Yeast Yeast 2003; 20: 905912. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.

1017

Research Article

Cloning and chromosomal mapping of URA3 genes of Pichia farinosa and P. sorbitophila encoding orotidine-5 -phosphate decarboxylase
Chise Suzuki1 *, Nao Yoshida2 , Etsuko Okano1 , Toshiyuki Kawasumi2 and Yutaka Kashiwagi1
1 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2 Japan Womens University, Faculty of Home Economics, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo

112-8681 Japan

*Correspondence to: Chise Suzuki, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. E-mail: csuzuki@affrc.go.jp

Abstract
The PfURA3 gene, which encodes orotidine-5 -phosphate decarboxylase, of osmotolerant yeast Pichia farinosa NFRI 3621, was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of the PfURA3 gene and its deduced amino acid sequence indicated that the gene encodes a protein (PfUra3p) of 267 amino acids. Pulsed-eld gel electrophoresis and subsequent Southern blot analysis showed that the genome of P. farinosa NFRI 3621 consisted of seven chromosomes, each approximately 1.12.2 Mb in size (11.8 Mb in total) and that PfURA3 was located on chromosome V. Pichia sorbitophila is considered as a synonym of P. farinosa. The genome of P. sorbitophila IFO10021 may consist of 12 chromosomes, each approximately 1.22.2 Mb in size. P. sorbitophila has two copies of URA3 genes, termed PsURA3 and PsURA30, which were located on chromosome VIII and III, respectively. The difference between PfURA3 and PsURA3 was only two amino acid substitutions, whereas that between PsURA3 and PsURA30 was six amino acid substitutions and the deletion of the C-terminal amino acid by a stop codon insertion. The sequences of PfURA3, PsURA3 and PsURA30 have been deposited in the DDBJ data library under Accession Nos AB071417, AB109042 and AB109043, respectively. Copyright 2003 John Wiley & Sons, Ltd. Keywords: URA3 ; Pichia farinosa ; Pichia sorbitophila ; CHEF; osmotolerant yeast

Received: 25 October 2002 Accepted: 18 May 2003

Introduction
The URA3 gene encodes orotidine-5 -phosphate decarboxylase (ODCase) (orotidine-5 -phosphate carboxylase; EC 4.1.1.23), which catalyses the last step in the biosynthesis of pyrimidine in Saccharomyces cerevisiae as well as in several other yeasts. In S. cerevisiae, the URA3 gene is one of the most commonly used markers for the selection of transformants (Botstein et al., 1979). The URA3 gene is particularly convenient since there is a method for counter-selection of the ura3 marker; wild-type strains of yeast cannot grow on media containing the pyrimidine analogue 5-uoro-orotic acid (5-FOA), whereas ura3 mutants grow normally on such media (Boeke et al., 1984).
Copyright 2003 John Wiley & Sons, Ltd.

In this study, we describe the cloning and sequencing of the URA3 genes of Pichia farinosa NFRI 3621 and Pichia sorbitophila IFO 10021. P. farinosa NFRI 3621 produces a killer toxin, termed salt-mediated killer toxin (SMKT; Suzuki and Nikkuni, 1994). Expression of the killer gene SMK1 is lethal in S. cerevisiae (Suzuki et al., 2000). Therefore, the expression system in P. farinosa is necessary to elucidate the immunity mechanism of SMKT, by which killer strains are not killed by own toxins. In addition, SMKT shows increasing killing activity against S. cerevisiae with increased NaCl concentration and the secretion of SMKT is post-translationally controlled by NaCl (Suzuki, 1999). In fact, P. farinosa is highly resistant to osmotic stress in general and to salt

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stress in particular. Thus, the expression system in P. farinosa is important for our understanding of the killer mechanism and to use this yeast as a host in functional expression of heterologous genes under the control of NaCl. The phylogenetic position of P. farinosa is close to the Candida species, as suggested by cladograms established from 18S and 25S rRNA sequence comparisons (Souciet et al., 2000). Non-universal decoding is widely distributed not only in Candida species but also in some hemiascomycetous yeasts. The CUG codon was shown to specify Ser instead of Leu in P. sorbitophila (de Montigny et al., 2000; Tekaia et al., 2000), which is considered as a synonym of P. farinosa according to Kurtzman (1998). We previously provided in vivo evidence that the CUG codon is translated as unmodied Ser in P. farinosa (Suzuki et al., 2002). Therefore, we were interested in comparison of two strains, P. farinosa and P. sorbitophila, in terms of chromosomal mapping and localization of the URA3 genes. In this study, cloning and sequence analysis of URA3 genes of P. farinosa (PfURA3 ) and P. sorbitophila (PsURA3 and PsURA30 ) are described and their genomes were compared by using pulsedeld gel electrophoresis followed by Southern blot analysis.

described previously (Adams et al., 1998). The P. farinosa genomic DNA library was prepared by ligation of partially digested genomic DNA fragments into YCplac 111 with LEU2 marker (Gietz and Sugino, 1988). A series of nested deletions in pNYU1 was generated by limited Exo III nuclease digestion according to the suppliers instructions (Nippon Gene, Japan). The cloned fragment was sequenced with universal and walking primers by using an ABI Prism Big Dye Terminator Cycle Sequencing kit and the ABI Prism 310 Genetic Analyser (PE Applied Biosystems, USA). To clone URA3 homologues of P. sorbitophila, 4 kb and 6 kb Hpa I fragments of genome DNA of P. sorbitophila were subcloned in YCplac 22 with the TRP1 marker (Gietz and Sugino, 1988), resulting in pCS279 and pCS280, respectively. Clones that hybridized with the PfURA3 probe were isolated by colony hybridization. GenBank/DDBJ databases were searched for URA3 homologues using the BLAST algorithm (Altschul et al., 1990). Multiplesequence alignments and a phylogenetic tree were obtained using the ClustalW program (Thompson et al., 1994).

Material and methods Strains and media

Yeast strains P. farinosa NFRI 3621, P. sorbitophila IFO 10061 (CBS7064) and S. cerevisiae W303-1A (MAT a ade2-1 his3-4, 15 leu2-3, 112 trp1-1 ura3-1 can1-100 ) were used in this study. YPD medium containing 1% yeast extract, 2% peptone and 2% glucose was used for culture of yeast strains. Selective media contained 0.67% yeast nitrogen base (Difco), 2% glucose and appropriate supplements as needed. Escherichia coli DH5 was used for cloning and amplication of DNA.

Chromosomal DNA preparation and pulsed-eld gel electrophoresis

Chromosomal DNA was prepared as described previously (Carle and Olson, 1985). Pulsed-eld gel electrophoresis was done using a CHEF Mapper pulsed-eld electrophoresis system (Bio-Rad). The 1% agarose gels were prepared from Certied Megabase agarose (Bio-Rad) and then run in 0.5 TBE (45 mM Tris-borate and 1 mM EDTA) at 14 C at an initial switch time of 90.5 s, nal switch time of 265.9 s with a linear gradient (run time 38 h 27 min, angle 120 , voltage 4.5 V/cm). To separate chromosomes IIII of P. sorbitophila, the gel was run at an initial switch time of 114.5 s, nal switch time of 122.15 s, for 45 h, and to separate chromosomes VIX, at an initial switch time of 201.8 s, nal switch time of 210.3 s for 69 h. The size of the chromosomes was estimated by using the chromosomal DNA size marker of Hansenula wingei (Bio-Rad). Chromosomal bands were stained with ethidium bromide and visualized using an image analyser (Typhoon 9200, Amersham Biosciences).
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DNA manipulations
Yeast transformation by electroporation and yeast DNA isolation were carried out using standard procedures (Guthrie, 1991). Molecular manipulations were performed using standard methods described previously (Sambrook et al., 1989). Yeast colony polymerase chain reaction (PCR) was applied as
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Southern blot analysis and colony hybridization

The separated chromosomes were transferred onto nylon membrane (Zeta probe, Bio-Rad) by using vacuum blot apparatus (Vacugene, Pharmacia). For probes for hybridization, a 1 kb Bam HI Hin dIII fragment of pNYD9 (PfURA3 ), a 0.5 kb Sal I Eco RI fragment of pCS279 and a 1.5 kb Xba I-fragment of pCS280 were labelled with uorescein (GeneImage, Amersham Pharmacia) and used according to the suppliers instructions. Colony hybridization was performed as described previously (Sambrook et al., 1989).

Results and discussion

Transformation of S. cerevisiae W303-1A with the P. farinosa genomic DNA library constructed in the YCplac 111 allowed the isolation of three Ura+ transformants, out of 20 000, which could grow in a selective uracil- and leucine-free medium. Three plasmids, pNYU1, pNYU2 and pNYU3, were recovered from the three Ura+ Leu+ clones and conrmed to complement the uracil auxotrophy of S. cerevisiae W303-1A, indicating the presence of the PfURA3. Subsequent analysis of pNYU1, pNYU2 and pNYU3 showed that they shared common restriction fragments. Therefore, pNYU1 was further characterized (Figure 1A). A series of nested deletions in pNYU1 were generated. One of the deletion plasmids, pNYD9, which contained only 1.4 kb insertion fragment, retained the ability to complement the ura3 mutation. The 1.4 kb fragment of pNYD9 was sequenced on both strands. The sequence analysis of pNYD9 revealed a complete open reading frame (ORF) of 801 bp, which could encode a polypeptide of 267 amino acid residues (Figure 2). We separated the chromosomes of P. farinosa NFRI 3621 and those of P. sorbitophila IFO 10061, which is considered a synonym of P. farinosa (Kurtzman, 1998). The genome of P. sorbitophila CBS 7064 reportedly consists of seven chromosomes (Sychrova et al., 2000). We found that P. farinosa NFRI 3621 consisted of seven chromosomal bands, which were uniformly stained by ethidium bromide (Figure 3A). These bands were labelled chromosomes IVII, numbered according to increasing size, which were estimated as 1.1, 1.2, 1.5, 1.8, 1.9, 2.1 and 2.2 Mb, respectively, with a
Copyright 2003 John Wiley & Sons, Ltd.

Figure 1. Restriction maps and subcloning of PfURA3 (A), PsURA3 and PsURA30 (B). Regions homologous to the URA3 are represented as shaded boxes with arrows and those used for probes are indicated as hatched boxes

total of 11.8 Mb. Under the same conditions, chromosomes of P. sorbitophila separated into approximately seven bands, with sizes estimated at 1.2, 1.4, 1.5, 1.8, 1.9, 2.0 and 2.2 Mb. However, under a different condition, the band of 1.2 Mb separated into two bands of 1.13 and 1.16 Mb and the bands between 1.8 and 2.0 Mb separated into four bands, suggesting that the bands with higher intensities were duplicates (Figure 3A). Therefore, the genome of P. sorbitophila may consist of 12 chromosomes if the chromosomes of similar size are not homologous chromosomes of heterozygous diploid. The karyotyping and heterozygosity of this strain remain to be investigated. We estimated the localization of PfURA3 by using Southern blot analysis of uorescein-labelled PfURA3. In P. farinosa, the probe hybridized to the band corresponding to chromosome V (Figure 3B), whereas in P. sorbitophila the probe hybridized to that of chromosomes III and VIII (Figure 3B). The band of chromosome VIII more strongly hybridized to the PfURA3 probe than that of chromosome III, suggesting differences in sequences between the two genes. Genomic Southern blot analysis showed that P. sorbitophila had two copies of URA3 genes. The 4 kb and 6 kb Hpa I fragments, the former more strongly hybridized to the PfURA3 probe than the latter, were cloned from chromosomal
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Figure 2. Nucleotide and deduced amino acid sequences of PfURA3, PsURA3 and PsURA30. Nucleotide sequence of 1.3 kb insertion of pNYD9, which carries the entire PfURA3, and corresponding regions of PsURA3 and PsURA30 are aligned. Conserved bases in the three sequences are indicated by asterisks below the sequence. The deduced amino acid sequence of PfUra3p is indicated when those of PsUra3p and PsUra30p are identical. Codons where amino acid substitution occurs are boxed and the amino acid residues are indicated. Numbering of the nucleotide starts with A in the rst Met codon of the open reading frame, and that of the amino acid residues starts with this Met. Ser139 encoded by the CTG codon is enclosed by a circle

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Figure 2. Continued

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Figure 2. Continued

DNA. The restriction map of the 4 kb and 6 kb Hpa I fragments is shown in Figure 1B. The genes homologous to URA3 encoded in the 4 kb and 6 kb Hpa I fragments were termed PsURA3 and PsURA30, respectively. The nucleotide sequences of PsURA3 and PsURA30 and their deduced amino acid sequences were aligned with those of PfURA3 (Figure 2). Most of nucleotide substitutions were observed at the third codon and thus the identity between PfUra3p and PsUra3p was 99.2%, with only two amino acid substitutions, Asp to Glu and Glu to Asp. The identity between PsUra3p and PsUra30p was 97.7%, with six amino acid substitutions and a deletion of the C-terminal Gln by a stop codon insertion. The calculated molecular masses of PfUra3p, PsUra3p and PsUra30p were all 29.6 kDa, and their isoelectoric points were 6.1, 6.1 and 6.8, respectively. PsURA3 complemented the uracil auxotrophy of S. cerevisiae, whereas PsURA30 did not (data not shown). We estimated the localization of PsURA3 and PsURA30 on chromosomes by using 0.5 kb Sal I Eco RI fragment upstream of PsURA3 and 1.5 kb Xba I fragment downstream of PsURA30. The Sal I Eco RI fragment hybridized to the band corresponding to chromosome VIII, whereas the Xba I fragment hybridized to that of chromosome III (Figure 3B), suggesting that PsURA3 and PsURA30 localize on chromosome VIII and III, respectively. This was consistent with the result that PCR products were amplied from DNA
Copyright 2003 John Wiley & Sons, Ltd.

template extracted from the band of chromosome VIII using PsURA3-specic primers but not from the band of chromosome III (data not shown). The amino acid sequences of PfUra3p, PsUra3p and PsUra30p shared signicant similarities to those of other Ura3 proteins of yeasts. The phylogenetic tree is shown in Figure 4. Of the Candida species analysed, C. maltosa, C. albicans and C. tropicalis contained Q9 as the major ubiquinone and decode CUG as serine, whereas C. boidinii possessed Q7. P. farinosa and P. sorbitophila, possessing Q9 and decoding CUG as serine, are closely related to Candida species with Q9 (Sugita and Nakase, 1999). In P. farinosa, the CTG codon encodes Ser instead of Leu (Suzuki et al., 2002). Our results show that the 139th amino acid residue encoded by CTG in PfURA3, PsURA3 and PsURA30 was Ser (Figure 2). The Glu138 of Ura3p of S. cerevisiae corresponding to the Ser139 of PfUra3p is located in an -helix and the side chain faces the outside of the molecule in the crystal structure (Miller et al., 2000). PfURA3 and PsURA3 were functional in S. cerevisiae, even though the CTG codon was translated to Leu, suggesting that the side chain of the Ser139 does not contribute to the stability of the protein structure or to the activity of the enzyme. Consistent with this result, the amino acid residues corresponding to Ser139 were not conserved in various URA3 homologues (data not shown). Our results suggest that these genes can be used as a
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P. farinosa P. sorbitophila
XI, XII IX, X VII VI V IV III 1.37 II I IV, V III VIII VII VI

Mb 27-3.13 235

1.81 1.66

1.05

II I

B P. farinosa probe PfURA3

P. sorbitophila PfURA3 Sal I-Eco RI Xba I 5' PsURA3 3' PsURA30

VII VI V IV III

VIII

VIII

II I

III

III

Figure 3. Separation chromosomes and Southern hybridization using PfURA3 and PsURA3-specic probes. (A) Chromosomes of P. farinosa and P. sorbitophila. (B) Southern blot analysis of chromosomes of P. farinosa and P. sorbitophila. Chromosomal DNAs were separated in a 1% agarose gel using a pulsed-eld gel electrophoresis, stained by ethidium bromide and hybridized with uorescein-labelled probes, as described in Materials and methods. H. wingei chromosomal DNA was run alongside as a marker

Figure 4. Phylogenetic relationships of PfUra3p, PsUar3p and PsUar30p, and 10 other Ura3p of yeasts. The phylogenetic tree was constructed by ClustalW software based on the amino acid sequences of orotidine-5 -phosphate decarboxylases that were obtained from the GenBank/DDBJ database, using the following Accession Nos: S. cerevisiae (X00191-1; Rose and Botstein, 1983); Pichia ohmeri (Z35100-1; Piredda and Gaillardin, 1994); Pichia stipitis (U08629-1; Yang et al., 1994); Candida maltosa (D12720-1; Ohkuma et al., 1993); Candida tropicalis (AF040702-1; Su et al., 1998); Candida albicans (X14198-1; Losberger and Ernst, 1989); Candida boidinii (M86236-1; Sakai et al., 1992); Debaryomyces hansenii (AY033329-2; Bansal et al., 2001); Kluyveromyces lactis (D00431-1; Mizukami and Hishinuma, 1988); Kluyveromyces marxianus (Z21934-1; Bergkamp et al., 1993)

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Acknowledgement
This work was supported in part by the Pioneer Program from the Ministry of Agriculture, Forestry and Fisheries, Japan.
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