Current Genetics (1984) 8 : 265-270

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© Springer-Verlag 1984

Cloning of a yeast dihydrofolate reductase gene in Escherichia coli* Kamalendu Nath and Edward W. Baptist 1 Department of Biology, C. W. Post Center, Long Island University, Greenvale, New York 11548, USA

Summary. The dihydrofolate reductase gene of Saccharornyces cerevisiae has been isolated by selection of trimethoprim resistant Escherichia coli transformed with a gene bank of yeast DNA in plasmid pBR322. From a 9.2 kilobase pair BamHI DNA fragment this gene has been localized to a 1.76 kb fragment, the restriction map of which appears different from those reported for the E. coli and the mouse dihydrofolate reductase genes. The enzyme encoded by the chimeric plasmid was established as yeast dihydrofolate reductase by its sensitivity to antifolates in vivo through growth studies and in vitro by enzyme assay. Since, the expression of this gene occurs independent of its orientation within the chimeric plasmid, the 1.76 kb fragment may contain functional regulatory sequences in addition to the structural sequences for yeast dihydrofolate reductase.

Key words: DHF Reductase - Cloning - Trimethoprim Yeast

Introduction The enzyme dihydrofolate reductase (EC 1.5.1.3) carries out an NADPH-dependent reduction reaction of dihydrofolate to tetra-hydrofolate essential for the biosynthesis of thymidine, purines and glycine. This enzyme, in

* This work was carried out in part at Merck & Co., Rahway, New Jersey, USA and at Southern Biotech, Inc., Tampa, Florida, USA l Current address: Department of Zoology, University of Georgia, Athens, Georgia 30602, USA Offprint requests to: K. Nath

general is the principal target of folate analgos, such as trimethoprim, methotrexate and others (Burchall and Hitchings 1965; Futterman 1957). The folate analogs are clinically important as antibacterial, antiprotozoal, immunosupressant and antineoplastic agents. These chemotherapeutic effects of folate analogs are mediated by an inhibition in the activity of dihydrofolate reductase (Blakely 1969). The dihydrofolate reductase in higher eucaryotes have a much lower affinity for the folate analogs as compared to the enzyme from procaryotes (Burchall and Hitching 1965). On the genomic level, the dihydrofolate reductase structural gene, which is colinear with the coding sequences in Escherichia coli (Rood et al. 1980; Smith and Calvo 1980) and bacteriophages T4 and T5 (Hanggi and Zachau 1980; Peter et al. 1979) is segmented in such higher eucaryote as mouse (Chang et al. 1978; Nunberg et al. 1980). Thus, while the mouse dihydrofolate reductase gene contains five large intervening DNA sequences (Nunberg et al. 1980), the E. coli gene contains none (Smith and Calvo 1980). Consequently, the expression of the E. coli dihydrofolate reductase gene can be readily obtained through genomic cloning (Rood et al. 1980), while the expression of the mouse enzyme requires the cloning of complementary DNA copy of the messenger RNA (Chang et al. 1978). It was of interest to determine if the yeast dthydrofolate reductase which is similar to the mouse enzyme in its trimethoprim resistance property could be expressed in E. coli rendering this organism resistant to concentrations of trimethoprim which are otherwise inhibitory to its growth. A segmented gene is less likely to be expressed in E. coli and would require the construction of a complementary DNA copy of the functional messenger RNA. We report here, the successful expression of yeast dihydrofolate reductase gene as a cloned, genomic DNA fragment in E. coli.

266

K. Nath and E. W. Baptist: Cloning of a yeast FOL gene grown in Luria broth agar containing ampicillin and then replicated on to ML agar containing trimethoprim. The preparation of Southern blots (Southern 1975) and hybridization of such blots with radioactive DNA probes prepared by nick translation (Maniatis et al. 1975; Rigby et al. 1977)was performed by established procedures (Denhardt 1966; Wahl et al. 1979). The analysis of the chimeric DNA in large number of transformants was performed by an alkaline denaturation dependent minllysate procedure of Birnboim and Doly (1979). Dihydrofolate reductase activity was measured in crude extracts by modification of a procedure described by Baccanari et al. (1975). The crude extract was a 30,000 g supernatant (20 rain at 4 °C) of exponentially growing washed cells lysed by sonication. To facilitate lysis of yeast, these cells were pretreated with glusulase and washed repeatedly. Restriction maps were constructed by comparison of DNA fragments generated by single and multiple restriction enzyme digestions to known standards (Nath and Bollon 1977; Nath 1981).

Fig. 1. Cloned DNA fragment that confer trimethoprim resistance to E. coli. Chimeric DNA from four trimethoprim resistance clones (pYDR - 1 through -4) was cleaved with BamHl and analyzed on a 0.4% horizontal agarose gel. The electrophoresis was performed at 20 mAmp for 17.5 h utilizing a pH 8.0 buffer that contained 40 mM Tris, 20 mM sodium acetate, 5 mM EDTA and 0.5 #g/ml of ethidium bromide. A, B, C, D represent bacteriophage XDNA cleaved with EcoRI, Sinai, BgllI and HindlII respectively, and E represents BamHI treated pBR312 DNA. The slot designated pBR322 represents BamHI treated pBR322 DNA

Materials and methods

Chemicals. Restriction endonucleases were obtained commercially from Bethesda Research Laboratories, Inc., New England Biolabs, Miles and Boehringer Mannheim. Bacterial alkaline phosphatase (BAPF) was purchased from Worthington whereas trimethoprim, aminopterin and methotrexate were purchased from Sigma. The radioisotope [32p]d-CTP was purchased from New England Nuclear. Organisms. Saccharomyees cerevisiae strain XJBI-IC (aade2, met1, ural) obtained from Yeast Genetic Stock Center, Berkeley, CA USA, was grown either in Y medium or yeast nitrogen base minimal medium as described previously (Nath and Bollon 1976). Escherichia eoli strain RR1 was grown either in Luria broth (Miller 1972) or in one of the following two minimal media: ML (Curtiss III et al. 1968), Vogel Bonner medium E (Vogel and Bonner 1956), supplemented with glucose, leucine, proline and thiamine. Methods. Yeast DNA was isolated by a cesium chloride density gradient method, described previously (Nath and BoUon 1976). Transformation experiments were carried out in E. coli RRt with chimeric DNA constructed as described (Nath and Bollon 1977) except that the linear plasmid DNA was treated with alkaline phosphatase to remove 5'-phosphate and prevent religation of the ends to form pBR322 circles. The selection for the yeast dihydrofolate reductase gene was based on the difference in trimethoprim sensivity between the E. coli and the yeast enzymes. The transformed E. eoli were first

Results

Yeast dihydrofolate reductase gene in E. coli A gene bank of S. cerevisiae generated by cleavage of yeast DNA with BamHI and insertion into the BamHI site of pBR322 DNA, that was present in about 11,000 transformed E. coil RR1 was screened for the presence o f the yeast dihydrofolate reductase gene by the ability o f the transformants to grow in minimal agar containing trimethoprim at concentrations that is inhibitory to the growth of RR1 (20 ~g/ml). Five transformants showed resistance to trimethoprim and plasmids from 4 of these isolates that retransformed RR1 to trimethoprim resistance with 100% efficiency showed the presence of a 9.2 kb DNA insert (Fig. 1). The fifth isolate containing a 4 kb insert and unable to retransform RR1 to trimethoprim resistance was presumed to be a/z2 coli mutant and utilized as control. The 9.2 kb insert in one transformant 1 pYDR9.2/1 was present in an orientation that was opposite to that present in the other three (Fig. 2A). Of the seven enzymes used for the construction of a restriction map o f the 9.2 kb insert, HindlII and PstI had 4 sites, SalI had 2 and the remaining 4 endonucleases had one site each (Fig. 2B). Hybridization of Southern blots with [32p] probes (pYDR9.2/1, pYDR9.2/4) confirmed that the inserts were indeed of yeast origin. BamHI treated whole yeast DNA showed hybridization with a DNA size of only 9.2 kb and no larger. All DNA fragments of the

The transformants described here were not constructed in the present laboratory, hence, instead of assigning them two letter laboratory abbreviations, functional descriptive abbreviation of YDR for yeast dihydrofolate reductase together with the fragment sizes have been used

K. Nath and E. W. Baptist: Cloning of a yeast FOL gene B

B _I_ BR322 q- P

YDR 9.2. P

pBR322-L

-ii

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Pv A s

A

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A=Ava I ; B=Bom HI; H= Hind TIl'; P=Pst I ; S=SoI I

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267

pYDR 9 . 2

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B Fig. 2A, B. Map of the BamHI yeast DNA insert. A Orientation - /1 through /4 represents the chimeric DNA pYDRg.2/1 through 4. The position of the 9.2 kb insert has been kept fixed while that of pBR322 has been switched in the two orientations. B Map of 9.2 kb insert - The numbers represent restriction fragment sizes in kilobase pairs

Table 1. Transformants constructed from Sail and HindlII treated pYDR9.2 DNA DNA source

pYDR9.2/1 pYDR9.2/4 pYDR9.2/1 pYDR9.2/4

Restriction enzyme used

Number of transformants scoreda

Number of cotransformantsb Tmpr c

Tcr

SalI Sail HindIII HindIII

5,274 3,862 7,510 6,793

5,040 38 5 50

71 58 1,702 1,162

a The selection was for 20 #g/ml of ampicillin resistant transformants b The ampicillin resistant transformants were replicated to test for resistance to 20 ~zg/ml of trimethoprim (Trapr) and 15 ug/ml of tetracycline (Tor) c The transformants from the SalI chimera grew within 24 h after replication onto trimethoprim minimal agar medium, those from the HindIII chimera required more than 24 h for growth

restriction map construct in Fig. 2 were identified in the whole yeast DNA treated with the corresponding endonucleases. However, no such hybridization occured in Southern blots of whole E. coli RR1 DNA treated similarly.

Fig. 3. Characterization of a subfragment of 9.2 kb yeast DNA that confers trimethoprim resistance in E. coli RR1. Slots 1, 2 and 3 represent 1.4 #g of pYDR1.76/11, 1.5 ~zgof pBR322 and 0.64 /sg of pYDR1.76/41 DNA respectively. Slots X, Y and Z represent 0.33 t~g of HindIII treated bactriophage XDNA, 0.8 t~g of HaelII treated bacteriophage PM2 DNA and 0.20 ~zgof EcoRI treated XDNA respectively. Electrophoresis was performed on a horizontal 1.2% agarose slab gel at 15 mAmp for 20 h

Isolation o f a shortened D N A fragment Since SalI and HindIII delineates overlapping DNA fragments of about 2 kb each, together representing the entire 9.2 kb BamHI DNA fragment (Fig. 2B), pYDRg.2/ 1 and pYDRg.2/4 DNA were digested with either SalI or HindIII, ligated with pBR322 that was pretreated with the same restriction endonuclease and phosphatase, and transformed in RR1. The cotransformation efficiency of these transformants for trimethoprim resistance, shown in Table 1, was in excess of 95% for chimeric DNA constructed from SalI treated pYDR9.2/1 but less than 1% for all other constructs. When screened b y a mini-lysate procedure, the smallest DNA fragment in the trimethoprim resistant clones o f Table i was a 1.76 kb BamHI/SalI DNA fragment (Fig. 2B) present only in SalI constructs (Fig. 4). None o f the HindIII constructs yielded fragments smaller than 1.76 kb. Two SalI constructs, pYDR1.76/11 and pYDR1.76/ 41 derived from pYDR9.2/1 and pYDRg.2/4 respectively were used for further analysis (Fig. 3). The 1.76 kb DNA fragment is present in opposite orientation in these constructs (Fig. 4). p Y D R 1 . 7 6 / l l probably arose by deletion of larger SalI DNA segment in pYDRg.2/1 (Fig. 2A) and recircularization o f the smaller SalI fragm e n t containing most o f pBR322 (Fig. 4). pYDR1.76/ 41 on the other hand probably arose from the insertion

268

K. Nath and E. W. Baptist: Cloning of a yeast FOL gene Barn HI Sal T

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Table 2, Comparison of antifolate concentrations required for growth inhibition

L HindTIT I ~ pBR522* I i Sale I ~,,, pBR522

pYDR 1.76/11 pYDR 1"76/41

Fig. 4. Restriction map of the 1.76 kb yeast DNA subfragment. The 1.76 kb yeast DNA insert can be seen to be present in two opposite orientations, when compared with the markers on pBR322 such as HindIII site above. The pBR322* lacks a 0.28 kb BamHl/SalI pBR322 DNA fragment

i.O

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E. coli/pBR322 E. coli mutant 5 mutant 6 E. coli pYDR9.2/1 pYDR9.2/4 S. cerevisiae X2180-1A

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Trimethoprim

Aminopterin

50%

50% total

total 0.5

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30 50

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100 500

750 2,500

50 > 250 50 250

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>2,500

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E. eoli was grown at 37 °C and S. cerevisiae at 30 °C as described in the legend to Fig. 5 This concentration was obtained from constructing a series of growth curves (similar to the one presented in Fig. 5 for one such concentration) at increasing concentrations of the antifolate

0.5

E 0 0~ l.f')

Inhibitory concentrationsb 0zg/ml)

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t

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Effect o f antifolates on growth

Aminopterin 0.5

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z 0.2

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2

5

4

5

Time (hours)

Fig. 5. Effect of antifolates on growth rates. E. coli strain RR1 containing pBR322 (P) or pYDR9.2/1 (D) and E. coli trimethoprim-resistant mutant (M) were grown in minimal medium. S. cerevisiae X2180-1A (Y) was grown in a minimal nitrogen base medium. All exponentially growing cultures were subdivided at A590 of 0.1 or less and grown further with or without trimethoprim (t) or aminopterin (a). The measured absorbancy was normalized to an absorbancy of 0.1 for the initial measurement (time = 0)

o f a 2.04 kb Sail DNA fragment, derived from pYDR9.2/ 4 (Fig. 2A) into the SalI site of pBR322; the 2.04 kb insert consisting of a 1.76 kb SalI/BamHI yeast DNA fragment and a 0.28 kb BamHI/SalI pBR322 DNA fragment (Fig. 4). Nonetheless, the two chimeric plasmid appear identical with respect to conferring trimethopfim resistance and presence o f the six restriction sites shown in Fig. 4.

Growth inhibition studies with a spectrum of antifolates in disc tests indicated that the various pYDR clones were similar to yeast but distinctly different from E. coli RR1 (with or without pBR322) and its trimethoprim resistant mutants. In direct growth measurement studies presented in Fig. 5, whereas RR1 containing pBR322 (P) and its mutant (M) were inhibited by 10 pg/ml of t r i m e t h o p d m , clone pYDR9,2/1 (D) and yeast (Y) remained unaffected b y this or higher concentrations of trimethopfim. In the presence of trimethoprim, the doubling time for RR1 and its mutant increased by a factor of 5 and 2 respectively. In the presence of 10 pg/ml of aminopterin however, the growth of RR1 mutant (M) remained unaffected while the doubling time for clone pYDR9.2/1 (D) increased by a factor of 2, similar to that for RR1 (P) and yeast (51). F r o m growth curves constructed with increasing concentrations of antifolates it was observed that the trimethopfim concentrations required for a 50% inhibition in the growth rate of E. coli, increased several hundred folds when it contained the yeast gene: 200 times in case of pYDR9.2/1 and 100 times in case o f pYDR9.2/4 (Table 2). The E. coli mutants showed sensivity to trimethoprim that was in between E. coli and pYDR clones. However, the mutants were highly resistant to aminopterin. The sensitivity of E. coli to aminopterin, on the other hand was unaffected by the presence of pYDR since the yeast enzyme is inhibited by lower concentrations o f aminopterin (Table 2).

K. Nath and E. W. Baptist : Cloning of a yeast FOL gene

269 Measurement o f dihydrofolate reductase activity

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TMP [nM] Fig. 6. Effect of increasing concentrations of trimethoprim in the assay mixture on dihydrofolate reductase activities. The 1.0 ml standard dihydrofolate reductase assay mixture contained 100 mM Tris-HC1, pH 7.2, 12 mM 2-mercaptoethanol, 60 ~M NADPH, 50 ~M dihydrofolate and 10 to 250/~1 of crude extract. All components of the reaction except dihydrofolate were mixed in a 1 ml cuvette and the decrease in A34 o was followed during a 5 min preincubation period. After addition of substrate, the reaction was followed for a minimum of 5 min. Trimethoprim (TMP) was added after 5 min preincubation along with the substrate. The result was the same when TMP was added during the preincubation period, pBR322 and pYDR9.2/1 represent extracts from 17. coli RR1 containing the .respective plasmids

Table 3. Measurements of dihydrofolate reductase activity in cell extract Crude extract

E. coli/pBR322 + trimethoprim E. coli/pYDR9.2/1 + trimetboprim S. cerevisiae + trimethoprim

Enzyme activity in cultures grown in Minimal mediaa

Enriched mediab

Inhibitiond

Inhibition

mU/mg c %

mU/mg

2.92 0.07 29,6 25.3 5.16 4,28

%

98

8.70 0.00

100

17

18.1 3.65

80

15

1.41 1.17

17

a E. coli was grown in Vogel and Bonner Sulfate medium E supplemented with 20 ~zg/ml of ampicillin. S. cerevisiae was grown in yeast nitrogen base media supplemented with 3% glucose b E. co//was grown in Luria broth supplemented with 20 tzg/ml ampicillin and S. cerevisiae was grown in y media supplemented with 3% glucose c One unit of enzymatic activity is the amount of enzyme that reduces 1 umole of dihydrofolate/min at 25 °C. mU = milliunits d By the addition of 40 nM trimethoprim in the reaction mixture

Measurements of dihydrofolate reductase activity in crude extracts indicated the presence o f an enzyme in E. coli p Y D R that resembled the enzyme present in yeast. As shown in Fig. 6 enzyme assay performed in the presence of increasing concentrations of trimethoprim indicated that 40 n M of this antifolate brings about an almost complete inhibition in the enzyme activity in t?. coli (pBR322) but a 20% inhibition of the enzyme in yeast and E. coli pYDR. This low level inhibition o f enzyme activity in yeast and E. c o l i p Y D R did not change substantially upto 500 n M trimethoprim. Besides similar insensivity to antifolate, the enzyme in E. coli p Y D R follows nutritional control similar to the enzyme in yeast. As shown in Table 3, upon growth in nutritionally enriched medium the enzyme activity in E. coli increased by about 300%, while it decreased by about 70% in yeast. Upon growth in enriched medium the total enzyme activity in pYDR decreased by about 40%. However, most of the remaining activity in p Y D R clone was of E. coli in origin, since, 80% o f the activity was inhibited by trimethoprim. This suggests a similar control for yeast dihydrofolate reductase gene, whether present in yeast or in E. coli.

Discussion

The growth inhibition and enzyme activity studies with antifolates presented here indicate that the yeast dihydrofolate reductase gene can be successfully cloned in E. coli by a m e t h o d that selects for the resistance characteristics conferred upon it by a donor gene product. Obviously, the dihydrofolate reductase gene of yeast differs from that of mouse even though the proteins expressed by both have similar drug resistance properties. The smaller subcloned yeast DNA fragment of 1.76 kb is more than sufficient for the 780 base pairs required for the amino acid sequence of the yeast dihydrofolate reductase, reported to be of about 26,000 molecular weight (Wu et al. 1980) as well as the p r o m o t o r sequence which may occupy up to several hundred base pairs at the 5'-end, common in eucaryotic genes. Although expression o f any eucaryotic genomic DNA fragment in t?. coli does not absolutely rule out the presence of intervening sequences, it does suggest that any such sequences in yeast dihydrofolate reductase gene, if present must be extremely small as compared to the mouse dihydrofolate reductase gene whose average coding sequences o f about 120 nucleotides are separated by approximately 7 kb o f intervening DNA sequences (Nunberg et al. 1980). A comparison o f the restriction profiles o f the various reported dihydrofolate reductase genes suggest that this

270 gene in the lower eucaryote, S. cerevisiae, is unlike that of mouse, a higher eucaryote, and superficially similar to that of the procaryote E. coli. The dihydrofolate reductase gene of mouse is about 42 kb long and contains about 5 intervening DNA sequences (Nunberg et al. 1980). The cDNA for the coding sequence of the mouse gene contains none of the restriction sites described for yeast, however, numerous sites for EcoRI, BarnHI, HindlII and PstI, amongst others are present in the intervening DNA sequences (Nunberg et al. 1980). The BamHI DNA fragment of yeast is similar to that reported for E. coli strains RSO and C600 (Rood et al. 1980; Smith and Calvo 1980) in that in both species it is a 8 to 9 kb BamHI DNA fragment, having two Sail sites in each. However, there are more restriction sites present on the yeast DNA fragment than on E. coli, such as 4 HindlII site in yeast but none in E. coli. The smaller DNA fragments, 1.76 kb of yeast and 1.6 kb of E. coli differ greatly not only in the total number of sites but also in the actual location of these sites. The arrangements of restriction sites on another dihydrofolate reductase gene frQm bacteriophage T4 (Hanggi and Zachau 1980) are quite different from those in E. coli or S. cerevisiae. Cloning of the yeast dihydrofolate reductase gene and its expression in E. coli offer the possibility of a convenient source of large amounts of this enzyme. Detailed studies of the yeast enzyme and its reactivity to antifolates will provide useful comparison to those carried out in great numbers with the enzymes from procaryotes and eucaryotes.

K. Nath and E. W. Baptist: Cloning of a yeast FOL gene

References Baccanari D, Phillips A, Smith S, Sinski D, Burchall J (1975) Biochemistry 14:5267-5273 Birnboim HC, Dolly J (1979) Nucleic Acids Res 7:1513-1523 Blakely RI (1969) The biochemistry of folic acid and related ptefidines. North-Holland Publishing Company, Amsterdam Burchall JJ, Hitchings GH (1965) Mol Pharmacol 1 :126-136 Chang ACY, Nunherg JH, Kaufman RJ, Erlich HA, Schimke RT, Cohen SN (1978) Nature 275:617-624 Curtiss R III, Charamella LJ, Stallions DR, Mays JA (1968) Bacteriol Rev 32:320-348 Denhardt DT (1966) Biochem Biophys Res Commun 23:641646 Futterman S (1957) J Biol Chem 228:1031-1038 Hanggi UJ, Zachau HG (1980) Gene 9:271-285 Maniatis T, Jeffrey A, Kleid DG (1975) Proc Natl Acad Sci USA 72:1184-1188 Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor, New York Nath K (1981) Arch Biochem Biophys 212:611-617 Nath K, Bollon AP (1976) Gen Genet 147:153-168 Nath K, Bollon AP (1977) J Biol Chem 252:6562-6571 Nunberg JH, Kaufman RJ, Chang ACY, Cohen SN, Schimke RT (1980) Cell 19:355-364 Peter G, Hanggi UJ, Zachan HG (1979) Mol Gen Genet 175: 333-341 Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:237-251 Rood Jt, Laired AJ, WilliamsJW (1980) Gene 8:255-265 Smith DR, Calvo JM (1980) Nucleic Acids Res 8:2255-2274 Southern EM (1975) J Mol Biol 98:503-517 Vogel HJ, Bonner DM (1956) J Biol Chem 218:97-106 Wahl GM, Stern M, Stark GR (1979) Proc Natl Acad Sci USA 76:3683-3687 Wu J, Florance JR, Hoogsteen K (1980) Fed Proc (abs) 39:1771

Acknowledgements. Technical assistance in part was provided by Mr. Albert B. Lenny at Merck & Co. This work is currently supported by grants from C. W. Post Research Committee. A preliminary report of this work was presented at the 10th International Conference on Yeast Genetics and Molecular Biology held at Louvain-la-Neuve(Belgium) in 1980.

Communicated by M. S. Esposito Received October 24, 1984 / Accepted January 25, 1984

Cloning of a yeast dihydrofolate reductase gene in Escherichia coli.

The dihydrofolate reductase gene of Saccharomyces cerevisiae has been isolated by selection of trimethoprim resistant Escherichia coli transformed wit...
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