Nucleotide sequence and characterization of a Gene conferring resistance to ethionine in yeast Saccharomyces cerevisiae

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 71, No. 4, 211-215. 1991

Nucleotide Sequence and Characterization of a Gene Conferring Resistance to Ethionine in Yeast Saccharomyces cerevisiae N A O F U M I S H I O M I , ~ H I D E K I F U K U D A , ~ YASUKI F U K U D A , 2 K O U S A K U M U R A T A , 3. AND A K I R A K I M U R A 3

Engineering Research Laboratories, Kanegafuchi Chemical Industry Co., Ltd., 1-8 Miyamae-machi, Takasagocho, Takasago, Hyogo 676, t Chukyo Community College, 2216 Toki-cho, Mizunami, Gifu 509-61, 2 and Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, 3 Japan Received 24 October 1990/Accepted 9 January 1991 The nucleotide sequence of a D N A fragment, when present on a multi-copy plasmid, conferring ethionine resistance to Saccharomyces cerevisiae cells was determined. The fragment contained one long open reading frame and the frame was confirmed to be an ethionine resistant gene caused by frame-shift mutation. Other than ethionine resistance, an increased dosage of the gene directed overaccumulation in cells of S-adenosyl-Lmethionine. The protein deduced from the open reading frame consisted of 617 amino acid residues with a calculated molecular weight of 67, 977.

cerevisiae cells was performed according to the methods of CaC12 (7) and lithium acetate (8), respectively. Plasmids pER1, pER2, pER3 and pER4 were constructed by inserting HindlII partial digests of pYSMH1 into HindlII site of YEpl3 (Fig. 1). Plasmid pER5 was constructed as follows: the HindlII fragment [3.0 killo-base (kb)] from pER4 was inserted into the HindlII site of a vector plasmid pUC19 (9). The 2.2 kb Pstl fragment was then cut out from the resultant plasmid and was inserted into the PstI site of pJDB207. For the construction of plasmid pER6 with a gene disrupted by frame-shift mutation, pER4 was digested with NcoI and ligated after blunting the NcoI cohesive ends by using a D N A Blunting Kit (Fig. 1). Plasmids pER7 and pER8 with genes disrupted by the insertion of an irrelevant 2 D N A fragment were constructed as follows: HindllI fragment (3.0kb) having kpnI and BssHII sites was cut out from pER4 and inserted into the Hindlll site of a vector plasmid pBR322. Kpnl [1,503 base pair (bp)] and BssHIl (1,501 bp) 2 D N A fragments were then inserted into the corresponding sites of the Hindlll fragment on pBR322. The entire HindlII fragment with Kpnl or BssHII D N A fragment was again cut out with the HindlII site of YEpl3 (Fig. 1). Ethionine resistance and S A M accumulation In order to check their resistance to ethionine, the yeast cells were aerobically grown at 30°C in 3.0 ml SD medium ( l . 0 × 1 8 c m test tube) supplemented with 2.0raM D,tethionine. Growth was turbidimetrically determined at 610 nm (A610) on a Hitachi U-1100 spectrophotometer. For the accumulation of SAM, the yeast cells were aerobically grown at 30°C to late log phase (A6t0=5-6) in 50ml SD medium (5dl Sakaguchi flask) containing 5.0raM zmethionine. Extraction and analysis of SAM in cells were carried out as described previously (1). D N A sequencing and analysis The HindIII fragment from pER4 was subcloned into M13mpl8 and M13mp19. Single-stranded DNAs were prepared according to the method of Messing (10) and subjected to sequencing by the dideoxy method (11). The nucleotide sequence was analyzed by computer with software termed "DNASIS" (Hitachi Co., Ltd., Tokyo). Searches for open reading frames (ORF) and restriction sites, calculation of molecular weight (M.W.) and measurement of hydropathy

In earlier studies, we had cloned a D N A fragment conferring yeast Saccharomyces cerevisiae cells resistance to ethionine, a methionine analogue, onto a vector plasmid YEp13 and the hybrid plasmid was designated pYSMH1 (1). Other than the resistance to ethionine, the D N A fragment enhanced the accumulation of S-adenosyl-Lmethionine (SAM) in cells, especially when the yeast cells with pYSMH1 were grown in a medium containing Lmethionine (2). Preliminary enzymatic analysis of extracts prepared from yeast cells with pYSMH 1 indicated that the D N A fragment might direct the synthesis of methionine adenosyltransferase, SAMI or SAM2 (2). However, the result was apparently inconsistent with the observation of Cherest et al. (3) that S. cerevisiae cells could acquire ethionine resistance through disruption of the gene for SAM1 or SAM2. In order to identify the product of the gene for ethionine resistance on pYSMH1 and to get further insight into the nature of ethionine resistance in S. cerevisiae cells, the nucleotide sequence of the gene was determined and compared with those of SAM1 (4) and SAM2 (5) genes. MATERIALS AND METHODS Strains and m e d i u m Yeast S. cerevisiae DKD-5D-H (MA Ta, leu2-3, leu2-112, trpl, his3), a gift from Dr. Y. Oshima, Escherichia coli DH1 (F', gyrA96, recA1, relA1, endA1, thil, hsclR17, supE44, 2 ) and JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, 2 , lac-proAB), [F, traD36, proAB, laclU-Z M15]) were used as hosts of plasmids. Yeast cells were aerobically grown at 30°C in SD minimal medium (2.0O//ooglucose, 0 . 6 8 ~ yeast nitrogen base w / o amino acids; p H 5 . 0 ) supplemented with 2 0 ~ g / m l L-tryptophan and 20 p g / m l L-histidine. Construction o f plasmids Vector plasmids YEpI3 and pJDB207 (6), a gift from Dr. J. D. Beggs, were used for subcloning of a gene for ethionine resistance on p Y S M H I . Vector plasmids were digested with restriction endonucleases, dephosphorylated with alkaline phosphatase and then ligated with various D N A fragments by using a Ligation Kit. Transformation of E. coli and S.

* Corresponding author. 211

212

SHIOMI ET AL.

J. FERMENT. BIOENG.,

(average window size: 6 amino acids) were also performed by using the " D N A S I S " system. H o m o l o g i e s in nucleotide and amino acid sequences were inspected in E M B L Data Banks in " D N A S I S " software. Northern blotting Log phase (A610=4) cells (5 × 108 cells) grown aerobically at 30°C in SD medium with and without a 2.0 m M n,L-ethionine supplement were washed once in chilled 0.85o/oo saline solution and total R N A was prepared according to the m e t h o d of Elder et al. (12). Northern blotting was carried out by the m e t h o d o f Maniatis et al. (13). R N A s were electrophoresed in 1.0% agarose gel with M O P S buffer (20 m M M O P S , 5.0 m M CH3COONa, 1.0 m M E D T A , 0.00210/oo diethylpyrocarbonate), transferred to a H y b o n d - N filter and then hybridized with a probe in a hybridization buffer containing 500/00 f o r m a m i d e at 42°C. The filter was rinsed twice with 2 × SSPE (0.37 M NaCl, 0.25 M NaH2PO4, 25 m M E D T A ) buffer containing 0.1~oo sodium dodecyl sulfate (SDS), twice with 1 × SSPE containing 0.1% SDS, once with 0.5 × S S P E containing 0.1% SDS at 55°C and then exposed to Fuji X RAY Film for 2 0 h at r o o m temperature. The D N A probe was labelled with [(r-32P]dCTP using a R a n d o m Primer D N A Labelling Kit. The ribosomal R N A s were used as size markers. Ultraviolet microscopy Yeast cells were aerobically grown at 30°C in SD m e d i u m with and without 5.0 m M Lmethionine or 2.0 m M n,L-ethionine, washed once in chilled 0.85~00 saline solution and then suspended in the same solution. P h o t o g r a p h s were taken by an ultraviolet microscope, Model U V M P (Ernst Leitz, Wetler). The wave length employed was 260 nm with a bandlength o f 3 mm and a film, K o d a k Spectrum Analysis no. 1 (Eastman K o d a k Rochester) was used. Cell suspension was placed on a vycor slide and the cells were exposed to UV for 10 min at r o o m temperature. Chemicals Kits used for ligation, blunting, rand o m primer D N A labelling, plasmids ( M 1 3 m p l 8 and M 1 3 m p l 9 ) and restriction enzymes (except for BssHII) were purchased from T a k a r a Shuzo Co., Ltd., Japan. BssHII was from N i p p o n Gene Co., Ltd., Japan. H y b o n d - N filter and [(~-32p]dCTP (3,000 C i / m m o l ) were from A m e r s h a m - J a p a n Co., Ltd.

= H

2Kb

I

PBsK

I pYSMHI

ii

pERI

II

I

HSH

I

I~/

I

I

H

I

I

I

pER3 pER4

EP

E I

pER2

pER5

E l 'r I

160

I

+

130

]

-

ND

i

-

I0

+

140

I

I

SAM

+

I II

]

NO

pER6

ND H

K

K

H

pER7

15 H

Bs

Bs

H

I ~ t I 12 Trimming of DNA insert in plasmid pYSMH1. Procedures for plasmid construction are described in Materials and Methods. [], Chromosomal DNA fragment with a gene for ethionine resistance; =, vector YEpl3 fragment; x, position of frame-shift mutation; [ , 2DNA fragment inserted for mutation. Restriction enzymes used were: H, HindII1; P, Pstl; E, EcoRI; S, Sall; Bs, BssHII; K, Kpnl and N, Ncol. Et r, susceptibility to ethionine (+, resistant; , sensitive); SAM, SAM contents in cells (mg/g of dry cells). ND, not determined. pER8

I.

FIG. 1.

P s t I / H i n d I I I fragment located on the left hand end o f p Y S M H 1 is required for the expression o f ethionine resistance. Nucleotide sequence of a gene for ethionine resistance To locate the region of O R F of a gene for ethionine resistance, nucleotide sequence of HindIII fragment (3.0 kb) (Fig. 2A) o f pER4 was determined (Fig. 3) by the sequencing strategy shown in Fig. 2B. A computer search for O R F showed the existence of only one possible O R F region, which begins at + 1 and ends at + 1,851, in the nucleotide sequence determined (Fig. 2C). To confirm that the O R F is for the gene responsible for ethionine resistance, S. cerevisiae D K D - 5 D - H cells were t r a n s f o r m e d with plasmid pER6, pER7 and pER8 causing disruption in the O R F at 0

I

l

RESULTS Subcloning of a gene for ethionine resistance The hybrid plasmid p Y S M H 1 contains a c h r o m o s o m a l D N A fragment (6.5 kb) responsible for ethionine resistance of S. cerevisiae D K D - 5 D - H cells in the B a m H I site o f the vector plasmid Y E p l 3 (1). To locate the gene for ethionine resistance in the D N A fragment and to determine the nucleotide sequence o f the gene, the c h r o m o s o m a l D N A fragment in pYSMH1 was subcloned onto various plasmids (Fig. l). Plasmids p E R l , pER2, pER3 and pER4 contained various HindIII fragments generated by partial digestion of p Y S M H l on a vector plasmid Y E p l 3 . W h e n these plasmids were used to t r a n s f o r m S. cerevisiae D K D - 5 D - H cells, t r a n s f o r m a n t s with pER1 or pER4 became resistant to ethionine, whereas t r a n s f o r m a n t s with pER2 or pER3 were sensitive to the chemical. Therefore, the gene for ethionine resistance was thought to be located in a 3.0 kb HindIII fragment on the left hand end o f p Y S M H I . Furthermore, t r a n s f o r m e d cells o f S. cerevisiae D K D - 5 D - H cells with pER5, which contains a 2.2 kb p s t l / H i n d l I I c h r o m o s o m a l D N A fragment on pJDB207, showed no resistance to ethionine. The result indicates that the 0.8 kb

N

~1

I

H

Bp

I

2

I

P

~,

I

I

Pv

3Kb

I

i

I

BsK SaPv N

I

I

I

I

/

Sp H x I i

I

A

i

B

•

: i

_~

: q

•

m t

ii

•

i

,i

C

D

1 I

508 I I II

I II I!

ii

I I II I I

/

FIG. 2. A, Restriction m a p of the HindIIl fragment from pER4. Restriction enzymes used were: Bp, BspHI; Sa, SauI; Sp, SplI and Pv, PvuII. Other abbreviations for restriction enzymes are shown in Fig. 1. B, Sequencing strategy of the HindIII fragment. Extent and

direction of DNA sequencing are indicated by horizontal arrows. C, ORF. Vertical bars represent positions of ATG codons. D, Probe DNA used for Northern blot.

VoL. 71, 1991

NUCLEOTIDE SEQUENCE OF ETHIONINE RESISTANCE GENE

-443 -332

GATCTTTTAGACCATTGCTTCTTGTTACCGGAGGCATGCGACCTG~CATCGGGATGTCTTAT~G~CAAATCTCTCGACTTTTG AGCTTGTTCTCCTCTGCTTATTGTTCGAGATGATGTTTATTTACATATTCTTTGACGT~T~TATCGG~T~TGCATGCGCATTCGGGTTAGCCCCGTTATAGGGAAAA

-221

TGAAAAAAAAAAT~TAAAAAAAAAAAGACTAAAAAAATAG~GAGCACGGC~CTTAAAAAGAAAAAAAAAATTCATGAT~CTGCAAAATAGTAAAG~CCCTGGCAAA

-110

AGAAATAAATCCGGT~GGG~GATAGACCGGCTTAAAGAGTACTTTTTCAGATTTGTC~GAGTCTGTGTTAGCGGTG~G~GATAG~G~G~GACGAAAG~GACT

1

213

ATGTCTAAAC~TTTAGTCATACCACC~CGACAG~GATCATCGATTATCTACTCCACCAGTGTCGGAAAGGCAGGGCTTTTCACGCCTGCAGACTACATCCCACAGGAG M S K Q F S H T T N D R R S S I I Y S T S V G K A G L F T P A D Y I P Q E

112

TCAG~GAAAACTT~TTGAGGGCG~GAGC~GAGGGTAGTG~G~G~CCTTCCTATACCGGC~TGACGATGAGACGGAGAGGG~GGTG~TACCATTCGTTGTTA S E E N L I E G E E Q E G S E E E P S Y T G N D D E T E R E G E Y H S L L

223

GATGCC~C~TTCGCGGACATTGC~C~G~GCGTGGC~C~GGTTATGACTCTCACGACCGT~GCGGTTGCTTGACG~G~CGGGACCTGCT~TAGAC~CAAA D A N N S R T L Q Q E A W Q Q G Y D S H D R K R L L D E E R D L L I D N K

334

CTGCTCTCTC~CACGGC~CGGTGGGGGAGATATAGAAAGTCACGGACATGGCC~GC~TTGGACCGGACGAGG~GA~GACCAGCTGAGATTGCAAATACGTGGGAG L L S Q H G N G G G D I E S H G H G Q A I G P D E E E R P A E I A N T W E

445

AGCGCGATCGAGAGTGGCCAGA/~AATCAGCAC~CTTTT~GAGAGAAACGC~GTGATCACGATG~TGCGTTGCCGCT~TCTTCACCTTTATCTTGC~TTCGTTG S A I E S G Q K I S T T F K R E T Q V I T M N A L P L I F T F I L Q N S L

556

TCACTAGCATCTATTTTCTCCGTCTCACATTTAGGGACGAAAGAGCTAGGTGGTGTTACACTCGGTTCTATGACTGCT~CATCACGGGTCTTGCTGCTATTC~GGTCTG S L A S I F S V S H L G T K E L G G V T L G S M T A N I T G L A A I Q G L

667

TGTACATGTCTGGACACACTGTGTGCGCAGGCATACGGTGCCAAAAACTACCACTTGGTGGGTGTGCTAGTGCAGAGATGTGCCGTGATCACCATCTTGGCGTTCTTGCCA C T C L D T L C A Q A Y G A K N Y H L V G V L V Q R C A V I T I L A F L P

778

ATGATGTATGTTTGGTTTGTTTGGTCGGAAAAGATCCTAGCACT~TGATTCCGGAGAGAGAACTATGCGCGCTAGCGGCT~CTATCTACGTGT~CCGCATTCGGTGTG M M Y V W F V W S E K I L A L M I P E R E L C A L A A N Y L R V T A F G V

889

CCAGGATTCATCCTTTTTG~TGTGGT~GAGGTTCCTAC~TGTC~GGTATATTCCATGCATCCAC~TCGTGCTCTTTGTGTGCGCACCCTTG~CGCATTGATG~C P G F I L F E C G K R F L Q C Q G I F H A S T I V L F V C A P L N A L M N

1000

TACTTACTTGTTTGG~TGAC~GATTGGGATTGGGTACCTTGGTGCGCCATTATCGGTTGTGATC~CTACTGGTTGATGACGCTCGGATTACT~TATACGC~TGACC Y L L V W N D K I G I G Y L G A P L S V V I N Y W L M T L G L L I Y A M T

1111

ACC~GCAC~GGAGAGACCACTCAAATGCTGG~TGGTATCATCCCT~GG~C~GCATTT~G~CTGGCGT~GATGATTAACCTAGCTATTCCCGGCGTGGTGATG T K H K E R P L K C W N G I I P K E Q A F K N W R K M I N L A I P G V V M

1222

GTGGAGGCAGAGTTCCTCGGCTTTG~GTGTTGAC~TTTTCGCTTCCCATCTGGGCACCGATGCCTTGGGCGCTCAGTCGATTGTGGCTACGATTGCGTCTCTTGCATAC V E A E F L G F E V L T I F A S H L G T D A L G A Q S I V A T I A S L A Y

1333

C~GT~CCTTTCTCTATCTCCGTTTCTACCAGTACACGTGTGGCC~TTTTATCGGCGCGTCGCTATACGACAGCTGCATGATCACGTGCCGCGTGTCCTTATTGTTGTCC Q V P F S I S V S T S T R V A N F I G A S L Y D S C M ~ T C R V S L L L S

1444

TTTGTGTGCTCCTC~TG~CATGTTCGTTATCTGCCGTTAT~GG~CAAATCGC~GTCTATTTTCTACTGAGAGCGCTGTAGTGAAGATGGTCGTGGACACACTACCT F V C S S M N M F V I C R Y K E Q I A S L F S T E S A V V K M V V D T L P

1555

CTTCTTGCGTTCATGC~TTATTCGATGCCTTTAATGCGTCCACCGCCGGATGCCTACGTGGTC~GGGAGACAAAAAATAGGTGGGTACATC~CCTAGTCGCATTCTAC L L A F M Q L F D A F N A S T A G C L R G Q G R Q K I G G Y I N L V A F Y

1666

TGTCTAGGTGTGCCCATGGCATATGTGTTAGCATTCCTGTATCATCTGGGTGTAGGCGGCTTATGGTTGGGTAT~CTAGCGCGTTGGT~TGATGAGTGTGTGTC~GGA C L G V P M A Y V L A F L Y H L G V G G L W L G I T S A L V M M S V C Q G

1777

TACGCCGTTTTTCATGGTGACAGACGCCGTATTCTCGGAGCGGCACGC~GCGC~TGCTGAGACCCATACATCATAAAACTCCTTCAGGGTCGAATGACGTATAGGGGAG Y A V F H G D R R R I L G A A R K R N A E T H T S * * *

1888

ATGCACACCCCCACCCCCGC~CACACCCAGTCTTCTAGCC~TTATGTTGGT~GT~TTTT~GGGTTTCGCCATGTGGCATGCCACCGGC~T~TACACGTGCCC

1999

GTACGGCACCAAACGGGCATGCATTTCTGCATTTGAAAAAGTTTGCTGCCCTTCATGTGCTTTTATGTATGCATACACATACACATTCGTGCATACATTCATTTATATA1%

2110

TATAGAGTGTGCATCGTGCATATGATTATTTATTACCACTTTTTTTGTTTTTGATTTTGTTCGTCAAAAGG~GAG~GGCATATCG~GGGTGTAATGAGAAAAGCTT

FIG. 3.

Nucleotide and deduced amino acid sequences. Nucleotide sequence was determined using chromosomal DNA fragment

(Sau3A1/Hindlll) of pER4 and numbered from + 1, the presumed initiation codon. TATA-Iike sequences were underlined and potential transcription termination sequences were double-underlined.

NcoI, Kpnl a n d BssHII sites. All o f t h e t r a n s f o r m a n t s car-

PstI t o NcoI site in t h e HindllI f r a g m e n t (3.0 k b ) (Fig. 2 A )

r y i n g t h e s e p l a s m i d s were f o u n d to b e sensitive t o e t h i o n i n e (Fig. 1). T a k e n t o g e t h e r w i t h t h e fact t h a t t h e 0.8 k b Pstl/HindIII f r a g m e n t at t h e left h a n d e n d o f p Y S M H 1 (Figs. 1 a n d 2 A ) is n e c e s s a r y f o r t h e e x p r e s s i o n of ethionine resistance, the result obtained here indicates t h a t t h e g e n e o f i n t e r e s t n e e d s t h e r e g i o n at least f r o m t h e

o f p E R 4 . T h e n , we a s s u m e d t h a t t h e O R F o f t h e g e n e f o r e t h i o n i n e r e s i s t a n c e w a s c o n t a i n e d in t h e l o n g e s t s e q u e n c e , w h i c h b e g i n s at + 1 A T G a n d e n d s at + 1 , 8 5 2 T A A (Fig. 3), a n d we h e r e a f t e r call this p u t a t i v e g e n e ERC1 ( E t h i o n i n e R e s i s t a n c e C o n f e r r i n g gene). T h e O R F o f ERC1 c o n t a i n s c h a r a c t e r i s t i c s e q u e n c e s be-

TABLE 1. Phe Leu

Ile Met Val

UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG

13 16 10 18 9 7 19

Ser

Pro

8

15 22 6 19

Thr

8 4 4

Ala

23

UCU UCC UCA UCG CCU CCC CCA CCG ACU AC C ACA ACG GUC GCC GCA GCG

9 10 6 8 5 3 6 3 4 13 10 10 11 8 20 14

Codon usage in ERC1 Tyr End End His Gin Asn Lys Asp Glu

UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG

7 14 0 0 9

5 20 5 10 15 7 14 5 14 21 17

Cys End Trp Arg

Ser Arg Gly

UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG

8 8

0 9 7 4

0 3 8

5 9

2 22 12 9

7

214

SHIOMI ET AL.

.1. FERMENr. BIOEN(;.,

+5 "~

0

-5

l~"

r~ '

L

I

0

120

I

'

I,

l

240 360 480 Amino ocid residues

I

600

FIG. 4. Hydropathy analysis of corresponding protein of ERCI. Index: hydropathic index. ing found in yeast genes. Two kinds of promoter sequences (Goldberg Hogness box (14)-like sequences) were found at - 3 5 8 T A T A A and - 2 3 2 T A T A . A conserved A X X A T G X X T sequence for the translation initiation site located between - 2 and 6. A potential termination sequence T A T G T . . . T T T proposed by Zeret and Sherman (15) was found downstream of the stop codon T A A ( + 1,852). M.W. of protein directed by E R C 1 was calculated to be 67,977 from the deduced amino acid sequence (Fig. 3). Many unpreferred codons were used in E R C 1 and the codon bias index determined by the method of Benneten and Hall (16) was 0.3, suggesting that transcription of E R C 1 is not very efficient in S. cerevisiae DKD-5D-H (Table 1). Hydropathy determined by the method of Kyte and Doolittle (17) indicated that the N-terminal region of the protein was more hydrophobic compared with other regions (Fig. 4). Homology searches with EMBL Data Banks indicated that there were no genes homologous with ERC1. Northern blot analysis of a gene for ethionine resistance Cells of S. cerevisiae DKD-5D-H with pER4 were grown on SD medium with and without 2.0 mM D,tethionine. RNA was extracted from both cells and used for the hybridization with a labelled 0.65 kb K p n I / N c o l fragment (Fig. 5). The labelled probe was found to hybridize with RNA, which had a molecular size of approximately

A 1

B 2

J

1

i

2

~ W

.-el~- 23 s ~ " ~ 16 S

O

FIG. 5. Northern blot analysis of ethionine resistance gene transcript. Probe used for Northern blotting is shown in Fig. 2D while the positions of ribosomal E. coil RNAs used as size markers are shown on the right of photographs. RNAs used for blotting were prepared from S. cerevisiae DKD-5D-H cells with pER4 grown on SD medium in the absence (A) or presence (B) of 2.0raM o,c-ethionine. Lane 1, agarose gel electrophoresis of total RNA, lane 2, autoradiogram after Northern blotting with a probe•

,.3

/

/

A

B

C

FIG. 6. Localization of SAM. Ultraviolet micrographs were laken using cells of S. cerevisiae DKD-SD-H with pYSMH 1 grown on SD medium containing: A, none; B, 5.0 mM L-methionine and (', 2.0raM D,L-ethionine. Bars in photographs represent 2.01ml in length. 2.3 kb, prepared from cells grown in the presence of D,Lethionine (Fig. 5B). On the other hand, no hybridization bands were observed in the case of RNA prepared from cells grown in the absence of D,t-ethionine (Fig. 5A). Judging from the size of the putative ORF (Fig. 2C), the observed 2.3 kb band was thought to be a result of hybridization between m R N A of a gene for ethionine resistance and the probe used. The a m o u n t of m R N A of a gene for ethionine resistance specifically increased when S. cerevisiae DKD-5D-H cells with pER4 were grown in SD medium containing D,t-ethionine (Fig. 5B), but not increased when the same cells were grown in the absence of D,L-ethionine (Fig. 5A). Relationship between ethionine resistance and SAM accumulation To examine whether E R C 1 has a function enhancing SAM accumulation, SAM contents were analyzed in six transformants carrying various subcloned plasmids (Fig. l). The a m o u n t of SAM in transformants with pER1 or pER4 carrying a gene for ethionine resistance were extremely high and the amounts were almost the same as that of S. cerevisiae DKD-5D-H cells with pYSMHI (130-160rag of dry cells). On the other hand, transformants with pER3, pER7 or pER8, in which the gene for ethionine resistance is disrupted, showed a very low level of SAM and the contents were almost the same as that of S. cerevisiae DKD-5D-H with YEpl3 (10-15 mg/g of dry cells). The results of the study on SAM accumulation clearly indicated that, in addition to ethionine resistance, the E R C 1 product has a capability of enhancing intracellular accumulation of SAM. The cells of S. cerevisiae DKD-5D-H with pYSMH1 were found Io contain a large amount of UV-absorbing material, when they were examined with UV-microscopy (Fig. 6). The contents of UV-absorbing material appreciably increased, when S. cerevisiae DKD-5D-H cells with pYSMH1 were grown in SD medium supplemented with L-methionine (Fig. 6B) or D,L-ethionine (Fig. 6C). The UV-absorbing material accumulated in the presence of ]methionine has been determined to be SAM (2). DISCUSSIO N Ethionine resistant mutants have been isolated from

VOL. 71, 1991

NUCLEOTIDE SEQUENCE OF ETHIONINE RESISTANCE GENE

several kinds o f p r o k a r y o t i c and e u k a r y o t i c cells. In m o s t o f these m u t a n t s , the gene for m e t h i o n i n e a d e n o s y l t r a n s ferase is d i s r u p t e d and these m u t a n t s are repressed to f o r m S - a d e n o s y l - L - m e t h i o n i n e ( S A M ) f r o m L-methionine and a d e n o s i n e 5'-triphosphate. H o w e v e r , s o m e m u t a n t s h a v i n g a capability to o v e r p r o d u c e S A M were o f t e n f o u n d a m o n g e t h i o n i n e resistant m u t a n t s , especially those o f p r o karyotes. F o r e x a m p l e , yeast m u t a n t s h a v i n g d a m a g e d eth2-7 (18) or eth3-1 (19) can o v e r p r o d u c e S A M , a l t h o u g h these yeast m u t a n t s are resistant to ethionine. Thus, e t h i o n i n e resistant p h e n o t y p e s have two distinct features relating to the synthesis o f S A M . H o w e v e r , no plausible exp l a n a t i o n s have been offered to fully explain the m e c h a nism for the e n h a n c e d a c c u m u l a t i o n o f S A M by e t h i o n i n e resistant m u t a n t s so far. T o genetically elucidate the relationship between e t h i o n i n e resistance and S A M a c c u m u l a t i o n , we cloned a yeast S. cerevisiae D N A f r a g m e n t that, w h e n present in cells o n a m u l t i c o p y p l a s m i d , c o n f e r r e d b o t h p h e n o t y p e s , e t h i o n i n e resistance and S A M a c c u m u l a t i o n (1). W e also d e t e r m i n e d the n u c l e o t i d e s e q u e n c e o f the gene responsible for the e t h i o n i n e resistance (Fig. 3). T h e n u c l e o t i d e seq u e n c e o f the gene was different f r o m those o f yeast S. cerevisiae S A M 1 (4) and S A M 2 (5), a l t h o u g h o u r p r e v i o u s results suggested that o u r gene m i g h t e n c o d e S A M 1 or S A M 2 (2). F u r t h e r m o r e , the c o m m o n sequence G A A A A C T G T G G f o u n d in u p s t r e a m regulating regions ( 200 300) o f genes r e q u i r e d for m e t h i o n i n e and S A M syntheses (MET3, MET25, S A M 1 and S A M 2 ) (5) was not f o u n d in the n u c l e o t i d e sequences o f our gene for e t h i o n i n e resistance. H o w e v e r , the p h o t o g r a p h s (Fig. 6) o f S. cerevisiae D K D - 5 D - H cells with a gene for e t h i o n i n e resistance, have led us to suggest that S A M [and possibly also S A E (Sa d e n o s y l e t h i o n i n e ) ] a c c u m u l a t e d in certain organelles in the yeast cells. Similar p h o t o g r a p h s o f S. cerevisiae cells a c c u m u l a t i n g S A M h a v e been r e p o r t e d by N a k a m u r a and Schlenk (20) and they c o n c l u d e d that S A M was a c c u m u lated in vacuoles. In o r d e r to specify the p r o d u c t o f o u r gene for e t h i o n i n e resistance, we are c o n c e n t r a t i n g o u r efforts on the e l u c i d a t i o n o f protein factors i n v o l v e d in the t r a n s p o r t o f S A M (and also S A E ) across the v a c u o l e m e m brane, a l t h o u g h h y d r o p a t h y analysis o f o u r gene for e t h i o n i n e resistance indicated no a m i n o acid sequence h o m o l o g i e s for p e r m e a s e s r e p o r t e d currently. ACKNOWLEDGMENTS

We thank Dr. T. lto, Kyoto University, for his help in UV microscopy study. We also thank Dr. J. D. Beggs, Edinburgh University, for providing plasmid pJDB207 and Drs. T. Nakamura and Y. Ishino, Takara Shuzo Co., Ltd., in nucleotide sequence analysis. We are grateful to Mrs. Y. Nojima, Kanegafuchi Chemical Industry, Co., Ltd., and Y. lnoue, Kyoto University, for their help with this study. REFERENCES 1. Shiomi, N., Fukuda, H., Morikawa, H., Fukuda, Y., and

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