Plant Physiol. (1992) 99, 1729-1731 0032-0889/92/99/1 729/03/$01 .

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Received for publication March 23, 1992 Accepted April 9, 1992

Plant Gene Register

Nucleotide Sequence of the Gene for a Glutathione S-Transferase from Cell Suspension Cultures of Silene cucubalus1
Ralf Prandi and Toni M. Kutchan* Lehrstuhl fur Pharmazeutische Biologie, Universitat Munchen, Karlstrasse 29, D-8000 Munchen 2, Germany
Glutathione S-transferases (4) are involved in numerous plant physiological processes such as herbicide resistance (8), senescing flower petal response to ethylene (7), host response to pathogenic attack (2), and mammalian systems in the detoxification of xenobiotics. It has also been proposed that human glutathione S-transferase interacts with mercury, copper, and cadmium, therefore becoming a part of the heavy metal detoxification system (1). The gene sequences of only a limited number of members of this multigene family have been reported in the literature. To date, there is sequence information available for a maize gene (3, 10) and two wheat genes (gstAl [2] and gstA2 [6]) encoding various glutathione S-transferase isozymes. We report here the nucleotide sequence of a gene coding for a constitutive glutathione Stransferase from cell suspension cultures of the Alpen Leimkraut, Silene cucubalus (Fig. 1, Table I). The gene sequence contained 2507 nucleotides from the 5'-flanking region and 1191 nucleotides from the 3'-flanking region. The open reading frame was interrupted by two introns, intron I being 1719 nucleotides long and intron II 116 nucleotides long. When a 2513-nucleotide segment of the 5'-untranslated region was fused in front of a gene for 13-glucuronidase and transformed into Nicotiana plumbaginifolia protoplasts, promoter activity equal to 70% of the 35S cauliflower mosaic virus promoter was detected. The nucleotide sequence within the reading frame was 51.9% homologous to the wheat gstA2 gene when compared by the Needleman-Wunsch algorithm (9).
Table I. Characteristics of the Gene for Glutathione S-Transferase from Cell Suspension Cultures of S. cucubalus Organism: Silene cucubalus; cell suspension cultures. Location on Chromosome: Unknown. Gene, Function, Pathway: GST; glutathione S-transferase; transfer cysteine sulfur of glutathione to select herbicides; in vivo function not known. Clone Type:
Genomic. Sources: Genomic library in X Charon 4A, constructed from genomic DNA from cultured cells. Sequencing Techniques: Supercoil sequencing, unidirectional deletion subcloning and complete dideoxy sequencing of both strands. Method of Identification: Nucleotide sequence comparison of exons with the cDNA clone for glutathione S-transferase (5). Expression Characteristics: Transient expression assays in Nicotiana plumbaginifolia protoplasts of 2513 nucleotides of the 5'-noncoding region (position -2497 to +16) revealed promoter activity equal to 70% of the 35S cauliflower mosaic virus promoter.

Regulation: Unknown. (G + C) Content: (35%); within reading frame (47%). Sequence Homologies: Wheat gstA2 gene (51.9%, reading frame; 37.2%, 5'-noncoding
region) (6). Structural Features of Gene: TATA box, transcriptional start site, translational start site, translational stop codon. Introns denoted by brackets in Figure 1. Antibodies: Not available. Subcellular Localization of Gene Product: Cytosolic. GenBank Accession No.:
M84969

l This work was supported by a grant from the Bundesminister fur Forschung und Technologie, Bonn, Germany, and by a grant from the Korber Stiftung, Hamburg, Germany, to Professor M.H. Zenk.

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Plant

Physiol. Vol. 99,

1992

-2550 -2475 -2400 -2325 -2250 -2175 -2100 -2025 -1950 -1875

GAATTCACCTGACGGTTGCACA

AGATTGGAGAAGCCACAAAAAGTTATCCTTGTAGATGAGCCTAGCTTTTTCCCGCAACTGATCTATAGTCTACAA
ACAGATGAGATAATATAAGGTTCATCCCAAACCAAGGAATCCTTGGAGCCAAAGAAGTGAGTTTTGCATTTTCTG

TACAACCACCTGAATGTTTGTAATCAAATTGAGTGTGGTGCAACATTTATTAAGTGGTTCCTCAGTTACTACTGA
TGTAAAACTGCTATTCAGATTTTCAGGAACAATCATTTTGGAATAATACAAATCTAATTCATGTGAGTTCTTATA GTATCACAGTACAAAATCGATCGAAGAAGGTAAATGAGACAAAGCAAACAACTGATACGTAGTTCACAAGCACAC GAGATACCATATCAGCAGGTTCACCCAATCCAAACCCAAATGAATCAAAATTAGGGTTTCGCCTTGCCCAAAATT CAGCAGTTACTTCAAATCACAGTCAAATAATAGGACAAGATACCATTAATGTACTAATAAATAAAATCGGTAACC AAAATCAACAAGAAAGCAAGCATCCAACAAACAAAAACAATAAAGAGTAACAGCGGAAGGACGATAAAAGGGAAA AAAGGGACCTGAAGTTATCAATAGGGAAAGGACTGAGTTGAACATCGTCGAGACATCAACTTTTTTTCGCCATCG TCGATAGGAAACGACCCTAAATCAATTATTGATAATAATAATTATTAGAGTTTGAGTAGAAAACCTTAGATCTGA ACAAAGGGGGGAACCTGTGGTGTTAAGAGTAACAGCGGAAGGAAGATAAGAAACCCGGTGAATAATGGATGTGGG

-1800 -1725
-1650 -1575

TAATGACGACGAGGACGACGG'T2GAAACATGGGTTAGGTTAGAGTGTTGGTGGTGGCAGAGTAGAACAACGGTGAA
AGAGGGGTTAGGTTAGAGTGTTGAGTGAATGTGTGATAGAGAATGTGAGATATATCGTATACGCAGGCGTTAGTA ATTTTAAGCCAATTTTGGAGAAATTAAGACATCAGAGTTTAATATGTTTGATGTTGTTTAAAGAGCACGAACAAT GGACATCAGTTTTAAGAATTATCCGATGTCAAAATAATTATATACACATCGGGTTTTTTCAGAACTTTGATGTCT ATGATTTATACATCAGTTTTTTTAATAATCAGATGTCTATAAAGTGATGTCTATTACGATTTTTGTAGTAGTGTG ACCTAGTAGACGAGAGCCTCTAGGACCCAATATATGGACGATATTATTATCGGGAATGCTAATGTTGTGAAATGG GTAATCCTTGTGCCTAAGCAATTGTTGTTCTTACTATCTAATCTTAATAATGGCATGACTTTATATATGTGATTT GCATGTGTGACCCAATCCCCTTTGACTCTCTTTATTATATTGTTTACATCGTATTTTCTAGACTATACAAACCAA

-1500 -1425 -1350 -1275 -1200 -1125 -1050

-975 -900

-825 -750 -675 -600 -525

ACCAAACCCAAATCGATAATTCGACCTTGATAGAAATTCACCATAGCAACTCATGCGTAGTAGACATTTTCTCTG TGGATTCGACCCCTTAATACTACATTAATTTGTTGTGCCTAGGGTATCTTTGAATAGGTGTGCGATAGCCTATCA GGTAAACTCTTTCTCCTCGGCCCGAACCAAACTTAGAAGCCCATGTATACACTCCGTAGTAAACCTGGCCATGGG ACAAGCTGGGGTTGATTCGAGCCAACCTGCGTCGGGGCTGGCCCTAAACCAGCGTGAGTCGCGTGGCAAGGCAAG GATCGAGTCGGATGGGGCTTATTCATGCTTGGCCGTAAATCAGCTATCAGCCTGTGCTACCAATCATTTTATTTA TTTATTTATTTATTTTAGCAGGCTTGGATAGGTCGGGCCTAACACTCGAGTCTCGACTCAGCCTGGTTCAGGGCT GGTTAAGCCCAGCTTTTGGCCGGCCAGACGAGCTTTGGMTCGACAAACCCGCAAGAAGGGTTGGTTTAGGCCCC GGTTGACAATTATTTGGTCTCGCCTATTCCATAGTATATACTCTCTTTGCCTGTCATTTGTTTGCCTTTAATTTT

-450 -375 -300

-225

AACACAAAGACCAAGGAAATGGGAGGGGTCAATTATTAGATGACAAGTTGACCAAATTAAGTGTAAATGCTCCAT
TTATCCATCGAAAATATTTCTAAAATGAAMGACAAACMTTMCTGMAAAACCTAAAATTAAACAAACGACCG

GGACGAAGTGAGTAATTAAGTACATAGTATAACTTACAAATCAAGAATTTTCTCCAATCMCGTCAGAGTATAC

GTATCCACGTTTCGAAGACCAATCAAGATGCAACTTCTTAACAAGTTCCACTCCATTAAAAAGTCAACTAACATT

-150
-75

AAAAATTAGTCGTCCATAGTACATGCTAAAACAACTCATTCCAGGAAACGAATATACTATACAATAATGGTGGGA
TTTAATTATAATATTGTCACAAAACTATATACTAATTCAACATA CCACCCTCCTAGAATACGTGATT

+1 +76 +151

ATCTCATTTCATTACATAATAATTCAAAACAAATAATAGCAAATAATAATAA,rACGATCAAGGTGCATGGAAAC
M T

I
F

K V H G N
E F
V

CCAAGGTCCACCGCGACTCAACGTGTCTTGGTTGCGCTTTATGAGAAACACCTTGAATTTGAGTTTGTACCCATT
P
R S G

T A T
A G G

Q R V L V A L Y
H K

K N

GACATGGGTGCTGGTGGTCACAAACAACCGTCTTACCTCGCCCTTAACMATGTTCGTGTCTTTTTTTTTAAAAT
D M

P S Y L A L

+226 +301 +376 +451 +526 +601

+676 +751 +826 +901 +976 +1051 +1126 +1201 +1276 +1351 +1426 +1501

TCATATTTTGTTATTTATGAGTAGAGGTATCTATAAGTCAACCATTTATAAGTCAAGTTCCGACTTTCTATCTTA
GATAGATTAGCATTATAAATCAAAATGATATTGAAATCACGACTTTCTTAGGTATCTATAGTCAACCATTTAT

AAGTTTGATCTGAATGGAGTACACAACTGTTAATCTATCTCTTTGATCTACATAAACATTTTATTTTTGTATTTA TACTAGATTTAAGTAGTGTATGAAATTTTTTACCATGGAACTGACCCGGGCAACCAGTAGACCAGCTTGATTATT
ATTCTAACGATCTTACTCTTAGTGCTAGAAAAGAGGTAGATTTGCGAACMTCTAAAATGAAAATTGTGACGAGA

ACTACACCAAATAATTTGACTTTTAGAGTAAAAACAACTGCCATTAATTTAGTAAGTCATTTTGCTTTATATTAT
CATAATTCAGATCAAAGACTAGCGAATCTTTCGTCATTATTTGAAATGAAACTTG MAAMCGAGMAT ATATTACTAAATCTAATGACTTTTAGGTTTTMTACAGCATCGMTACGTMTCTGTTTMGAAATTGAGGGCTA CTATGTTTTTAGAGATTTAATCATGTTCCCATCTATATGGCMTATGGATATATCATTTTCTCTTTAAATTATTT GGGATATTTACTTAGTACCTCTATGTTTTTTCT7TM CTGTTTTATAAGGTTCATCCCCTTTTTTGCACTAAAA

AACCTTGGTGGACTACAACTCATGGTCACGAATTTAAAAGTAACCATTTCTTTTCTAAATTAATCGTCTTTTTT
GAGATCTTCGATTTTAAAAAGAAAAATAAATCTCCGTTTTATGAACTCATCGCAAGAATCACGGTCGATCAACTT TGTAACATGAAAACGTGGAATAGAAGGATACGTTAGATTTAGATTGAATATAATAATATGGGTAAGGCCCAAGAG CCCATGTTGGAAACCAGTAGGTCTTGCTTGTGACGGGTCGGATCTTACGACGGGTATTTTGTGAGAGGAAATAGG TAGATGAGACAAGGTGGGCACCCCCCATGTGCTTCTCCACTCACCCTTTATGAGTATTTTGTGAGAGAAAATGGT ATCCGTCACTCATTTGTGACGGATAGTGTCCGTCACAAATGAGMTTTGTGGTTGGAAACAGAGTATGTGGATCT
GGCATTGATTCTTCAATTAGTGCTTATGACATGGTCCCATATTAACTTTGGAGTTGTACAATGCAAGAACAATCA

AGTTAATTAAACAACTCTACCCATATACGCATTATATTTTAATCAAAGAACAATTATGGCATTTTCATTTCATGT

Figure 1. (Legend appears on facing page.)

GLUTATHIONE S-TRANSFERASE GENE FROM SILENE CUCUBALUS

1731

+1576
+1651 +1726 +1801 +1876 +1951

GAATTTGGCCTCATCAACATTTATGATAAATTGATAAGTGTAGCGCAAAAGTGTTAATTAAGTCCTTTAACTTTG TTCACCGATCAATAATTATCTATGGTTATACTTTGTGAAGGTAATTATTTTCACCAAGAAAAAATAATTTGTGAA GACAATGAATAAATATTGACTGGGACGAATAAGTAATTGCTAAAGTTAATAGGAGTACTAAGCACTAAGTGTAGA TTAATTTCATAATCCGTATTAGATAAGTCAATTTCGGAACTTATAAGGGTTGTAAGCAAGACTTAATCTCTGAAG

ATAGATGAACTTATAATTTGTTCCTTCTAATGTATGAATACA-CCATTTGGTCAAGTGCCTGCTCTTGAGGATGG
P F G

Q V P A L

AGAAATCAAGCTTTTTGaAATTTTCCCTTAACTCATTCATTATTCCTATTTAGTCACACTTTGTTTATTTTCAT
E I K L F E
S R

+202 6

ATTTTAAAAAAAAATAGAAGGAAATTACCAATTTCTAACAAATGGCATTCTCAAAC&GTCAAGAGCTATACA
A
I T

+2101 +2176

AAGTACTTAGCATACACACACGATCACCAAAACGAGGGAACCTCATTGATTCACAAGGAAAAACACGAAATGGCG
K Y L A Y T H D H Q N E G T S L I H K E K H E M A GCCCAGTTAGTCTGGGAGGAGGTAGAAGCTCATCAGTTTGACCCAGTGGCGAGTAAACTTGCATGGGAGCTTGTT A Q L V W E E V E A H Q F D P V A S K L A W E L V TTCAAGGGTATATTTGGAATGCAGACTGATACGACTGTGGTTGAGGAGAATGAGGCTAAGTTAGCTAAGGTCTTG

+2251
+232 6 +2401

G I

M O T

V V

GATGTCTATGAGGCGCGATTGACCGAGTCCGAGTACTTGGGTGCCAATGACTCCTTCACATTAGTTGATCTTCAT
D V Y E A R L T E S E Y L G A N D S F T L V D L H CACTTGCCACTCTTGGGTTACTTAATGGGGACTCAAGTGAAGAAGTTGTTTGAGGAGAGGGCTCATGTGAGTGCA H L P L L G Y L M G T Q V K K L F E E R A H V S A

+2476
+2 551 +2626 +2701 +2776 +2 851 +292 6 +3 001 +3 076

TGGTGTAAGAAAATCTTGGCTAGGCCTTCTTGGGAGAAGACCTTGGCTCTTCAAAAGCAAGCTiTTAATTATG
W

C K K I L A R P S W E K T L A L Q K Q S * ATGAAATATTTGATTAGCTTTCATTTAAGTCAAAAATGAAATTAATATTTCCAATTTCTAGTTTGTTTTGATTGT TGTATTGTGTTGTTGCTTGCCTACTTTACCTTGGTGTATTTTGTTCAAGTTGTGTTTATCATGCGAATAATAATA CTGGATCAGAGTTCTTTGCGTTTTTTTACTTTGAATTTATGTTATTTAATCATGTGATATATGTATAACTGTATG

TTTGTCTAATGGACCATATATAATACTCCCTCCCATCCTCTATTTTCTTCCCTATTTCCTAAAACGGATTATTCA

+3151
+3226 +330 1 +337 6 +34 51 +3526 +3 601 +3676

GGTTTTCTTCCCCTTTCTATATTTGGTAACTTTTACTCTTATTTTATTCATCCCTCTCTCCTATCACACAACCCC ACTCAACTCTTTTACTTCTATTTTATTCCTTCCTTTACTATTTGTGGCCCACAATTTATTATTTAACTCCTAATT ATTCATTCCTCTCTCCTATTCCCAAACCCACCCAACTTTTTATCAATATTATACTCCTTCCCTTAATCTTTGTGC CCAAAAGAAAAGGGAAGAAAATAGAGGATGGGAGGGAGTAGTCATGTTATCAACCTTTATTAAATCAGTTGATGA CGTGTTTCCTTTGTGTTTACACTATCCGGATTTTATCTAGATTCGAGAAGAGTAGGACCATCAAAACGGTAAAAC AAGTGTAGCATATACGACTGCCATTTATAATTATGTCGTTAAAGATTAGATTAGTAAATGCTTCTAATAAGTATG ATGACAAAGATTAGATTAGTAAATGCTTCTAATAAGTATGATGACAAGGCCGACAGAAATAATTATGCAAATGTA CTCCCTATATTCAATCAATTATTGTTTCGATAATTGTCAAATTAACTATCATCATACATAATTAAAAGAAATTTC GACGACGCCACCTACTCATAAGAATTAAAGTCAAACTCATGAGTCAGAGTCATGACATGCAGGGCCGNCCCTATA GGGTGTTTAGGTTGGCAATTGACAAGGGCCCAAGTCTAGAAGGGGCCCATTAGTAAGAAATTAGAGTCAAATAAA ATTTATTACATGTAAAATTCATATTAAAGGAAGTAGTCTAGTGGTTTTCTATCTTGCTAAGTCATAAGAGATCTT

GTGTTTTATTCCTCATGGCTTCTTTTCACCATTTCTACTTTTTTTTTTTTTGGAATTC

Figure 1. Nucleotide sequence of the gene for glutathione S-transferase from S. cucubalus. TATA box, squared; introns, brackets; transcriptional start, 1; translational start, underlined; translational stop, double underlined.
LITERATURE CITED 1. Almar MM, Dierickx PJ (1990) In vitro interaction of mercury, copper (II) and cadmium with human glutathione transferase 7r. Chem Pathol Pharmacol Res Commun 69: 99-102 2. Dudler R, Hertig C, Rebmann G, Bull J, Mauch F (1991) A pathogen- induced wheat gene encodes a protein homologous to glutathione-S-transferase. Mol Plant-Microbe Interact 4: 14-18 3. Grove G, Zarlengo RP, Timmerman KP, Li N-q, Tam MF, Tu C-PD (1988) Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acids Res 16: 425-438 4. Ketterer B, Meyer D, Clark AG (1988) Soluble glutathione transferase isozymes. In H Sies, B Ketterer, eds, Glutathione Conjugation. Academic Press, London, UK, pp 73-135 5. Kutchan TM, Hochberger A (1992) Nucleotide sequence of a cDNA encoding a constitutively expressed glutathione S-transferase from cell suspension cultures of Silene cucubalus. Plant Physiol 99: 789-790 6. Mauch F, Hertig C, Rebmann G, Bull J, Dudler R (1991) A wheat glutathione-S-transferase gene with transposon-like sequences in the promoter region. Plant Mol Biol 16: 1089-1091 7. Meyer RC Jr, Goldsbrough PB, Woodson WR (1991) An ethylene- responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases. Plant Mol Biol 17: 277-281 8. Mozer TJ, Tiemeier DC, Jaworski EG (1983) Purification and characterization of corn glutathione S-transferase. Biochemistry 22: 1068- 1072 9. Needleman SB, Wunsch CD (1970) General method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48: 443-453 10. Shah DM, Hironaka CM, Wiegand RC, Harding El, Krivi GG, Tiemeier DC (1986) Structural analysis of a maize gene coding for glutathione-S-transferase involved in herbicide detoxification. Plant Mol Biol 6: 203-211