (Suárez et al., 1991a).

Strains

The following A. nidulans strains were used: biA1; pabaA1; yA2 pantoB100; fwA1 pyroA4 areA102; uapA24 pabaA1; uapA24 azgA4 pabaA1; uap-100 biA1; uap-100 areA102 pabaA1; uapC201biA1; fwA1 uap-100 uapC201 biA1; uapC201 areA102 pabaA1; azgA4 pabaA1; azgA4 areA102 pyroA4 pabaA1; fwA1 uapC201 areA102 azgA4 pyroA4 pabaA1; uaY207 pabaA1; uapC201 uapC401 pabaA1; uapC201 uapC401 uapA24 pabaA1; uapC201 uapC401 azgA4 pabaA1; uapC201 uapC401 uapA24 azgA4 pabaA1; uapC201 areA102 argB2 pabaA1; biA1, pabaA1, pyroA4, pantoB100 and argB2 indicate auxotrophies for biotin, p-aminobenzoic acid, pyridoxine, pantothenic acid, and arginine, respectively. yA2 and fwA1 result in yellow and fawn conidia, respectively. All these markers do not affect the regulation of gene products involved in purine catabolism. areA102 is described in the Introduction and has also been described previously (Kudla et al., 1990). uapC201 and uapC401 are described in this article. uapA24, azgA4, and uaY207 (Scazzocchio et al., 1982; Suarez et al., 1991a, 1991b) are non-revertible loss-of-function mutations in the corresponding genes. uap-100 is an up-promoter regulatory mutation described previously (Arst and Scazzocchio, 1975; Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993). Double and triple mutants were constructed by standard A. nidulans genetic techniques (Pontecorvo et al., 1953; McCully and Forbes, 1965).

Purine Transport Assays

Purine uptake was assayed by a novel method.() This method measures uptake in germlings. These are conidiospores incubated for a short time in nutrient media and thus committed to germination and nuclear division but where the germinal tube is not yet visible. Preliminary work in a variety of systems, including purine permeation, demonstrated that germlings showed the same response to induction as mycelia.This method avoids all the complications of uptake measurements in a mycelial mat (Scazzocchio and Arst, 1975). The appropriate strains of A. nidulans were grown for 3 days on complete media at 37 °C. Conidiospores were collected and used to inoculate liquid cultures of 200 ml of minimal medium with 5 m M urea as sole nitrogen source, all necessary supplements, and 27 µ M of 2-thiouric acid as a gratuitous inducer. Before inoculation the spores are counted and the concentration adjusted so as to give a final concentration of 5 10conidiospores/ml in the culture media. Inoculated cultures were incubated at 120 revolutions/min for 210 min at 37 °C. At the end of this incubation step, conidiospores were filtered through millipore filters (0.45 µm) and washed with minimal medium plus supplements. Washed conidia were resuspended in 10 ml of minimal medium plus supplements. This gives a concentration of 10conidia/ml. 0.5 ml-aliquots of this suspension were distributed in 2-ml Eppendorf tubes and equilibrated for 10 min at 37 °C. Uptake was initiated by the addition of radiolabeled uric acid or hypoxanthine at a final concentration of 20 µ M. Uptake assays were terminated by the addition of cold uric acid or hypoxanthine, at a final concentration of 0.8 m M, after 15, 30, 60, and 120 s. Conidiospores were then washed three times with 2 ml of ice-cold minimal medium plus 0.8 m M uric acid or hypoxanthine, boiled in 5% trichloroacetic acid, and soluble radioactivity was measured by liquid scintillation counting. The uptake rates of uric acid and hypoxanthine were linear throughout the sampling period. We have also determined that, under the above conditions, uptake is directly proportional with the number of conidiospores in the suspension. The actual viable conidiospore number was determined, for each assay, by removing and plating on complete medium, serial dilutions of an aliquot obtained before the final washing step. Initial uptake rates are expressed in p M substrate incorporated/min 10viable conidiospores. Uniformly labeled [8-C]uric acid (specific activity 1.94 GBq/mmol) and [G-H]hypoxanthine (specific activity 244 GBq/mmol) were obtained from Isotopchim, Ganagobie-Peyruis, France and Amersham International, United Kingdom.

Transformation Techniques

A. nidulans and E. coli transformations were performed as described by Tilburn et al. (1983) and Hanahan (1983), respectively.

Nucleic Acid Extraction and Manipulations

Total RNA and genomic DNA isolation from A. nidulans strains has been previously described by Lockington et al. (1985). Plasmid isolation from E. coli strains and DNA manipulations were performed as described by Sambrook et al. (1989).

Nucleic Acid Hybridizations

Southern and Northern blot analyses were performed, basically, as described by Sambrook et al. (1989), in 0.5 M sodium orthophosphate, pH 7, 1 m M EDTA, 7% sodium dodecyl sulfate, and 1% bovine serum albumin, at 65 °C for 18 h. [P]ATP-labeled DNA molecules used as gene-specific probes were prepared by using random hexanucleotide primers following the supplier's instructions (Amersham). Cloning of the uapC gene involved screening of a EMBL4 genomic library of A. nidulans (a gift from C. M. Lazarus) in the same buffer as that described for Northern or Southern blots, at 58 °C, and filters were then washed twice in 2 SSC, at 58 °C.

DNA Sequencing

DNA was sequenced using the dideoxynucleotide chain termination method (Sanger et al., 1977) on pAN903 and bluescript KSII+ double-stranded subclones of the uapC locus (see Fig. 3) and a series of oligonucleotides specific for both strands of the uapC locus. The oligonucleotides used were the following: 5`-CTGCAGCCTCTCGAAGC-3` (O1), 5`-ACCGTCAATCCCCGCCC3` (O2), 5`-CGATCAATCTCGACGTA3` (O3), 5`-GCGCATGCTGCAATCCC3` (O4), 5`-CGAAGTTCCTACCACCG3` (O5), 5`-CGGTGGTAGGAACTTCG-3` (O6), 5`-CCCAGATGAAGGATGCG-3` (O7), 5`-CATTCAGGGTGGCGTCC-3` (O8), 5`- GATAACGCCGTTGTTCTG-3` (O9), 5`- CGTTCCTCTTCTCCTCCG-3` (O10), 5`-GGAGGAGGATGTTGTGG-3` (O11), 5`-GACCAGAGGAACCAGGC-3` (O12), 5`-CTTTCATTGCGTTGCCG-3` (O13), 5`-GGAGTTAGCAAAGCG-3` (O14), 5`-GC-CGTTCGTGTGCGAGC-3` (O15), 5`-GATCCCACTGGTCCTCG-3` (O16), and the T3 and T7 primers (Pharmacia).


Figure 3: Physical map of the uapC gene region cloned in pAN903. Coding sequences are shown as white bars. Non-coding sequences, including a short intron, are shown as black bars. The direction of transcription is indicated by an arrow. uapC subclones used in determining the uapC nucleotide sequence and a nearly complete uapC cDNA clone used in determining the presence of introns (see ``Experimental Procedures'') are shown by gray and hatched bars, respectively. The sequencing strategy for both strands is shown with arrows (internal primers: filled triangles, universal; T7 and T3 primers, open triangles). S, SalI; P, PstI; Xb, XbaI; Xh, XhoI; H, HindIII;



cDNA Clones

5 µg of total RNA extracted from induced wild-type mycelia were denatured by heating for 5 min at 70 °C and reverse transcribed, in the presence of 1 unit of RNasin (Boehringer Mannheim), with 20 units of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim), using the oligonucleotide O3, for 1 h at 45 °C. The first strand thus synthesized was directly annealed with the oligonucleotide O12, 1 unit of Taq DNA polymerase was added, and reiterative polymerase chain reaction cycles were performed as described in the protocol of the GeneAmp DNA Amplification Reagent kit 5 (Perkin-Elmer-Cetus Instruments). The program used included 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 2 min, and elongation at 72 °C for 3 min. The resulting DNA product consisted of 1793 bp() and included the complete 5`-non-coding region of the uapC transcript and a nearly complete uapC open reading frame (the last four, C-terminal, codons are missing). This cDNA fragment was cloned by standard procedures in bluescript KSII+ digested with the SmaI restriction enzyme and sequenced as described previously for the genomic uapC clones. To map the 3` end of the uapC transcript, a reverse transcription reaction was performed, as described above, using an oligo(dT) primer, and then the DNA single strand products were amplified with oligonucleotide O11 in the presence of radiolabeled [S]dATP (Amersham). The size of the radiolabeled uapC-specific DNA product was estimated (139 and 142 bp) by 6% polyacrylamide-urea gel electrophoresis, using as size markers a uapC DNA sequence reaction with the same oligonucleotide (O11).

5` Primer Extension

Primer extension was performed using 5 µg of either non-induced or induced total RNA and the oligonucleotide O4 in the presence of 20 units of reverse transcriptase, 1 unit of RNasin, a ``cold'' nucleoside mixture (20 µ M) of dATP, dCTP, dGTP, and dTTP, and 5 µCi of radiolabeled [S]dATP, in a standard extension buffer, for 1 h at 45 °C. The size of the synthesized products was estimated by standard polyacrylamide-urea gel electrophoresis, using a sequence reaction as DNA size ladder.

Sequence Analyses

DNA and protein sequence analyses were performed with the DNA Strider1.2 program (Marck, 1988). DNA and protein searches, comparisons, and alignments were performed using the FASTA (Pearson and Lipman, 1988), BLAST (Altschul et al., 1990), GAP, BESTFIT, PILEUP, and PRETTYBOX (Smith and Waterman, 1981; Devereux et al., 1984) programs and the data bases SwissProt, PIR, and the translation of the EMBL/GenBank nucleotide sequences.


RESULTS

Genetic Identification of the uapC Gene: Isolation of uapC Gain-of-function Mutants

Arst and Scazzocchio (1975) previously described the isolation of uap-100, defined as an initiator constitutive, up-promoter mutation in the uapA gene. This mutation was later shown to be a 164-bp tandem duplication located 103 bp upstream from the uapA translation start resulting in a pronounced increase in the steady state uapA mRNA levels under both non-induced and induced conditions (Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993). The uap-100 mutation was selected as a mutation suppressing the inability of an areA102 mutant to grow on uric acid.

Extragenic revertants of a uapA24 (loss-of-function) areA102 double mutant selected on uric acid or xanthine should include gain-of-function mutations, analogous to uap-100, in the gene(s) coding for the secondary permease(s) which are not expressed in an areA102 background. Indeed, we isolated a number of suppressors which defined uapC, a new gene involved in uric acid-xanthine uptake. The mutation uapC201 exemplifies this class of suppressors (see Fig. 1); it was mapped by mitotic haploidization (McCully and Forbes, 1965) to chromosome I but recombines freely with uapA mutations (results not shown). Four phenotypically identical revertant mutations were closely linked to uapC201 (results not shown). By analogy to the uap-100 mutation, the specific suppression of the areA102 allele might result from regulatory mutations in the promoter of the uapC gene. In agreement with this assumption, we showed (results not shown) that: (i) in an areAbackground, the uapC201 mutation leads to increased sensitivity to 2-thioxanthine and 2-thiouric acid even in the presence of strongly repressing concentrations (10 m M) of ammonium as a nitrogen source; (ii) a uapC201 uapA24 double mutant grows clearly better than a uapA24 single mutant on uric acid or xanthine as nitrogen sources; and (iii) a uapC201 uap-100 double mutant shows an additive phenotype in respect to 2-thioxanthine (or 2-thiouric acid) sensitivity suggesting that two different uptake systems are involved. These results strongly suggested that the uapC gene codes for a permease able to take up, at least, uric acid, xanthine, 2-thioxanthine, and 2-thiouric acid. As areA102 eliminates the residual growth of azgAmutants on hypoxanthine, adenine, or guanine as nitrogen source, it could be asked whether the UapC permease is also responsible for the residual uptake of these purines. A triple mutant, areA102 azgA4 (loss-of-function) uapC201, was constructed and tested on various purines as nitrogen sources. Fig. 1shows that uapC201 suppresses partially the inability of an azgA4 areA102 double mutant to utilize hypoxanthine (and also adenine and guanine, results not shown) as sole nitrogen source and leads to reduced resistance to 8-azaguanine (not shown).


Figure 1: Growth tests of a uapC gain-of-function mutant. The strains tested (see diagram) are described under ``Results'' and ``Discussion.'' Complete genotypes are described under ``Experimental Procedures.'' Petri dishes contain 1% glucose-minimal medium plus necessary supplements (see ``Experimental Procedures'') and either 3.3 m M ammonium tartrate ( N), or 10 m M uric acid ( UA), or 10 m M hypoxanthine ( H), as sole nitrogen source. Growth was scored after 48 h at 37 °C.



Thus, our results show that: (i) uapC codes for a permease able to transport all naturally occurring purines, thus overlapping the specificities of the uapA and azgA gene products; (ii) uapC gene expression is under areA control and is absent (as judged by growth tests) in strains carrying the areA102 allele; and (iii) uapC gain-of-function mutations, such as uapC201, specifically but partially suppress the effect of the areA102 allele.

Isolation and Genetic Characterization of uapC Loss-of-function Mutants

In order to substantiate the role of the uapC gene in purine uptake, we isolated loss-of-function mutants. A uapC201 areA102 mutant strain, which is sensitive to 0.1 mg/ml 2-thioxanthine in the presence of 3 m M ammonium as nitrogen source, was mutagenized by UV light or N-methyl- N-nitro- N-nitrosoguanidine and 2-thioxanthine-resistant colonies were selected as described previously (Alderson and Scazzocchio, 1967; Diallinas and Scazzocchio, 1989). Such mutants could, in principle, be the result of loss-of-function mutations in a number of genes involved in purine catabolism ( uaY, hxA, hxB, cnx; Diallinas and Scazzocchio, 1989), as well as, loss-of-function mutations in the uapC or areA genes. However, very simple growth tests previously described (Alderson and Scazzocchio, 1968; Diallinas and Scazzocchio, 1989) allowed us to distinguish uptake mutants (and thus potential uapCmutants) from all other classes. These mutants should not grow on uric acid or xanthine, grow less well than wild-type strains on hypoxanthine, adenine, or guanine, and show resistance to 2-thioxanthine, 2-thiouric acid, and allopurinol. Five strains with the expected phenotype were isolated. In order to investigate whether the putative uapCmutations ( uapC401, uapC402, uapC403, uapC404, and uapC405) were genetically linked to the uapC gene (identified by the uapC gain-of-function mutations), we crossed all putative uapCuapC201 areA102 mutants to a uapCareA102 strain. Only uapCuapC201areA102 recombinants should be able to grow on uric acid as a nitrogen source. All putative uapCmutations showed very tight linkage to the uapC201 mutation; mutations uapC403, uapC404, uapC405 did not recombine with mutation uapC201 while mutations uapC401 and uapC402 recombined at low frequencies (10to 10).

We also crossed the putative uapCmutants with a wild-type strain ( uapCuapC201areA102 uapCareA, see ``Experimental Procedures'' for full genotype of strains) and in no case isolated strains showing a uapC201 phenotype among the progeny tested (100 progeny/cross). These crosses further confirmed the tight genetic linkage of the uapC loss-of-function and gain-of-function mutations.

The uapCmutations were placed in an areAbackground and all possible combinations of double and triple mutants carrying the uapA, azgA , and uapC loss-of-function mutations were constructed. Fig. 2 shows some crucial growth tests. Our results confirm that the uapC gene codes for a wide specificity purine permease overlapping the uptake activities of the uapA and azgA permeases. As uapCsingle mutations impair growth on hypoxanthine (and adenine and guanine) considerably less than azgAmutations and on uric acid (and xanthine) less than uapAmutations, but eliminate the leaky growth of strains carrying either uapAor azgAsingle mutations, it can be concluded that the uapC gene codes for a permease of wide specificity but having lower capacity for transport.

Purine Transport in Wild-type and Mutant Strains

We examined the rates of purine uptake in wild-type and all single, double, and triple purine permease loss-of-function mutants described above. Radiolabeled uric acid and hypoxanthine initial uptake rates were determined by a novel method using germlings (germinated conidiospores) incubated under induced conditions (see ``Experimental Procedures''). Table I shows a typical experiment of a series performed with higly consistent results. The rate of uptake of hypoxanthine is about five times higher than the rate of uptake of uric acid. Transport rates of uric acid and hypoxanthine uptake in mutant strains were in perfect agreement with the growth tests presented above. Uric acid incorporation is not altered in azgAstrains. It is nearly completely eliminated in strains carrying both uapA and uapC loss-of-function mutations. uapA and uapC single mutants show approximately 30 and 50-55% uric acid uptake rates, respectively, as compared to the wild-type. Hypoxanthine incorporation is dramatically reduced in azgAmutants (8-10% of wild-type rates). It is not altered in uapAsingle mutants. The rate of uptake of uapCand uapAuapCmutants is not distinguishable from the wild-type rate. However, the contribution to hypoxanthine uptake of the uapC permease is revealed by the azgAuapCdouble mutant (and the azgAuapC-uapAtriple mutant). While the contribution to hypoxanthine uptake of the UapC permease is masked by the much more efficient AzgA permease, it becomes obvious in its absence. This was predicted from the growth tests. Fig. 2 shows that in a uapCsingle mutant strain growth on hypoxanthine as nitrogen source is not impaired. However, the residual growth of an azgAstrain is abolished by the introduction of an uapCmutation.

While we have measured the uptake rates only for hypoxanthine and uric acid, results cited above show that UapA is specific for oxydized purines and their analogues, AzgA for hypoxanthine, adenine, guanine, and their analogues while UapC can accept all these purines. The uptake rates and the growth tests indicate, however, that UapC plays a more important role in the uptake of the oxydized than the reduced purines.

Molecular Cloning of the uapC Gene

Diallinas (1989) observed that in Southern blot analyses of A. nidulans DNAs, a uapA-specific probe detects weakly hybridizing non- uapA sequences. Given the close functional relationship of the uapA and uapC gene products, we considered the possibility that a uapA clone might cross-hybridize with uapC DNA sequences thus allowing the cloning of uapC by screening an A. nidulans genomic EMBL4 bank with a uapA-radiolabeled probe at low stringency hybridization conditions. We screened 5000 clones and isolated six, non- uapA, positively hybridizing plaques (see ``Experimental Procedures''). Restriction and Southern blot analyses identified two overlapping clones corresponding to the same genomic region of A. nidulans (results not shown). The restriction profiles of these clones was different from that expected if the phages contained the uapA genomic region of A. nidulans.

We demonstrated that this region contains the uapC gene as follows. First, we have shown that a vector carrying the 3.3-kb SalI- SalI genomic fragment of A. nidulans DNA present in clone LAN903 is sufficient to complement a uapCloss-of-function mutation. To this end, we constructed plasmid pAN903, carrying this fragment cloned into Bluescript KSII+ (Fig. 3). This plasmid was used to transform both a strain which cannot take up and utilize uric acid, uapC401 uapC201 uapA24, and a strain that cannot take up or utilize either uric acid or hypoxanthine ( uapC401 uapC201 uapA24 azgA4, see ``Experimental Procedures'' for full genotypes). Transformants were selected, respectively, on uric acid and on either uric acid or hypoxanthine as nitrogen sources. In each experiment, plasmid pAN503 (Diallinas and Scazzocchio, 1989) carrying an intact uapA gene was used as a positive control. The plasmid containing the putative uapC insert, pAN903, transformed both recipient strains for growth on uric acid at low frequencies comparable to those obtained with the plasmid pAN503 (about 10 transformants/µg of DNA). As expected, pAN903 also transformed the uapC401 uapC201 uapA24 azgA4 recipient strain for growth on hypoxanthine. Southern blot analysis of transformants obtained from the latter strain showed that heterologous integration of a single copy of pAN903 in the genome of A. nidulans is sufficient to complement the mutations carried by this strain for growth on uric acid or hypoxanthine (results not shown). This, together with sequencing data (see below), strongly suggests that the entire uapC gene is included in the 3.3-kb SalI- SalI restriction fragment of pAN903.

We further established the identity of the pAN903 insert by inactivation of the resident A. nidulans gene. A KSII+ vector carrying a 0.7-kb insert isolated from the PstI- PstI 1.6-kb restriction fragment (by removing the internal 0.9-kb XhoI- XhoI restriction fragment; see Fig. 3) was used, together with a plasmid carrying the argB gene, to cotransform a uapC201areA102argB2 strain (complete genotype under ``Experimental Procedures''). This strain utilizes uric acid as nitrogen source only because the uapC201 gain-of-function mutation suppresses the phenotype of areA102 on uric acid (see above). Thus the utilization of uric acid by this strain is strictly dependent on the integrity of the UapC permease. Among 1500 argBtransformants analyzed, two showed an apparent uapCphenotype (scored by lack of growth on uric acid as a nitrogen source and resistance to the analogue 2-thioxanthine). Southern blot analysis (not shown) confirmed that the recipient genomic 1.6-kb PstI- PstI restriction fragment was disrupted. Sequence analysis (see below) has confirmed that the disrupted sequences map within the uapC open reading frame. Both inactivation and complementation studies show that the sequences cloned contain the uapC gene. Further evidence as to the identity of the pAN903 insert came from studies on the regulation of expression and the sequence of the uapC gene described below.

Regulation of uapC Message Accumulation

We studied uapC message accumulation in wild-type and mutant strains grown under different physiological conditions by Northern blot analyses, as described under ``Experimental Procedures.'' Our results are summarized in Fig. 4. Fig. 4 a shows that (i) uapC maximum message accumulation is dependent on a functional uaY product and (ii) ammonium drastically represses uapC message accumulation. Fig. 4 b shows that uapC message accumulation is (i) weakly (2-3-fold) inducible by the gratuitous inducer 2-thiouric acid, (ii) increased (5-fold) in a uapC201 mutant strain, (iii) drastically diminished in an areA102 mutant strain, and (iv) significantly higher in a uapC201 areA102 double mutant than in an areA102 single mutant (but still lower than in a wild-type strain). These results further confirm that we have cloned the uapC gene and reveal how uapC message accumulation is regulated in response to pathway-specific induction and nitrogen metabolite repression.


Figure 4: Northern blot Analyses of uapC message accumulation. Approximately 5 µg of total RNA were loaded in each lane as described under ``Results'' and ``Experimental Procedures.'' Panel a, wild-type ( lanes 1-3) and uaY207 (loss-of-function) mutant ( lanes 4-6) strains grown under non-inducing ( lanes 1 and 4), inducing-repressing ( lanes 2 and 5), and inducing ( lanes 3 and 6) conditions, as described previously (Suárez et al., 1991b). Panel b, wild-type ( lanes 1 and 2), uapC201 mutant ( lanes 3 and 4), uapC201 areA102 mutant ( lanes 5 and 6), and areA102 mutant ( lanes 7 and 8) strains grown under non-inducing ( lanes 1, 3, 5, and 7) and inducing ( lanes 2, 4, 6, and 8) conditions. Below both panels a and b, are the same blots, washed and rehybridized using the A. nidulans actin gene (Fidel et al., 1988) as a probe.



Sequence of the uapC Gene

We have shown that the entire uapC gene is included within the 3.3-kb SalI- SalI restriction fragment of pAN903. The complete sequence of this DNA fragment was established for both strands (see Fig. 5). The sequencing strategy is described under ``Experimental Procedures.'' Our results revealed a single long open reading frame of 1737 bp interrupted by a single putative short intron of 46 bp recognized by the 5` and 3` consensus sequences of fungal introns (Gurr et al., 1987) whose existence is confirmed below. The direction of this open reading frame is consistent with the direction of uapC transcription established by Northern blot analysis using single-stranded radiolabeled probes (results not shown). The high GC content of the uapC sequence is in agreement with all previously characterized A. nidulans genes (Lloyd and Sharp, 1991).

The 5`-non-coding region of the uapC gene contains consensus sequences of putative regulatory importance (see Fig. 5). A 5`-TATA-3`box (Proudfoot, 1979) is found at position -113/-110 from the putative start of translation. The consensus sequence 5`-WGATAR-3` (both in the coding and non-coding strand) is found five times (positions -189/-184, -212/217, -250/-245, -524/529, and -562/567). These are candidate target sequences for AreA binding (Fu and Marzluf, 1990a; 1990b).() The sequence 5`-CGGTGCCGCCCG-3` at positions -280/-269 is a candidate sequence for the target site of the UaY regulatory protein as deduced by in vitro protection and interference studies conducted with fusion proteins containing the UaY zinc finger.() Finally, a sequence of unknown significance, 5`-TTTGCATTA-3` at positions -180/-172 is also found, similarly positioned (-125/-117), in the uapA 5`-non-coding region (Gorfinkiel et al., 1993).


Figure 5: Nucleotide Sequence of the uapC gene and amino acid sequence of its putative translation product. The nucleotide sequence shown was determined by sequencing both strands of a genomic clone and a nearly complete cDNA clone (see ``Experimental Procedures''). Restriction sites PstI and XbaI are indicated to allow the orientation of this sequence with respect to the physical map shown in Fig. 1. Putative AreA binding 5`-GATA-3` sites are shown by horizontal arrows. A putative UaY binding 5`-CGGTXCCCG-3` site is indicated by an open box. A 5`-TATA-3` box and a sequence similar to a uapA 5` non-coding sequence are shown overlined. The translation start codon and the intron 5` and 3` consensus sequences are shown underlined, and the translation stop codon is indicated with an asterisk. The major start and stop points of transcription are shown by black triangles while a possible minor start of transcription is shown by a white triangle. A putative 3` end polyadenylation signal is also underlined.



The 3`-non-coding region of the uapC gene contains the sequence 5`-TATGTA-3` which has been described previously as a 3`-polyadenylation signal in Saccharomyces cerevisiae (Russo et al., 1991; Irniger and Braus, 1994). However, its location downstream from the end of the uapC transcript (see below) and its absence from other genes makes it rather improbable to be involved in polyadenylation in A. nidulans.

Transcript Mapping of the uapC Gene

The 5` and 3` ends of the uapC transcript as well as the presence and position of a single short intron were established. The 5` start of uapC transcription was mapped by primer extension experiments using total RNAs extracted from both non-induced and induced wild-type strains and a uapC-specific oligonucleotide (O4) close to the start of uapC translation (see ``Experimental Procedures''). Fig. 6 shows a major start site corresponding to nucleotide -64 numbering from the ATG (see Fig. 5) for both non-induced and induced mycelia. Induction by 2-thiouric acid led to approximately 3-fold increased accumulation of uapC-specific primer extension products, a result in agreement with our Northern blot analyses (see Fig. 4, a and b). At position -80, another minor transcription startpoint is detected under non-inducing conditions. Within the transcribed sequence, an ATG codon is present 34 nucleotides upstream from the proposed start codon, but it leads to an open reading frame of only five amino acids.

The 3` end of the uapC transcript was mapped by determining the size of an appropriate cDNA clone. This was constructed by reverse transcribing induced uapC RNA with a 15-(dT) oligonucleotide and amplifying the first DNA strand with a uapC specific primer (O11), in the presence of radiolabeled [S]dATP (results not shown). Fig. 5shows the 3` end points of uapC transcription located 33 and 36 nucleotides downstream from the translation stop codon.

We have also obtained a uapC cDNA clone including a nearly complete (lacking only the last four, C-terminal, codons) uapC open reading frame (see ``Experimental Procedures''). This clone was completely sequenced (as described for genomic DNA sequencing) and allowed us to confirm the presence and the position of a 46-bp short intron predicted from genomic DNA sequencing. The position of this uapC cDNA clone is shown in Figs. 3 and 5. Thus, our results lead to an estimation of 1837/1840 nucleotides for the uapC major transcript, a size in agreement with that estimated from Northern blot analyses.

Analysis of the Predicted uapC Protein

The uapC open reading frame encodes a protein of 580 amino acids with a predicted molecular mass of 61251 Da (Fig. 5). Codon usage is not biased. As expected for a transporter, the UapC protein is extremely hydrophobic. The UapC hydropathy profile is practically superimposable on that described for the UapA protein (Gorfinkiel et al., 1993), independently of the algorithm and the ``window'' used to calculate the hydropathic character (Kyte and Doolittle, 1982; Eisenberg et al., 1982); it shows hydrophilic N and C termini and 12-14 (depending on the window used) putative, -helical, transmembrane segments. No putative glycosylation sites (Struck et al., 1978) were found in the UapC amino acid sequence.

We compared the UapC deduced amino acid sequence to that of all known proteins in data bases using the programs FASTA (Pearson and Lipman, 1988) and BLAST (Altschul et al., 1990) and further calculated similarity scores using the programs GCG/BESTFIT and GCG/GAP (Devereux et al., 1984). The most striking observation was the similarity of UapC to the UapA amino acid sequence (Fig. 7). These two purine permeases share 58.4% identical and 78.3% similar amino acid residues throughout their lengths. Identity reaches nearly 70% within hydrophobic segments and is lowest in the N- and C-terminal regions of the proteins. An extremely conserved hydrophilic region (amino acid residues 355-386 in UapC, 377-409 in UapA) is also present, and its putative functional significance is considered below. It is also of interest that the UapC protein lacks three acidic amino acid residues which comprise a significant part of the acidic amphipathic -helical region of UapA (residues 530-547; Gorfinkiel et al., 1993) overlapping with the last hydrophobic segment of the protein.

Fig. 8 shows other sequences providing significantly high scoring segment pairs with the UapC amino acid sequence. These are the YicE 48.9 kDa hypothetical protein of unknown function, encoded by an open reading frame 3` to the gene coding for the glutamine transporter gene of E. coli (34% identity overall; Burland et al., 1993), a putative polypeptide coded by an open reading frame in the 5` region of the cpe gene encoding an enterotoxin in C. perfrigens (31% identity; Brynestad et al., 1994), the Bacillus subtilis pyrimidine (uracil) permease (23% identity; Quinn et al. 1991; Turner et al., 1994), the Bacillus caldolyticus uracil permease (26% identity; Ghim and Neuhard, 1994), and the E. coli uracil permease (26% identity; X73586 in data bases). Interestingly, the segment showing the highest score between UapC (or UapA) and any of these bacterial transporters is a hydrophilic region in YicE (48% identity, 65% similarity over 52 amino acid residues) overlapping the best conserved hydrophilic region common to the UapC and UapA amino acid sequences described above (see Figs. 7 and 8).

A number of other transporters and membrane proteins show scattered regions with less significant amino acid sequence identity scores (19-21%; results not shown) when compared to the UapC or UapA sequences. Such similarities probably reflect common structural determinants in hydrophobic regions of membrane proteins.


DISCUSSION

The genetic identification, uptake studies, molecular cloning, regulation of expression, genomic and cDNA sequence and transcript map of the uapC gene, and an analysis of its putative translation product have been presented. Our results provide compelling evidence that the uapC gene codes for a wide specificity, low efficiency, permease transporting all natural purines as well as purine analogues. The specificity of the UapC transporter completely overlaps the transport specificities of UapA and UapC, the two specific, high efficiency purine permeases of A. nidulans.

It was described previously (Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993) that extragenic suppression of the inability of an areA102 mutant to grow on uric acid or xanthine as sole nitrogen source resulted from positive regulatory mutations in the promoter region of the uapA gene coding for the major uric acid xanthine permease. Here we describe extragenic suppressors of an areA102 mutant carrying a uapAloss-of-function mutation ( uapA24), for growth on uric acid, which define uapC, a gene mapping on chromosome I but recombining freely with the uapA gene. We constructed a series of double mutants carrying the uapC201 gain-of-function mutation and loss-of-function ( uapA24, azgA4) or other gain-of function ( uap100) mutations in the genes coding for the two specific purine permeases in A. nidulans and showed that uapC gain-of function mutations affect the transport of all purines and analogues transported by the uapA and azgA gene products. We further established the contribution of the uapC gene product to purine uptake by isolating and genetically characterizing a series of apparent uapCloss-of-function mutations, which were shown to be tightly linked to the uapC gain-of-function mutations.

An analysis of uric acid and hypoxanthine uptake directly confirmed the role of the uapC gene product as established by genetic studies. Both uptake studies and growth phenotypes showed that the UapC protein contributes significantly to uric acid uptake and weakly to hypoxanthine uptake. Transport assays showed that the UapC protein accounts for 30 and 10% of uric acid and hypoxanthine uptake rates, respectively. These studies also confirmed the roles of the AzgA and UapA permeases as established previously as specific for uric acid-xanthine and for hypoxanthine-adenine-guanine, respectively (Darlington and Scazzocchio, 1967; Arst and Scazzocchio, 1975; Scazzocchio and Arst, 1978).

The uapC gene was cloned by low stringency hybridization of an A. nidulans genomic library to a radiolabeled uapA-specific probe. The vector constructed (pAN903) from the clone isolated was shown to include a complete uapC gene by complementation of a uapCmutation and by insertional inactivation of the uapCresident gene. In Northern blots, the cloned sequence detects a 2.0-kb RNA transcript whose level of accumulation is differentially affected by uric acid induction and ammonium repression or by the presence of an uapC gain-of-function mutation, the areA102 allele or a uaY loss-of-function mutation. Finally, uapC sequencing has shown that this gene encodes a protein with a plausible structure for a transporter and a very similar amino acid sequence with the UapA permease.

Although uapC expression is regulated very similarly to uapA gene expression in response to ammonium repression and the presence of the areA102 allele (dramatically reduced transcript levels), it shows a different response to pathway-specific induction than the uapA gene. Non-induced uapC transcript levels are higher than uapA non-induced transcript levels (Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993). On the other hand, 2-thiouric acid induction affects more strongly uapA than uapC message accumulation (6-8-fold versus 2-3-fold; Diallinas and Scazzocchio, 1989; Gorfinkiel et al., 1993).() Interestingly, we observed that the putative UaY-binding site in the 5` regulatory region of the uapC gene shows differences with the UaY target sites established for the uapA, hxA (encoding purine hydroxylase I) or uaZ (encoding urate oxidase; Oestreicher and Scazzocchio, 1993) genes.The putative uapC UaY-binding site is 5`-CGGTGCCGCCCG-3` while the target sites in the regulatory regions of the other three genes conform to the sequence 5`-CGGAXGCCG-3`.

The sequence of genomic and cDNA uapC clones has been established and, together with experiments which mapped the 5` and 3` ends of the uapC transcript, it allowed us to determine the uapC transcript sequence, identify the uapC open reading frame, and to conduct a detailed, computer-aided analysis of its derived translation product. The UapA and UapC putative permeases share approximately 58% identical amino acids throughout their amino acid sequences. Similarity scores reach 78%. Both proteins are predicted, based on hydropathy profiles, to have extremely similar higher structures (see Fig. 7). Of greatest interest, is the presence of a long hydrophilic segment located between putative transmembrane domains 9 and 10 showing 70% identical (90% similar) amino acids residues in the UapA and UapC permeases. Another hydrophilic segment, located between putative transmembrane domains 5 and 6, also shows significant similarity (56% identity) while the remaining, shorter, hydrophilic segments are less similar in the two transporters. While sequence similarities of hydrophobic segments might provide clues to the structure or the topology of transport proteins, hydrophilic segments with important similarities should, in principle, be relevant to substrate recognition or to the mechanism of transport. If the UapC protein includes 14 transmembrane segments (using an 11-amino-acid ``window'' to calculate its hydropathy), the two long hydrophilic segments conserved in UapA and UapC would be exposed to the exterior of the cell and are thus good candidates to be involved in purine recognition and binding. The UapA and UapC permeases show overlapping specificities and, probably, different capacities for purine uptake. A few amino acid changes in these hydrophilic segments might be responsible for these differences. We have noted that the UapC permease lacks three acidic amino acid residues of the C-terminal, amphipathic domain present in the UapA permease. Other, more subtle, amino acid changes can be identified (for example, residue 372 in UapC is a glutamate, while the corresponding residue in UapA is an arginine) and might be of great help in the design of site-directed mutagenesis and domain-swapping experiments for testing structure-function relationships. The importance of the described hydrophilic region of greatest similarity in the UapA and UapC permeases is further suggested by the observation that this same region shows the highest similarity score with YicE, a non-identified open reading frame of E. coli which may well be a purine transporter (see below).


Figure 7: Amino acid sequence similarity alignment of the UapC and UapA permeases by the GCG/BESTFIT program. Identical amino acids are indicated by vertical lines, and similar amino acids are indicated by two dots or a single dot. Gaps are introduced by the program to maximize similarity scores. For both permeases, hydrophobic segments which are considered putative transmembrane domains are shown boxed. The UapC permease is shown on top.



We have also shown that the A. nidulans purine transporters are similar to pyrimidine transporters of E. coli and Bacillus species (see Fig. 8). 22 amino acid residues are identical and 42 additional amino acid residues are similar in all seven transporters analyzed. Pair-wise comparisons show amino acid identity scores from 22 to 60% (similarities from 51 to 78%) and suggest that this group of proteins might be made of two subgroups, the purine and the pyrimidine transporters. Interestingly, although the E. coli YicE protein is equally similar to the A. nidulans purine permeases (59% similarity) and to the bacterial pyrimidine transporters (53-58% similarity), it shares significantly more identical amino acid residues with the first (33-34% identity) than with the second (23-27% identity). This observation suggests that this protein of unknown function might be the first prokaryotic purine permease identified. If this is true, the alignment of the YicE and UapA or UapC sequences should be revealing for the identification of amino acid ``signature'' residues determining the ability of a transporter to take up purines in both prokaryotes and eukaryotes. Moreover, the putative product of an open reading frame of unknown function in C. perfrigens was also found to belong to the group of transporters described here. This open reading frame consists of a previously described 567-bp open reading frame plus 366 bp of what has been proposed to be its immediate 5` upstream region (Brynestad et al., 1994). We obtained a continuous 933-bp open reading frame (whose putative 311 amino acid translation product is shown in Fig. 8) by assuming a frameshift sequencing mistake, and adding an ``X'' to ``re-establish'' the reading frame, immediately upstream of the previously proposed ATG start codon. This makes an incomplete amino acid sequence of a protein with a significant identity similarity score (30-60%, respectively) with all the purine-pyrimidine transporters described here. Based on specific amino acid similarities, we believe that the C. perfrigens DNA sequence might correspond to an incomplete sequence of a purine transporter gene.


Figure 8: Alignment of the UapC permease sequence with all significantly similar (according to FASTA and BLAST programs) proteins from data bases (SwissProt, Pir, GenEmbl) using the program PILEUP. Identical amino acids are shown in black boxes and similar amino acids in gray boxes ( light or deep gray according degree of similarity). Gaps are introduced to maximize similarity. The protein sequences shown are the following: pyrp-Bs, pyrimidine transporter of B. subtilis (accession number in data bases P25982); pyrp-Bc, uracil transporter of B. caldolyticus (X76083), uraa; uracil transporter of E. coli (X73586); Uapa (X71807) and Uapc permeases of A. nidulans described in the text; Yice (P27432) a hydrophobic E. coli protein of unknown function; Cpx, a putative protein of unknown function from C. perfrigens (X71844).



We found no significant sequence similarities to other prokaryotic or eukaryotic proteins, including the purine-cytosine, allantoin, or allantoic acid transporters of S. cerevisiae (Rai et al., 1988; Yoo et al., 1992), or the nucleoside transporters of bacteria (Craig et al., 1994) or mammalian cells (Pajor and Wright, 1993; Huang et al., 1994). A purine-pyrimidine transporter has been recently identified in mammalian cells (Griffith and Jarvis, 1993) but the corresponding gene has not been cloned.

Conservation of transporter amino acid sequences has been previously described for the sugar facilitators of higher eukaryotes and the sugar active transporters of lower eukaryotes and prokaryotes (Marger and Saier, 1993). In contrast, amino acid transporters or facilitators of higher eukaryotes form many distinct groups which do not show significant sequence similarities with the family of fungal amino acid permeases, the plant, or the bacterial amino acid transporters (Sophianopoulou and Scazzocchio, 1989; Christensen, 1990; Reizer et al., 1993; Grenson, 1992).() It will be of great interest to know whether, in cases of transporter sequences conserved between prokaryotes and eykaryotes, functional complementation of prokaryote mutants can be achieved with eukaryotic genes and vice versa. This would imply conservation in structure and function of the proteins involved in transporter translocation (Ljungdahl et al., 1992; Green et al., 1992).

The fungal purine permeases and the bacterial pyrimidine transporters shown in Fig. 8exhibit nearly superimposable hydropathy profiles predicting 12-14 transmembrane segments (results not shown). Most prokaryotic and eukaryotic transporters are predicted to consist of a structural motif of 12 tightly packed transmembrane domains (Griffith et al., 1992; Saier, 1994). The sequence, showing neither significant similarities to any previously described family of transporters nor internal repeats, and the predicted structure, with possibly 14 putative transmembrane segments, of UapC and similar permeases described in this article, reveal a new family of prokaryotic and eukaryotic transporters.

  
Table: 62


FOOTNOTES

*
This work was supported by the Centre National de la Recherche Scientifique and by a European Union grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X79796.

§
Supported by a European Union ``Human Capital and Mobility'' Post-doctoral grant. To whom correspondence and reprint requests should be addressed. Tel.: 33-1-69416356; Fax: 33-1-69417808.

Supported by a grant from the Universidad de la Republica, Montevideo, Uruguay and a European Union grant.

**
Supported by a grant from the Universidad de la Republica Montevideo, Uruguay.

U. Tazebay, V. Sophianopoulou, C. Scazzocchio, and G. Diallinas, unpublished results.

The abbreviations used are: bp, base pair(s); kb, kilobase(s).

A. Ravagnani, T. Langdon, D. Gomez, V. Gavrias, H. N. Arst, Jr., C. Scazzocchio, and B. Cubero, unpublished results.

T. Suárez, M. Vieira de Queiroz, N. Oestreicher, and C. Scazzocchio, manuscript submitted for publication.

G. Diallinas, unpublished results.

V. Sophianopoulou and G. Diallinas, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Claire Rousseau for her excellent secretarial assistance, to Annie Glatigny and Teresa Suarez for their help in Northern blot analysis, and Fathia Mejjad for her technical assistance.


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