# Cloning of the yeast methionyl-tRNA synthetase gene

Franco Fasiolo, Jacques Bonnet, François Lacroute

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Franco Fasiolo, Jacques Bonnet, François Lacroute. Cloning of the yeast methionyl-tRNA synthetase gene. Journal of Biological Chemistry, 1981, 256 (5), pp.2324-2328. 10.1016/S0021-9258(19)69782-6. hal-05254403

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# Cloning of the Yeast Methionyl-tRNA Synthetase Gene*

(Received for publication, July 14, 1980, and in revised form, October 16, 1980)

Franco Fasiolo‡, Jacques Bonnet, and François Lacroute§

From the Laboratoire de Biochimie and §Laboratoire de Génétique Physiologique, Institute de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, 67084 Strasbourg Cedex, France

A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast *Escherichia coli* plasmid (pFL₁) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine. In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomal inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type. Measurement of the *K*<sub>m</sub> for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the *K*<sub>m</sub> for methionine to the same level as found for the wild type methionyl-tRNA synthetase. Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase. They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids.

The high efficiency of yeast transformation by hybrid plasmid vectors bearing part of the 2µ yeast plasmid, the *Escherichia coli* plasmid pBR322, and the inserted yeast DNA (1) enables one to clone easily any yeast gene for which one possesses a positive selection (resistance, release from auxotrophy, or thermosensitivity). As an approach to the study of the regulation of the aminoacyl-tRNA synthetases, we have decided to clone the yeast structural gene coding for the cytoplasmic methionyl-tRNA synthetase. We used as a recipient the mutant H.19.3.4 described by McLaughlin and Hartwell (2) which requires a high amount of methionine in the growth medium due to a *K*<sub>m</sub> alteration of the methionyl-tRNA synthetase. Despite initial controversy resulting from genetic analysis by Cherest *et al.* (3) regarding the segregation of the methionine auxotrophy and the temperature sensitivity, Unter (4) confirmed that this mutation was indeed in a single gene causing an alteration of the *K*<sub>m</sub> of the methionyl-tRNA synthetase which requires methionine at the permissive temperature and is temperature-sensitive for growth. We present here the experimental data showing that we have indeed cloned the methionyl-tRNA synthetase structural gene by complementing this mutation.

* This work was supported by grants from the Centre National de la Recherche Scientifique and the Fondation pour la Recherche Médicale Française. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom reprint requests should be addressed.

## MATERIALS AND METHODS

**Yeast and Bacteria**—F.F.1 sp 1, used as recipient for transformation has a *mes1.ura3* genotype and was constructed by recombining the H.19.3.4 mutant provided by McLaughlin and Hartwell (2) with *ura3* triple mutant isogenic to FL 100. This strain requires uracil and methionine for growth. The bacterial strains were all derivatives of *E. coli* BJ 5183.

**Genetic Techniques and Media**—Genetic techniques and media have been described (5). Biohazards associated with the experiments described in this publication have been examined previously by the French National Control Committee.

**Preparation of the Yeast DNA Pool**—Construction of the chimeric plasmid pFL₁ which has been used as a cloning vehicle has been described earlier (6). This plasmid (Fig. 1) possesses the 2µ EcoRI-D fragment of the yeast 2µ plasmid (7), a 1.1 kilobase HindIII fragment bearing the yeast URA3 gene (8) and the *E. coli* plasmid pBR322 (9). This plasmid has been opened by Bam HI restriction enzyme, treated by calf intestine alkaline phosphatase, and random yeast DNA fragments obtained by partial Sau IIIA digestion of DNA isolated from the strain FL 100 have been inserted in the Bam HI site by T₄ ligase. The ligation mixture has been used to transform, according to the procedure of Cohen *et al.* (10), BJ 5183, an efficiently transformable *E. coli* strain constructed by Bruno Jary. The comparison between BJ 5183 and other commonly used *E. coli* strains such as HB 101 shows that BJ 5183 is the easiest one to transform and displays less discrimination against high molecular weight plasmids.

**DNA Preparations**—In order to prepare DNA for transformation, the hybrid yeast *E. coli* plasmids (about 60,000 to 130,000 clones) carrying the yeast DNA pool were incubated onto L broth<fnref n="1" /> agar plates containing 20 µg/ml of ampicillin and incubated overnight at 37°C. The cells were washed off the plates into Sorvall centrifuge tubes. The cells were lysed with Triton X-100 according to the technique of Katz *et al.* (11), and the supercoiled plasmid DNA was separated from the bulk of the host chromosomal DNA by CsCl-ethidium bromide density gradient centrifugation. The ethidium bromide was extracted with isobutyl alcohol and the plasmid DNA carefully dialyzed against 10 mM Tris-1 mM EDTA buffer, pH 8.

Yeast DNA was isolated from the transformed cells by the method of Struhl *et al.* (12) and used to transform BJ 5183 for ampicillin resistance. Rapid plasmid DNA preparation was performed according to Rambach and Hogness (13).

**Transformations**—Yeast cells (*Saccharomyces cerevisiae*) were transformed according to the technique of Hinnen *et al.* (14) with minor modifications. A fresh logarithmic phase culture (250 ml) was concentrated to 1/10 of its volume by centrifugation and treated with zymolyase 60,000 (Kirin Brewery, Takasaki, Japan) in 1 M sorbitol until about 80% of the osmotic fragile cells were obtained (about 15 to 60 min). Spheroplasts were washed twice with 1 M sorbitol and resuspended in 0.6 ml of 1 M sorbitol, 10 mM Tris-HCl, 10 mM CaCl₂, pH 7.5. Plasmid DNA (50 µg in 0.2 ml of the above buffer) was added to 0.1 ml of the cell suspension and incubated for 15 min at room temperature. Then 3.5 ml of 20% polyethylene glycol 4000, 10 mM Tris-HCl, 10 mM CaCl₂ (pH 7.5) were added, and the spheroplasts were sedimented by centrifugation and resuspended in 1 ml of the sorbitol, Tris-HCl, CaCl₂ solution; the mixture was then added to 15 ml of melted minimal regeneration agar at 45°C and poured on solid minimal regeneration agar plates.

<footnote n="1">¹ L broth, 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1 mM NaOH, 1% gelose Difco.</footnote>
## RESULTS AND DISCUSSION

**Selection of Recombinant Plasmids**—A pool of yeast DNA fragments inserted into the hybrid *E. coli* yeast plasmid pFL₁, carrying the orotidine 5'-monophosphate decarboxylase gene from yeast (URA3), was used to complement a yeast strain (F.F.1 sp 1) defective for methionyl-tRNA synthetase and orotidine 5'-monophosphate decarboxylase. Spheroplasts from this strain were transformed by the yeast DNA pool and several colonies grew on plates deprived of methionine and uracil. In this way we avoided selecting revertants of the mutant methionyl-tRNA synthetase gene. We used three independent pools of yeast DNA in these experiments. Three colonies were obtained from the first pool, five from the second, and none from the third. Nothing grew on control plates where spheroplasts had been plated without in vitro DNA. The efficiency of transformation was tested by plating those spheroplasts which had been treated with a yeast DNA pool onto a medium supplemented with methionine; thus we estimate that about 6000 independently transformed clones had been obtained.

Two lines of evidence have indicated that the methionine phenotype (*Met*⁺) of these transformed colonies is carried by recombinant plasmids. First, DNA extracted from a single transformant from each of the two pools was able to transform *E. coli* BJ 5183 for ampicillin resistance (Amp⁺) (300 and 150 Amp⁺ colonies were obtained from the two clones). Moreover, when the plasmid DNAs extracted from the transformed *E. coli* were used to retransform the *mes1,ura3* strain, substantially higher frequencies of transformation resulted (1500 *Met*⁺ transformants/10 µg of plasmid DNA) in comparison with the original selection (3 to 5 *Met*⁺ transformants/50 µg of plasmid DNA). The second point which establishes the transmission of the *Met*⁺ phenotype by the recombinant plasmids has come from genetic analysis.

One *Met*⁺ transformant from each pool was crossed with the *mes1,ura3* recipients grown under selective conditions and the results of these crosses are given in Table I. It is clear that the two transformed clones show nonmendelian inheritance of the *Met*⁺ character. The genetic data also show that the *Met*⁺ character is sometimes lost at meiosis or during the last mitosis preceding it. *Met*⁺ character during mitosis under nonselective conditions was observed when *Met*⁺ clones were grown to stationary phase in complete medium; replica plating from YPG<fnref n="2" /> onto minimum medium indicated a loss of around 20%.

**Measurement of the Methionyl-tRNA Synthetase Activity in the Transformants**—The recipient strain used in our experiments harbors a methionyl-tRNA synthetase with a reduced affinity for methionine. Activity can therefore be restored either by increasing the concentration of the amino acid or the concentration of the enzyme or by decreasing the $K_m$ for methionine. The latter should be the case for the transformants described above if indeed the wild type gene has been cloned. In order to study the enzyme synthesized by the *Met*⁺ transformants, the aminoacylation of tRNA<sup>Met</sup> was measured in the crude extract. Table II shows that the transformants are overproducing a wild type methionyl-tRNA synthetase as deduced from the measurement of the relative specific activity and the $K_m$ values. The activity is approximately 30 times higher than that of the wild type. This might be expected when proteins are synthesized from genes which are introduced into autonomously replicating plasmids. The exact number of copies of plasmid DNA has not been determined, but we know from the literature that these can be as many as 80 copies (6, 17).

<figure><img src="image_2.png" /><figcaption>FIG. 1. Structure of the plasmid pFL₁. —, 2µ EcoRI-D fragment; ---, 1.1 kilobase yeast genomic fragment which codes for orotidine 5'-monophosphate decarboxylase (URA3); —, pBR322 plasmid. The length of the plasmid is 7.65 kilobases. The positions of restriction sites were taken from Sutcliffe (18), Cameron *et al.* (7), Bach *et al.* (8), and from our own data.</figcaption></figure>

Transformants were selected for the double phenotype, *Met*⁺,*URA*⁺. The frequency of transformation was measured by plating an aliquot of the hybrid plasmid carrying the yeast DNA pool on a minimal plate supplemented with methionine (5 mg), selecting therefore only for the *URA*⁺ phenotype born by each plasmid. Plates were incubated at 30°C. *E. coli* transformation was done according to Cohen *et al.* (10).

**Determination of Methionyl-tRNA Synthetase Aminoacylation Activity**—Aliquots of the cultures (50 ml) harvested in log phase were washed in extraction buffer and stored at -25°C. After thawing out, cells were resuspended in 500 µl of grinding buffer: 50 mM Tris-HCl (pH 7.4), 30 mM KCl, 2.5 mM glutathione. Then 500 µl of glass beads were added and the cells were ground by vortexing 2 min on a topbench vortexer at maximum speed. Then 1 ml of grinding buffer was added and the mixture was centrifuged 20 min at 5000 rpm. This crude supernate was used for the determination of methionyl-tRNA synthetase activity. Protein concentration was estimated according to Ehresmann *et al.* (15). Aminoacylations were carried out in the following medium: 50 mM Tris-HCl (pH 7.4), 30 mM KCl, 2.5 mM glutathione, 10 mM ATP, 12.5 mM MgCl₂, 0.1 mM [¹⁵S]methionine, 100 µg/ml of bovine serum albumin, 1.3 mg/ml of total tRNA, and 0.1 to 5 mg/ml of crude enzyme. At various time intervals 50-µl aliquots were quenched by 5% trichloroacetic acid and the precipitated aminoacylated-tRNA measured on Whatman paper discs.

**Agarose Gel Electrophoresis**—Restriction endonuclease-cleaved DNA was electrophoresed on horizontal, 0.8% agarose gels. About 1 µg of DNA was used per slot and electrophoresis was carried out at room temperature at 1 V/cm for 12-18 h. The electrophoresis buffer was 40 mM Tris-acetate (pH 7.9), 1 mM EDTA, and 5 mM sodium acetate. Ethidium bromide (1 µg/ml) was added to the gel and to the buffer. λ DNA digested with *Hind*III restriction enzyme was used as a reference marker.

DNA restriction digestions were performed as described by the enzyme suppliers (Bethesda Research Laboratories or Boehringer Mannheim).

**Nick-translation DNA Labeling and Hybridization**—Plasmid DNA containing the methionyl-tRNA synthetase gene was radioactively labeled by translation using DNA polymerase I (Boehringer Mannheim), 4 units/µg of DNA, DNase I (Boehringer Mannheim), 1 ng/µg of DNA, and [α-³²P]dATP, [α-³²P]dCTP, [α-³²P]dTTP (400 Ci/mmol). The reaction was terminated after 1 h at 15°C and chromatographed on a Sephadex G-50 column (1 ml) in 0.2 M Tris-HCl (pH 8.0), 0.2 M NaCl. Then 5 × 10⁷ cpm/200 µl were incorporated into DNA.

The 2 µg of total yeast DNA and 2 ng of DNA from two independent recombinant plasmids were digested with restriction endonuclease *Hind*III and transferred to nitrocellulose filters (16). For hybridization the radioactive probe was heated for 3 min at 80°C and diluted in 2 ml of hybridization solution (50% formamide, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, and 0.5% sodium dodecyl sulfate).

<footnote n="2">² YPG, 1% yeast extract, 2% peptone, 2% glucose.</footnote>
<caption>TABLE I</caption>

Segregation of the *Met*<sup>+</sup> character in the tetrads obtained from crosses of transformed yeast cells by a *mes1,ura3* strain

<table><caption>TABLE II</caption><thead><tr><th rowspan="2">Transformant</th><th colspan="5">No. of tetrads of each type</th></tr><tr><th>4 Met<sup>+</sup>:0 Met<sup>-</sup></th><th>3 Met<sup>+</sup>:1 Met<sup>-</sup></th><th>2 Met<sup>+</sup>:2 Met<sup>-</sup></th><th>1 Met<sup>+</sup>:3 Met<sup>-</sup></th><th>0 Met<sup>+</sup>:4 Met<sup>-</sup></th></tr></thead><tbody><tr><td>Clone 1</td><td>15</td><td>13</td><td>0</td><td>0</td><td>1</td></tr><tr><td>Clone 2</td><td>13</td><td>3</td><td>0</td><td>1</td><td>2</td></tr></tbody></table>

Measurement of the K<sub>m</sub> values for methionine in the wild type and yeast transformed cells

<table><thead><tr><th>Strain</th><th>Specific activity of the crude extract in arbitrary units</th><th>K<sub>m</sub> for methionine</th></tr><tr><td></td><td></td><td>μM</td></tr></thead><tbody><tr><td>Wild type</td><td>1<sup>a</sup></td><td>10 (± 1)</td></tr><tr><td>Mutant <i>mes1,ura3</i></td><td>Not detectable</td><td>2000 (± 500)</td></tr><tr><td>Transformed clone 1</td><td>30</td><td>10.6 (± 1)</td></tr><tr><td>Transformed clone 2</td><td>27</td><td>10 (± 1)</td></tr></tbody></table>

<sup>a</sup> The specific activity of the crude extract is 0.17 nmol of charged tRNA × min<sup>-1</sup> × mg<sup>-1</sup> of protein. Specific activity of the crude extract was measured using 100 µM methionine.

<figure><img src="image_3.png" /><figcaption>FIG. 2. Analysis of recombinant plasmids by agarose gel electrophoresis. DNA was isolated, digested with Eco RI, electro-phoresed on an 0.8% agarose gel, and stained with ethidium bromide. Lane 1, Hind III digest of λDNA; lanes 2 to 7, DNA from 6 subclones Amp' obtained from transformant 1; lanes 8 to 13, DNA from 6 subclones Amp' obtained from transformant 2; Kb, kilobases.</figcaption></figure>

These results rule out the possibility that the *Met*<sup>+</sup> phenotype is a consequence of cloning a gene acting on the regulation of the methionine-biosynthesis chain and causing an accumulation of methionine.

Analysis of the Plasmid DNAS—Six Amp' colonies from each transformant were selected for further analysis. Plasmid DNA was extracted and digested with Eco RI restriction

<figure><img src="image_4.png" /><figcaption>FIG. 3. Comparison of restriction enzyme digests of plasmid DNA isolated from clone 1 and clone 2. DNA was cleaved with the indicated restriction enzymes and the resulting fragments separated by electrophoresis on agarose gel. The sizes of HindIII-digested DNA markers shown on the left are listed in kilobases.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>FIG. 4. Schematic representation of cleaved plasmid DNA from clones 1 and 2 with the indicated restriction enzymes. The resulting fragments were separated by electrophoresis on a 5% polyacrylamide gel. A control of digested plasmid DNA (pFL<sub>1</sub>) with the corresponding restriction enzyme was run. The length of the bar represents 1 kilobase. Fragments in brackets have not been ordered.</figcaption></figure>

enzyme. If the inserted yeast DNA fragment does not contain any Eco RI sites, only two fragments will be obtained: i) the 2 µD fragment and ii) a fragment containing the remainder of the plasmid (5.6 kilobases) and the inserted yeast fragment. Obviously, the minimum length of the yeast sequence will be that coding for the structural gene of the yeast methionyl-tRNA synthetase. This can be estimated in the region of 2.3
<figure><img src="image_9.png" /></figure>

FIG. 5. Autoradiogram showing *Hind*III digest of plasmid DNA from clones 1 and 2. DNA was digested with *Hind*III and the resulting fragments were electrophoresed on a 0.8% agarose gel and transferred to nitrocellulose paper. The figure shows the hybridization with ³²P-labeled plasmid DNA isolated from clone 1. *Lanes* 1 and 4, pFL; plasmid-digested *Bam* HI and *Eco* RI restriction enzymes; *lane* 2, *Hind*III digest of plasmid DNA isolated from clone 1; *lane* 3, *Hind*III digest of plasmid DNA isolated from clone 2.

kilobases since the yeast methionyl-tRNA synthetase is a dimer of 2 × 86,000 daltons.<fnref n="3" />

Fig. 2 shows the restriction pattern of the plasmid DNA obtained with *Eco* RI. The several subclones of transformant 1 gave DNA fragments which were extremely varied in size, thus indicating a loss of DNA. For reasons which we do not explain, this loss of DNA is often observed during growth of yeast transformants. In contrast the second group of subclones gave a unique fragment of approximately 10 to 11 kilobases in addition to the 2µ D fragment. Only those subclones with 10 to 11 kilobases of *Eco* RI fragment were able to retransform yeast. One clone from each transformant was selected and called clone 1 and clone 2.

Plasmid DNAs from clones 1 and 2 were subjected to restriction digestion in order to estimate the total length of the methionyl-tRNA synthetase gene by summing up the common fragments obtained. Fig. 3 shows the digestion products obtained with *Hind*III, *Bgl* II, *Bam* HI, *Sal* I, *Pst* I, and *Xho* I restriction endonucleases. There are no *Bgl* II, *Bam* HI, *Pst* I, and *Sal* I sites in the inserted yeast DNA (this was deduced from the presence of high molecular weight forms and from the known restriction map of the plasmid vector) (see Fig. 1). Use of the *Bam* HI site in the plasmid vector to insert the *Sau* IIIA yeast DNA pool results in the loss of the *Bam* HI site in the recombinant plasmid. *Hind*III digestion yields 4 and 5 fragments, respectively, from plasmids 1 and 2. Only two of these are of the same size, but they belong to the plasmid vector. The absence of similar fragments in the cloned DNA results from the fact that the methionyl-tRNA synthetase gene has been inserted in two different orientations, as will be discussed.

Among those endonucleases recognizing tetranucleotide sequences, *Hpa* II and *Hae* III gave unexpectedly few fragments (2 to 4), whereas multiple *Taq* I sites are present in both plasmids. From the latter data a size of 2.8 kilobases was estimated for the methionyl-tRNA synthetase gene. We also calculated that the average size of cloned DNA was 3.5 kilobases in clone 1, and 4.5 kilobases in clone 2. Fig. 4 summarizes the digestion data obtained with this second class of endonucleases.

*Hind*III digestion products obtained from the two recombinant plasmids were transferred to nitrocellulose paper and hybridized to both of the two plasmid DNAs. Fig. 5 shows the results of the hybridization when the DNA from clone 1 is the radioactive probe. The radioactive pattern obtained resembles the cold *Hind*III digestion pattern of the two plasmid DNAs regarding the number and the relative electrophoretic mobility of the fragments. A striking feature which results from this analysis is that each *Hind*III fragment from clone 1 is complementary to that obtained from clone 2. This seems at first sight to be in contradiction to the absence of common fragments in the two inserted DNA segments. One way to solve this apparent paradox is to suggest that the orientation of the methionyl-tRNA synthetase gene in the plasmid vector is inverted in clone 1 relative to clone 2. A map of the *Hind*III sites of clone 1 and 2 is presented (Fig. 6).

The cloning of the yeast methionyl-tRNA synthetase gene opens some interesting future lines of research in addition to the fact that the protein can now be produced in larger amounts. First, sequencing of the leader region that precedes the structural gene should give some insight into the regulation of the genes, and second, sequencing of the structural gene offers a rapid route to the primary structure of the protein.

<figure><img src="image_10.png" /></figure>

FIG. 6. *Hind*III restriction map of the two cloned yeast DNA fragments coding for methionyl-tRNA synthetase. The arrows indicate the sites of cleavage. Shaded areas, plasmid vector DNA sequences; single line, yeast DNA sequences. The bar represents the length of 1 kilobase.

<footnote n="3">³ P. Walter and F. Fasiolo, unpublished experiments.</footnote>
*Acknowledgments*—We thank Prof. J. P. Ebel and Prof. Y. Bou-
langer for encouragement during this work. We acknowledge the help
of D. Barker in editing the manuscript.

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