Yeast 15, 1399–1402 (1999)

Molecular Cloning and Sequence Analysis ofZygosaccharomyces rouxii ADE2 Gene Encoding aPhosphoribosyl-aminoimidazole Carboxylase

HANA SYCHROVA1*, VALERIE BRAUN2 AND JEAN-LUC SOUCIET2

1Department of Membrane Transport, Institute of Physiology CzAcadSci, 142 20 Prague 4, Czech Republic2Laboratoire de Microbiologie et Genetique, Universite Louis Pasteur, UPRES A-7010-CNRS, 67083 Strasbourg,France

The nucleotide sequence of a 2·8 kb fragment containing the ADE2 gene of the osmotolerant yeast Zygosaccharo-myces rouxii has been determined. The gene was cloned from a Z. rouxii genomic DNA library by complementationof the Saccharomyces cerevisae ade2 mutant strain. The sequenced DNA fragment contains a 1710 bp open readingframe predicting a protein of 570 amino acids. The deduced amino acid sequence shares a high degree of homologywith Ade2p homologues in five other yeast species. The Gene Bank Accession No. of Z-ADE2 is AJ133372.Copyright ? 1999 John Wiley & Sons, Ltd.

— Zygosaccharomyces rouxii; ADE2; phosphoribosylaminoimidazole carboxylase

*Correspondence to: H. Sychrova, Institute of PhysiologyCzAcadSci, Videnska 1083, 142 20 Prague 4, Czech Republic.e-mail: sychrova@biomed.cas.czContract/grant sponsor: Grant Agency, Czech Republic;Contract/grant number: 204/98/0475.Contract/grant sponsor: European Community; Contract/grant

INTRODUCTION

Phosphoribosylaminoimidazole carboxylase(AIRC, EC 4.1.1.21) is one of the key enzymes inpurine biosynthesis. The yeast AIRCs are pro-posed to consist of two domains: a CO2 bindingdomain (N-part of AIRC, approximately the first400 amino acid residues) and a catalytic domain(C-part, remaining part of the polypeptide) (Hiepet al., 1993). Yeast mutant strains lacking thecorresponding enzyme activity are not able tosynthesize adenine and accumulate red pigment.Thus, they can be easily distinguished from whiteAde+ prototrophs. This phenomenon has beenused widely for construction of yeast plasmids

number: BIO-CT950161.

CCC 0749–503X/99/131399–04$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

containing AIRC-coding genes as selectionmarkers. Among the AIRCs from different yeastspecies, including Ade2p of Saccharomyces cerevi-siae (Stotz and Linder, 1990), Schwanniomycesoccidentalis (Gourdon et al., 1995), Candidaalbicans (Tsang et al., 1997), Ade1p of Pichiamethanolica (Hiep et al., 1993), and Ade6p ofSchizosaccharomyces pombe (Szankasi et al., 1988),a great homology was found. In the osmotolerantyeast Z. rouxii, the ADE2 gene could also be anideal marker gene for selection of transformants.Thus, cloning and sequencing of the ZrADE2gene, together with the isolation of the correspond-ing mutant strain, can be very useful inconstruction of vectors for genetic studies ofthis yeast.

In this paper we report the complete nucleotidesequence of a 2800 bp fragment containing the Z.rouxii ADE2 gene, and the comparison of deducedprimary sequences of all six yeast AIRCS is also

presented.

Received 26 March 1999Accepted 29 April 1999

1400 H. SYCHROVA, V. BRAUN AND J.-L. SOUCIET

MATERIALS AND METHODS

Strains, DNA manipulations and growthS. cerevisiae ade2 strains used for complemen-

tation were W303.1B (MATa leu2-3/112 ura3-1trp1-1 his3-11/15 ade2-1 can1-100 mal10) (Walliset al., 1989) and YPH250 (MATa leu2-Ä1 ura3-52lys2-801 trp1-Ä1 his3-Ä200 ade2-101) (Sikorskiand Hieter, 1989). The Zygosaccharomyces rouxiiCBS732 wild-type strain was used for DNA libraryconstruction. DNA manipulations, transformationof E. coli and yeast, yeast and bacteria growthwere performed following the standard protocols(Sambrook et al., 1989).

Z. rouxii genomic DNA libraryThe gene library was constructed by a standard

procedure. Isolated genomic DNA was partiallydigested by the Sau3A restriction enzyme, andDNA fragments of 2·5–4 and 4–7 kb, respectively,were separately ligated into the polylinker BamHIsite of the yeast multicopy YEp352 vector (Hillet al., 1986). The two ligation mixtures were trans-formed into E. coli and the resulting 60 000 col-onies were pooled and used to prepare plasmidDNA. The plasmid DNA was introduced back toE. coli and the presence of Z. rouxii DNA insertsin plasmids was verified by restriction analysis ofpDNAs from 24 independent colonies.

DNA sequencing and sequence analysesDNA sequencing was carried out using the

Thermo Sequenase radiolabelled terminator cyclesequencing kit (Amersham Life Science, Inc.,USA). The DNA insert was sequenced on bothstrands directly from YEp352 using first universalprimers of vector polylinker, followed by primerscorresponding to the internal sequence of theDNA insert. Version 9.1 of the GCG software

package (Genetic Computer Group, Madison, WI,

Copyright ? 1999 John Wiley & Sons, Ltd.

USA) was used for DNA and protein sequenceanalyses.

Figure 1. Restriction map of the 2·8 kb insert of Z. rouxii DNA in YEp352. The position andorientation of the ORF is represented by a shadowed box and an arrow.

RESULTS AND DISCUSSION

The Z. rouxii DNA library was used to transformthe W303 S. cerevisiae strain and the Ade+ trans-formants obtained were analysed further. Plasmidswere isolated from the Ade+ yeast colonies,amplified in E. coli, and the ability of selectedplasmids to complement the Ade" phenotype wasverified by transformation of another S. cerevisiaeade2 strain, YPH250. In all cases, the transform-ants containing selected plasmids were whiteand could grow on minimum medium withoutadenine.

The restriction analysis of plasmid insertsrevealed a common DNA insert of about 3 kb inwhich the ade2 complementing capacity was local-ized (see Figure 1). The nucleotide sequence of thefragment containing the ADE2 gene is presented inFigure 2. An open reading frame (ORF) runningfrom nucleotides 741 to 2450 corresponds to apolypeptide of 570 amino acid residues in length,with Mr 62·2 kDa and a predicted isoelectric pointof 6·96.

Comparison with other five known yeast hom-ologues from S. cerevisiae, S. pombe, C. albicans,S. occidentalis and P. methanolica revealed a highsimilarity at the protein level, as is shown in Figure3 and Table 1. The alignment of all AIRCs to-gether using the programme PileUp shows (Figure3) that several regions are highly conserved amongthe proteins, the most conserved being the regionof the putative catalytic domain (amino acids465–523). The estimation of percentages of identityand similarity between different enzymes obtainedwith the Bestfit programem (Table 1) showed thehighest level of identity between the AIRCs ofC. albicans and S. occidentallis, and Z. rouxii and

S. cerevisiae.

Yeast 15, 1399–1402 (1999)

1401ADE2 OF ZYGOSACCHAROMYCES ROUXII

Figure 2. Nucleotide sequence of Z. rouxii ADE2 gene. Thepredicted amino acid residues of the ORF are shown below thenucleotide sequence.

Copyright ? 1999 John Wiley & Sons, Ltd.

Figure 3. Amino acid alignment of ADE2 homologues amongC. albicans (Accession No. U75582), P. methanolica (X76529),S. cerevisiae (M59824), S. occidentalis (U23210), S. pombe(X14488) and Z. rouxii (AJ133372). The regions of 100%identity are shadowed.

The analysis of the Z-ADE2 promoter region(1–740 nt) revealed several putative TATA boxesbut no hexanucleotide sequence TGACTC wasfound. This sequence is a binding site of transcrip-tional factors Gcn4p (Hinnebusch, 1988), Bas1pand Bas2p (Daignan-Fornier and Fink, 1992) andis characteristic for promoters of genes encodingenzymes involved in adenine biosynthesis, and also

of those genes that are under general amino acidcontrol. The absence of TGACTC distinguishesthe Z. rouxii ADE2 from its homologues of

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1402 H. SYCHROVA, V. BRAUN AND J.-L. SOUCIET

C. alibcans, P. methanolica, S. cerevisiae andS. occidentalis, which all possess at least one copyof this sequence.

Codon usage analysis is biased to A or T in thewobble position of most ZrADE2 gene codons,which is in agreement with a Z. rouxii codonfrequency table (Nakamura et al., 1998). Three ofthe possible 61 codons (CGG, CGA, CCG) are notused at all in this gene but there is no strong codonusage bias as observed in some highly expressedyeast genes.

ACKNOWLEDGEMENTS

This work was supported by Grants 204/98/0475from the Grant Agency of the Czech Republic andBIO-CT950161 from the European Community.

Table 1. Pairwise comparison of the peptidic sequences of different yeast AIRCs.

C.a. Ade2p P.m. Ade1p S.c. Ade2p S.p. Ade6p S.o. Ade2p Z.r. Ade2p

C.a. Ade2p 100 76·0 72·7 59·7 81·3 71·8P.m. Ade1p 80·9 100 71·2 61·5 74·9 71·1S.c. Ade2p 77·3 76·6 100 58·2 73·1 79·0S.p. Ade6p 68·7 69·5 66·7 100 60·6 60·7S.o. Ade2p 85·3 80·9 78·6 69·5 100 70·2Z.r. Ade2p 77·7 76·9 84·6 68·9 76·3 100

The numbers indicate the percentage of identities (similarities) between the different proteins obtainedwith the Bestfit programme. C.a. Ade2p, AIRC of C. albicans (Accession No. U75582); P.m. Ade1p,AIRC of P. methanolica (X76529); S.c. Ade2p, AIRC of S. cerivisiae (M59824); S.p. Ade6p, AIRC ofS. pombe (X14488); S.o. Ade2p, AIRC of S. occidentalis (U23210); Z.r. Ade2p, AIRC of Z. rouxii(AJ133372).

REFERENCESDaignan-Fornier, B. and Fink, G. R. (1992). Co-

regulation of purine and histidine biosynthesis bytranscriptional activators BAS1 and BAS2. Proc. NatlAcad. Sci. U S A 89, 6746–6750.

Gourdon, P., Janatova, I., Meilhoc, E., Klein, R. D.,Costagliolo, P. and Masson, J. M. (1995). Sequenceanalysis of the ADE2 gene coding for phosphoribo-sylaminoimidazole carboxylase in Schwanniomycesoccidentalis. Yeast 11, 1289–1293.

Hiep, T. T., Kulikov, V. N., Noskov, V. N., Sizonenko,G. I., Chernoff, Y. O. and Pavlov, Y. I. (1993).The 5-aminoimidazole ribonucleotide-carboxylasestructural gene of the methylotrophic yeast Pichia

Copyright ? 1999 John Wiley & Sons, Ltd.

methanolica: cloning, sequencing and homologyanalysis. Yeast 9, 1251–1258.

Hill, J. E., Meyers, A. M., Koerner, T. J. and Tzagoloff,A. (1986). Yeast E. coli shuttle vectors with multipleunique restriction sites. Yeast 2, 163–167.

Hinnebusch, A. G. (1988). Mechanisms of gene regu-lation in the general control of amino acid biosyn-thesis in Saccharomyces cerevisiae. Microbiol. Rev. 52,248–273.

Nakamura, Y., Gojobori, T. and Ikemura, T. (1998).Codon usage tabulated from the international DNAsequence databases. Nucleic Acids Res 26, 334–343.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor Laboratory, New York.

Sikorski, R. S. and Hieter, P. (1989). A system of shuttlevectors and yeast host strains designed for efficientmanipulation of DNA in Saccharomyces cerevisiae.Genetics 122, 19–27.

Stotz, A. and Linder, P. (1990). The ADE2 gene fromSaccharomyces cerevisiae: sequence and new vectors.Gene 95, 91–98.

Szankasi, P., Heyer, W.-D., Schuchert, P. and Kohli, J.(1988). DNA sequence analysis of the ade6 gene ofSchizosaccharomyces pombe. J. Mol. Biol. 204, 917–925.

Tsang, W. P., Cao, B.-Y. and Wang, J. (1997). Sequenceanalysis of Candida albicans phosphoribosyl-aminoimidazole carboxylase (ADE2) gene. Yeast 13,673–676.

Wallis, J. W., Chrebet, G., Brodsky, M., Rolfe, M.and Rothstein, R. (1989). A hyper-recombinationmutation in S. cerevisiae identifies a novel eukaryotictopoisomerase. Cell 58, 409–419.

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