Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant

Gene 210 (1998) 195–201

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant Nobuko Hamasaki-Katagiri a, Yasuhiro Katagiri b, Celia White Tabor a, Herbert Tabor a,* a Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA b Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0830, USA Received 18 August 1997; received in revised form 27 November 1997; accepted 16 December 1997; Received by B. Dujon

Abstract Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [ Hamasaki-Katagiri, N., Tabor, C.W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35–43]. © 1998 Elsevier Science B.V. Keywords: Polyamine; Spermidine; Spermidine synthase; Spermine; Yeast

1. Introduction Spermine is the final product of the polyamine biosynthesis pathway in eukaryote cells. Spermine is synthesized by addition of the aminopropyl moiety from decarboxylated S-adenosylmethionine to spermidine, and this reaction is catalyzed by spermine synthase (spermidine aminopropyltransferase, EC 2.5.1.22) ( Table 1). * Corresponding author. Tel: +1 301 496 2562; Fax: +1 301 402 0240; e-mail: [email protected] Abbreviations: aa, amino acid(s); AC, GenBank Accession Number; ADH1, gene coding for alcohol dehydrogenase 1; ApR, ampicillin resistant; dcAdoMet, decarboxylated S-adenosylmethionine; D, deletion; HPLC, high-performance liquid chromatography; kb, kilobase(s) or 1000 base pairs; Leu+, leucine prototroph; OD , optical density at 600 600 nm; ORF, open reading frame; SD, minimum dextrose medium; SPE2, gene coding S-adenosylmethionine decarboxylase; SPE3, gene coding spermidine synthase; SPE4, gene coding spermine synthase; YPAD, yeast extract, peptone, adenine sulfate and dextrose medium. 0378-1119/98/$19.00 © 1998 Published by Elsevier Science B.V. PII S 03 7 8 -1 1 1 9 ( 9 8 ) 0 0 0 27 - 4

We have been studying polyamine biosynthesis in S. cerevisiae and have shown that spermidine or spermine is essential for this organism for growth (Balasundaram et al., 1991; Hamasaki-Katagiri et al., 1997). Although spermine is ubiquitously present at a significant level in eukaryote cells, the physiological roles of spermine and therefore those of spermine synthase are unclear. In order to test for the functions of spermine in S. cerevisiae, it was desirable to prepare a deletion mutant that was not able to convert spermidine to spermine, and to test whether this defect affected the growth of this organism. For this purpose, it was necessary to determine the chromosomal localization of SPE4, the gene encoding spermine synthase. A BLAST search (Altschul et al., 1990) of the amino acid sequence deduced from SPE3 gene (spermidine synthase) (Hamasaki-Katagiri et al., 1997) with the reported sequences for the entire S. cerevisiae genome in the databases showed 50% identity with an open reading frame (ORF ), listed as L9634.5, on the right arm of chromosome XII (Locus YSCL9634,

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Table 1 Biosynthesis of polyamines in S. cerevisiae

Waterston, 1996). [ The percentage identities were obtained using the GAP program of the Wisconsin Package (Genetics Computer Group, 1994). Sequences were obtained from the Saccharomyces Genome Database of Stanford University or GenBank (Benson et al., 1994).] A similarity between L9634.5 and the reported sequences for the mammalian spermidine synthases had already been noted by Waterston. We considered it unlikely, however, that this sequence on chromosome XII represented a divergent duplicate spermidine synthase, since we had previously shown that mutants lacking the SPE3 gene on chromosome XVI completely lacked spermidine synthase activity and spermidine. Instead, we thought that it would be possible that the L9634.5 gene codes for spermine synthase since spermidine synthase and spermine synthase catalyze similar reactions ( Table 1). In order to investigate whether this was indeed the case, we prepared a deletion mutant of this locus, and found that this mutant showed the phenotype predicted for a mutation in SPE4; namely no detectable spermine synthase and spermine.

2. Materials and methods 2.1. Growth of yeast cells The yeast strains and plasmids that were used in this work are listed in Tables 2 and 3. Cells were maintained in rich YPAD medium ( Wickner, 1991), and were grown in polyamine-free SD media when growth experiments, polyamine analyses or an enzyme assay were performed. Cultures were incubated, with shaking, in air at 30°C. Yeast strains were transformed by the lithium acetate

method (Ito et al., 1983; Becker and Lundblad, 1992). The growth of polyamine-deficient organisms was measured as described previously (Cohn et al., 1980; Balasundaram et al., 1991; Hamasaki-Katagiri et al., 1997). Growth was followed by measuring the OD 600 of the cultures, and the cultures were diluted whenever the OD was greater than 1. The OD values shown 600 600 in Fig. 2 have been corrected for these dilutions. 2.2. Construction of plasmids encoding yeast spermine synthase Based on the nucleotide sequence upstream and downstream to the L9634.5 ORF on S. cerevisiae chromosome XII in the database, an upstream primer (5∞-AGAAGCTTCA TATGGTTAAT AATTCACAGC ATCC-3∞) and a downstream primer (5∞- GAGGATCCAA TCAGCCATTG ATGGCATATT CGG-3∞) were synthesized with the introduction of the HindIII and NdeI sites (upstream primer) and the BamHI site (downstream primer) (underlined). PCR was performed using Pfu DNA polymerase (Stratagene) and chromosomal DNA from the wild-type strain 2602 as a template. The PCR product was digested with HindIII and BamHI, and the 1.0-kb fragment was subcloned into the HindIII–BamHI site of two vectors. Plasmid pSPE4.01 was obtained by subcloning this fragment into the cloning vector pBluescriptIIKS(+) (Stratagene), and sequenced by the dideoxy-mediated chain termination procedure with modified T7 DNA polymerase (Sequenase 2.0, US Biochemical Co., Tabor and Richardson, 1987) to confirm that the fragment contained no PCR-generated errors. Another plasmid, pSPE4.04, was obtained by inserting this fragment into

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N. Hamasaki-Katagiri et al. / Gene 210 (1998) 195–201 Table 2 S. cerevisiae strains used in this work Strain

Genotype

Reference or source

2602 Y504 Y505

MATa his6 leu2 ura3-52 SPE4 MATa his6 leu2 ura3-52 spe4D::LEU2 MATa his6 leu2 ura3-52 spe3D::URA3

R.B. Wickner This study This studya

aSpe3 deletion-URA3 insertion was constructed by inserting URA3 gene into a deletion between two NdeI sites in the SPE3 gene in pSPE3.07 (Hamasaki-Katagiri et al., 1997). Table 3 Plasmids used in this work Plasmid

Characteristics

Size of insert (kb)

Plasmid vector

Reference or source

pSPE4.01 pSPE4.03 pSPE4.04

ApR SPE4 ApR spe4D::LEU2 2m, ApR ADHpa URA3 SPE4

1.0 2.6 1.0

pBluescriptIIKS(+) pBluescriptIIKS(+) pVT101-U

This study This study This study

aADHp: ADH1 gene promoter.

the yeast expression vector, pVT101-U ( Vernet et al., 1987), containing the constitutive ADH1 gene promoter ( Tables 2 and 3). 2.3. Gene disruption Disruption of L9634.5 was carried out by the onestep gene disruption technique (Rothstein, 1991) (Fig. 1). pSPE4.01 was treated with AflII and NcoI to remove 45% of the L9634.5 ORF, while the LEU2 fragment was obtained by treating YEp351 ( Hill et al., 1986) with NarI and HpaI. After blunt-end ligation of these two fragments, the resulting plasmid, pSPE4.03, was digested with XbaI and XhoI to obtain a 2.6-kb fragment. This DNA fragment, containing the L9634.5 deletion-LEU2 insertion construct, was used to transform the SPE3 SPE4 leu2 strain, 2602. Six Leu+ transformants were selected, and their polyamine content was analyzed; five out of six isolates did not contain detectable amounts of spermine. Two Leu+ isolates that did not contain spermine were chosen, and the disrup-

tion of their chromosomal L9634.5 by homologous recombination was confirmed by PCR. One of these isolates, Y504 ( Tables 2 and 3), was crossed with a wildtype strain and the diploid contained spermine, indicating that the Spe4+ phenotype (i.e. containing spermine) was dominant. Tetrad analyses showed 2:2 cosegregation of spermine biosynthesis and leucine auxotrophy (data not shown). 2.4. Assay of spermine synthase Cell lysate was prepared by a French Press as previously described (Hamasaki-Katagiri et al., 1997), and more than 98% of the endogenous polyamines were removed by dialysis. Spermine synthase activity was measured using [14C ] spermidine, a precursor of spermine ( Table 1), as the substrate; the synthesized spermine was separated from spermidine by thin-layer chromatography on Polygram Ionex #25 SA-Na sheets (Ion Exchange/Silica Gel, Macherey-Nagel ). The assay mixture contained 0.1 M glycylglycine–NaOH (pH 9.0),

Fig. 1. Schematic structure of the SPE4 gene on the right arm of chromosome XII (above) and spe4 deletion-LEU2 insertion construct in plasmid pSPE4.03 (below). The ORF, represented by the thick bar, is 0.9 kb. The deletion was 0.4 kb, and the LEU2 insert (not drawn to scale) was 2.0 kb.

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0.1 M dithiothreitol, 0.2 mM decarboxylated S-adenosylmethionine, 0.5 nmol of [14C ] spermidine (50 nCi), and 1–2 ml of the cell lysate (final volume, 10 ml ). The mixture was incubated at 37°C for 15 min, and the reaction was stopped by the addition of 1 ml of 50% trichloroacetic acid. The chromatograms were developed with 2 M KCl, and radioactivity was detected using the PhosphoImager SF (Molecular Dynamics) (R =0.55 for f putrescine, 0.36 for spermidine and 0.20 for spermine). Spermidine synthase activity was measured in the same way, except that [14C ] putrescine, a precursor of spermidine ( Table 1), was used as the substrate. The protein concentration was assayed as described by Bradford (1976). Synthetic decarboxylated S-adenosylmethionine was kindly provided by Dr Giorgio Stramentinoli. 2.5. Determination of polyamine and decarboxylated Sadenosylmethionine levels Polyamines and decarboxylated S-adenosylmethionine were analyzed by modifications of the HPLC method described previously ( Tabor et al., 1973; Oshima, 1983; Balasundaram et al., 1991; HamasakiKatagiri et al., 1997).

3. Results and discussion 3.1. Characteristics of spe4 deletion mutant We had previously described mutant strains of S. cerevisiae that had a very low spermine synthase activity (Cohn et al., 1980). However, these mutants had a complicated history; i.e. they had been obtained after chemical and spontaneous mutagenesis and they had not been characterized further. We show here that the SPE4 gene, which codes for spermine synthase, is located on the right arm of chromosome XII of S. cerevisiae, and we constructed a deletion mutant lacking this gene. This deletion mutant has neither spermine synthase nor spermine. As described in Section 2.2, we also constructed a plasmid, pSPE4.04, containing the SPE4 gene. The ORF encodes a polypeptide of 300-aa residues with a calculated molecular weight of 34 090. In order to measure spermine synthase activity, it was critical to have a convenient system to separate spermidine from spermine. We developed a thin-layer chromatographic method to separate these two compounds (with R values of 0.36 for spermidine and 0.20 for spermine, f see Section 2.4). It had been difficult previously to obtain such a complete separation of spermidine and spermine in thin-layer or paper-chromatography systems. Since this method also separates putrescine from spermidine, this method is also useful for the assay of spermidine synthase.

No spermine synthase activity was found in the cell extracts of the spe4 deletion mutant, Y504 ( Table 4). Spermine was not detected in the cells of Y504 after growth in polyamine-deficient media ( Table 5). The presence of the spermine synthase expression plasmid (pSPE4.04) in the spe4 deletion mutant produced more than 100-fold higher spermine synthase activity than the activity in wild-type cells ( Table 4). The spermine level increased to twice the wild-type level. The discrepancy between the increase in the spermine synthase activity and the spermine level (100-fold versus twofold ) might be explained by the low level of decarboxylated S-adenosylmethionine in these cells ( Table 5). We note that spermidine synthase activity is increased in the spe4D mutant cells, but do not know the reason for this increase. This increase is not explained by derepression resulting from a lack of spermine in the mutant since the mutant that carries pSPE4.04 and contains an increased level of spermine also shows a high spermidine synthase activity ( Tables 4 and 5). It is noteworthy that the amount of decarboxylated S-adenosylmethionine in the spe4D cells was as low as the wild type ( Table 5). The level of decarboxylated Sadenosylmethionine is even lower in the spe4D strain containing pSPE4.04. We postulate that the decarboxylated S-adenosylmethionine level was so low in these strains not only because this compound is being used for formation of polyamines but also because S-adenosylmethionine decarboxylase is presumably repressed by spermidine or spermine (Mamont et al., 1982; Pegg, 1984). The spe4D strain had a normal morphology and grew as well as the wild-type strain in the absence of exogenous polyamines, whereas a spe3D strain stopped growing ( Fig. 2) and showed an altered morphology ( Hamasaki-Katagiri et al., 1997). Homozygous spe4D diploid cells sporulated normally in the absence of exogenous polyamines (data not shown). These findings suggest that spermine is not essential for the growth of S. cerevisiae. This is consistent with our previous findings that the cessation of the growth of the spe2D mutant and the loss of the ability of homozygous spe2 diploid cells to sporulate were both restored by the addition of spermidine alone, even though the spe2 mutants cannot synthesize spermine (Cohn et al., 1978; Balasundaram et al., 1991) ( Table 1). Our findings that the spe4 deletion mutant grows well, even though this strain contains no spermine, is also consistent with the reports of Samejima and his colleagues (Shirahata et al., 1993; Beppu et al., 1995) that the growth of rat hepatoma cells or weight gain of rats is not affected by treatment with a specific inhibitor for mammalian spermine synthase. The physiological role of spermine remains unclear at present.

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N. Hamasaki-Katagiri et al. / Gene 210 (1998) 195–201 Table 4 Spermidine synthase and spermine synthase activities of SPE4 and spe4D strains Strain

2602 Y504/pVT101-U Y504/pSPE4.04

Chromosomal genotype

Plasmid genotype

None None SPE4

SPE4 spe4D::LEU2 spe4D::LEU2

Specific activitya Spermidine synthase

Spermine synthase

107 856 619

40 <10b 6600

aSpecific activity: pmol of spermidine or spermine formed per min per mg protein. bThe limit of the assay was ca. 10 pmol of spermidine or spermine per min per mg protein. Table 5 Polyamine and decarboxylated S-adenosylmethionine levels of SPE4 and spe4D strainsa Strain

Chromosomal genotype

Plasmid genotype

Putrescine

Spermidine

Spermine

dcAdoMet

280 960 150

100 <5 200

70 30 <10

(nmol/g wet weight cells) 2602 Y504/pVT101-U Y504/pSPE4.04

SPE4 spe4D::LEU2 spe4D::LEU2

None None SPE4

20 80 50

aThe limit of detection was 25, 50, and 100 pmol of putrescine, spermidine and spermine (1.3 nmol per g wet weight cells for putrescine, 2.5 for spermidine and 5.0 for spermine), and the retention times for each polyamine were 26 min, 49 min, and 102 min, respectively. The limit of detection for dcAdoMet was 10 nmol per g wet weight cells, and the retention time was 64 min.

Fig. 2. Growth curve of spe4D, spe3D and wild-type strains after polyamine depletion. Y504 (spe4D) containing the vector plasmid, pVT101-U ($) or pSPE4.04 (#), Y505 (spe3D, &) and 2602 (wild type, +).

3.2. Comparison of amino acid sequence of S. cerevisiae spermine synthase with spermine synthase from other species and with spermidine synthases Fig. 3 shows the comparison of the amino acid sequence of S. cerevisiae spermine synthase (deduced from the SPE4 gene located on chromosome XII, AC U53879) and of S. cerevisiae spermidine synthase (deduced from the SPE3 gene located on chromosome XVI, AC U27519). The amino acid sequence of spermine synthase is 50% identical to that of spermidine synthase; the nucleotide sequences of the SPE4 and SPE3 genes

are 38% identical. Six consecutive amino acids, which are conserved among all known eukaryotic spermidine synthases, are perfectly conserved in yeast spermine synthase (GGGDGG at aa no. 95–100 of yeast spermine synthase, underlined in Fig. 3). These amino acids are also conserved in human and mouse spermine synthases, which are 96% identical to each other ( Korhonen et al., 1995; Francis, 1996). The similarity between spermidine synthase and spermine synthase is not too surprising since both enzymes catalyze a transfer of the aminopropyl moiety from decarboxylated Sadenosylmethionine to the primary amino group of the aminobutyl chain in a precursor compound; i.e. putrescine for spermidine synthase and spermidine for spermine synthase, respectively ( Table 1). In S-adenosylmethionine-dependent methyltransferases, there are amino acids at relatively defined intervals called the ‘S-adenosylmethionine binding motif ’, based on crystal structure analyses of several of these methyltransferases (Cheng et al., 1993; Vidgren et al., 1994; Cheng, 1995; Schluckebier et al., 1995). The six consecutive amino acids mentioned above overlap with this motif. We have previously shown that all known eukaryote spermidine synthases, which use decarboxylated Sadenosylmethionine as one of the substrates, have this motif (Hamasaki-Katagiri et al., 1997). This motif can also be found with a similar spacing in yeast spermine synthase (marked with asterisks in Fig. 3). This motif is mostly conserved even in mammalian spermine synthases, which have much less similarity to eukaryote spermidine synthases ( Table 6). Therefore, we postulate

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Fig. 3. Comparison of the amino acid sequences of S. cerevisiae spermine synthase ( Yspmsyn, AC U53879) and S. cerevisiae spermidine synthase ( Yspdsyn, AC U27519). The sequences were aligned by the GAP program of the Wisconsin Package (Genetics Computer Group, 1994). The numbers on the left side of the sequences refer to the position of the amino acid of spermine synthase. The six consecutive amino acids underlined are conserved among all known spermidine synthases. The amino acids that are postulated to interact with decarboxylated S-adenosylmethionine are indicated by asterisks (see Section 3.2). Table 6 Percentage identity of amino acid sequences of spermidine synthases and spermine syntheses from S. saccharomyces and human

YSpmsyn YSpdsyn HSpmsyn HSpdsyn

YSpmsyn

YSpdsyn

HSpmsyn

HSpdsynab

—

50 —

27 28 —

48 57 25 —

aYSpmsyn, yeast spermine synthase; YSpdsyn, yeast spermidine synthase; HSpmsyn, human spermine synthase, HSpdsyn: human spermidine synthase. bPutative spermidine synthases from a plant (Datura stramonium) have more than 50% identity to yeast spermine synthase as well as to yeast and human spermidine synthases. They have less than 30% identity to human spermine synthase.

that these amino acids in yeast spermine synthase interact with decarboxylated S-adenosylmethionine. A comparison of the amino acid sequences of spermine synthase and spermidine synthase from S. cerevisiae and human sources is summarized in Table 6. First, the yeast and human ( Wahlfors et al., 1990) spermidine synthases are 57% identical. In fact, the spermidine synthases from species as divergent as plants (Datura stramonium, Michael, 1996a,b) and mammals are strikingly similar (more than 50% identity). Thus, these genes have diverged very little since the ancestral eukaryotic spermidine synthase gene arose several billion years ago. Second, yeast spermidine synthase and yeast spermine synthase are 50% identical. Since convergent evolution

could not have produced this large an identity, the spermine synthase gene must have descended from the spermidine synthase gene by a gene duplication. The gene duplication probably occurred around the time that the eukaryotes arose because most prokaryotes do not contain spermine synthase. Finally, human spermine synthase is far more divergent from human spermidine synthase (25% identity) than yeast spermine synthase is from yeast spermidine synthase. These results suggest that either evolutionary changes in yeast spermine synthase have been constrained, or evolutionary changes in mammalian spermine synthases were accelerated possibly due to positive selection.

4. Conclusion We have identified and isolated S. cerevisiae SPE4, a gene coding for spermine synthase, on the right arm of S. cerevisiae chromosome XII. The amino acid sequence of this enzyme is highly similar to that of S. cerevisiae spermidine synthase. A deletion mutant of this gene lacked both spermine synthase activity and spermine. Both of these defects were restored by re-introduction of this gene. The SPE4 gene is not essential for the normal growth of S. cerevisiae. This is in striking contrast to the absolute requirement of this organism for spermidine.

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The physiological function of spermine in yeast is still unknown.

Acknowledgement We wish to thank Dr Thierry Vernet for the generous gift of pVT101-U.

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