Yeast 14, 1307–1310 (1998)

Molecular Cloning and Sequence Analysis of theSchizosaccharomyces pombe ade10+ Gene

CHRISTIAN LIEDTKE† AND HENNING SCHMIDT*

Institute of Genetics, Technische Universitat Braunschweig, Biozentrum, D-38106 Braunschweig, Germany

We have cloned and sequenced the Schizosaccharomyces pombe ade10 gene encoding 5-phosphoribosyl-4-carboxamide 5-aminoimidazole transformylase inosine monophosphate cyclohydrolase. The sequence has anuninterrupted open reading frame of 1755 nucleotides corresponding to 585 amino acid residues. The deduced aminoacid sequence shows a high degree of similarity to the purH gene product of many species, including Saccharomycescerevisiae, human, chicken and Escherichia coli. Moreover our data indicate that intrachromosomal recombinationin Schiz. pombe is enhanced if the ade10 gene product is defective. The sequence has been submitted to the EMBLdata library under Accession Number Y16419. ? 1998 John Wiley & Sons, Ltd.

— Schizosaccharomyces pombe; 5-phosphoribosyl-4-carboxamide 5-aminoimidazole (AICAR) trans-formylase; inosine monophosphate (IMP) cyclohydrolase; intrachromosomal recombination

CCC 0749–503X/98/141307–04 $17.50? 1998 John Wiley & Sons, Ltd.

For the not closely related budding yeast Sac-charomyces cerevisiae, all genes involved in theentire de novo purine biosynthesis are known as aresult of the genome sequencing project. Surpris-ingly, Saccharomyces has two ATIC-encodinggenes (Tibbetts and Appling, 1997). These twogenes, called ADE16 and ADE17, show 85% iden-tity to each other. Schiz. pombe appears to haveonly one such gene.

In this paper, we report the cloning and sequenc-ing of the ade10+ gene of Schiz. pombe; it is able tocomplement the ade10-3H mutation as well as theade10-18 mutation. We have recently isolated thelatter mutation in a screen for mutations whichincrease intrachromosomal recombination in themating-type region.

*Correspondence to: Henning Schmidt, Institute of Genetics,Technische Universitat Braunschweig, Spielmannstrasse 7,D-38106 Braunschweig, Germany. Tel: 49-531-3915775; Fax:49-531-3915765: E-mail: schmidt@alpha.bio.nat.tu-bs.de.†Present address: Rosenstiel Center Mailstop 029, BrandeisUniversity, 415 South Street, Waltham, MA 02254-9110, USA.

MATERIALS AND METHODS

The ade10-18 mutant was found in a screen formutants which show increased levels of intra-chromosomal crossing-over in the mating-typeregion of Schiz. pombe. The screen and the

INTRODUCTION

The ade10+ gene of Schizosaccharomyces pombeencodes 5-phosphoribosyl-4-carboxamide 5-aminoimidazole (AICAR) transformylase inosinemonophosphate (IMP) cyclohydrolase (ATIC)catalysing step 9 and step 10 of the de novo purinesynthesis (reviewed in Gutz et al., 1974). In Schiz.pombe 12 loci (ade1–ade10, gua1 and gua2) havebeen found to be involved in this pathway. Four ofthese genes have been cloned and sequenced: ade1(McKenzie et al., 1987), ade2 (Speiser et al., 1992),ade4 (Ludin et al., 1994) and ade6 (Szankasi et al.,1988). Genetic studies revealed that the ade10locus controls both AICAR transformylase (EC2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10)activity, suggesting that Ade10 is a bifunctionalenzyme (Whitehead et al., 1966).

Received 9 March 1998Accepted 24 April 1998

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? 1998 John Wiley & Sons, Ltd.

Figure 1. Nucleotide sequence of the Schiz. pombe ade10 gene.The predicted amino acid sequence of the deduced polypeptideis shown above the nucleotide sequence (EMBL accession no.Y16419).

properties of the mutants will be described else-where. The ade10 gene was cloned by functionalcomplementation using the Schiz. pombe genomiclibrary described in Barbet et al. (1992). Schiz.pombe strains were transformed according to Itoet al. (1983) and plasmids were rescued by themethod of Hoffman and Winston (1987). Forsequencing we cloned the ade10 gene into pUC18and created nested deletions with Exonuclease III(Pharmacia). Both strands were sequenced by thedideoxynucleotide chain termination method(Sanger et al., 1977) using Sequenase (USB).Sequence analysis was performed with the GCGpackage (Genetics Computer Group, Wisconsin,USA) or using the BLAST network service(National Center for Biotechnology Information).DNA manipulations were done according toSambrook et al. (1989).

RESULTS AND DISCUSSION

During mating-type switching it is essential thatvirtually no intrachromosomal crossing-over takesplace since otherwise a lethal deletion would occur.This topic is discussed in Schmidt and Gutz (1994).We constructed a strain in which such deletions areno longer lethal. In short, the essential gene fromthe L region, let1 was integrated into the ura4gene. Second, the ade6+ marker was inserted into anon-essential part of the L region. Therefore, adeletion of L is no longer lethal but can easily bedetected by means of the red colour, if the ade6+

gene is lost concomitant with the L region. Thedetails of this strain and the screen will bedescribed elsewhere (C. Liedtke and H. Schmidt,manuscript in preparation). With the help of thisstrain we isolated mutants which yielded signifi-cant numbers of deletions in the mating-typeregion of Schiz. pombe. Here we report on amutant (M18) which, besides showing such intra-chromosomal crossing-over, is unable to grow onminimal medium.

Tetrad analysis with the mutant M18 revealedcosegregation for both the hyper-recombinationphenotype and the auxotrophy. We used thegenomic Schiz. pombe library pURSP2 (Barbetet al., 1992) for functional complementation ofboth phenotypes and isolated a plasmid (pCL4)which complements the hyper-rec phenotype andthe auxotrophy of M18.

The plasmid pCL4 contains an insert of about3·5 kb in length. To sequence it we made a series of

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1309. ade10+

Figure 2. Alignment of the deduced amino acid sequence of the Schiz. pombe Ade10 protein withS. cerevisiae ADE16 (accession no. P54113), S. cerevisiae ADE17 (accession no. P38009), chickenpURH (accession no. P31335) and human pURH (accession no. P31939). Abbreviations for thespecies are: sp, Schizosaccharomyces pombe; sc, Saccharomyces cerevisiae; gg, Gallus gallus; hs, Homosapiens. Identical amino acids are marked with a black background, similar amino acids are markedwith a grey background. Dashes (gaps) have been introduced for optimal alignment.

deletions using Exonuclease III (Pharmacia)starting on both sides of the polycloning site ofpCL4.

The obtained sequence revealed an uninter-rupted open reading frame of 1755 nucleotidesencoding a putative protein of 585 amino acids. Asearch in the GenBank database with the BLASTprogram (Altschul et al., 1990) showed similarities

? 1998 John Wiley & Sons, Ltd.

of the ORF to proteins from different species allencoding ATIC, which catalyses step 9 and step 10in the de novo purine biosynthesis. The Schiz.pombe protein has homologies to ATIC from S.cerevisiae (69% identity to ADE16 and 71% iden-tity to ADE17, respectively), human (61% iden-tity), chicken (60% identity) and Escherichia coli(41% identity).

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ACKNOWLEDGEMENTS

We thank Cathrin Struck and Tobias Sperka forexpert technical assistance and Herbert Gutz forcritical reading of the manuscript. We are gratefulto Martin Lutzelberger for help with the align-ment. This work was supported by the DeutscheForschungsgemeinschaft, grant SCHM 786/2-2.

REFERENCES

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. andLipman, D. J. (1990). Basic local alignment searchtool. J. Mol. Biol. 215, 403–410.

Barbet, N., Muriel, M. J. and Carr, A. M. (1992).Versatile shuttle vectors and genomic libraries for usewith Schizosaccharomyces pombe. Gene 114, 59–66.

Gutz, H., Heslot, H., Leupold, U. and Loprieno, N.(1974). Schizosaccharomyces pombe. In King R. C.

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(Ed.), Handbook of Genetics, vol. 1, Plenum Press,New York, London, pp. 395–446.

Hoffman, C. S. and Winston, F. (1987). A ten-minuteDNA preparation from yeast efficiently releasesautonomous plasmids for transformation ofEscherichia coli. Gene 57, 267–272.

Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983).Transformation of intact yeast cells treated with alkalications. J. Bacteriol. 153, 163–168.

Kunz, B. A., Barclay, B. J., Game, J. C., Little, J. G. andHaynes, R. H. (1980). Induction of mitotic recombi-nation in yeast by starvation for thymine nucleotides.Proc. Natl. Acad. Sci. USA 77, 6057–6061.

Kunz, B. A., Taylor, G. R. and Haynes, R. H. (1986).Induction of intrachromosomal recombination inyeast by inhibition of thymidylate biosynthesis.Genetics 114, 375–392.

Ludin, K. M., Hilti, N. and Schweingruber, M. E.(1994). The ade4 gene of Schizosaccharomyces pombe:cloning, sequence and regulation. Curr. Genet. 25,465–468.

McKenzie, R., Schuchert, P. and Kilbey, B. (1987).Sequence of the bifunctional ade1 gene in the purinebiosynthetic pathway of the fission yeast Schizosac-charomyces pombe. Curr. Genet. 12, 591–597.

Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNAsequencing with chain terminating inhibitors. Proc.Natl. Acad. Sci. USA 74, 5463–5467.

Schmidt, H. and Gutz, H. (1994). The mating-typeswitch in yeasts. In Wessels, J. G. H. and Meinhardt,F. (Eds), The Mycota, vol. I. Springer-Verlag, Berlin,Heidelberg, pp. 283–294.

Speiser, D. M., Ortiz, D. F., Kreppel, L., Scheel, G.,McDonald, G. and Ow, D. W. (1992). Purine biosyn-thetic genes are required for cadmium tolerance inSchizosaccharomyces pombe. Mol. Cell. Biol. 12,5301–5310.

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

Tibbets, A. S. and Appling, D. R. (1997). Saccharo-myces cerevisiae expresses two genes encodingisozymes of 5-aminoimidazole-4-carboxamide ribo-nucleotide transformylase. Arch. Biochem. Biophys.340, 195–200.

Whitehead, E., Nagy, M. and Heslot, H. (1966). Inter-actions entre la biosynthese des purins nucleotides etcelle de l’histidine chez le Schizosaccharomyces pombe.C. R. Hebd. Seances Acad. Sci. Ser. D. Sci. Nat. 263,819–821.

In Schiz. pombe the corresponding enzyme isencoded by the ade10+ gene (Gutz et al., 1974).The identities mentioned above suggested that wehad cloned the ade10+ gene. Therefore, we trans-formed the plasmid pCL4 into the originalade10-3H mutant of Schiz. pombe. The trans-formed cells were able to grow on minimal mediumthus proving that pCL4 contains the ade10+ gene.Obviously, our mutant M18 also has a defect inade10. The nucleotide sequence of the Schiz.pombe ade10+ gene is presented in Figure 1; analignment with Ade10 homologues of S. cerevisiae,human and chicken is given in Figure 2.

Independently of our work, Yoshioka, Kato andOkayama (unpublished data) have recently sub-mitted to the data library a partial cDNA sequence(Accession Number D89243) which corresponds tothe C-terminal part of Ade10.

Our data suggest that there is a correlationbetween the block in purine biosynthesis and theincreased frequency of mitotic intrachromosomalcrossing-over. Presumably starvation for adenylatecaused by the ade10-M18 mutation leads to animbalance of the nucleotide pool which is recombi-nogenic. A similar observation was made forthymidylate starvation in S. cerevisiae (Kunz et al.,1980, 1986). This condition proved also to berecombinogenic and led to increased mitoticcrossing-over and gene conversion.

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