Plant Molecular Biology 22: 171-176, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium.

Update section

Short communication

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Nucleotide sequence of a genomic gene encoding tritin, a ribosome- inactivating protein from Triticum aestivum

Noriyuki Habuka, Jiro Kataoka, Masashi Miyano, Hideaki Tsuge, Hideo Ago and Masana Noma Life Science Research Laboratory, Japan Tobacco, Inc., 6-2 Umegaoka, Midori-ku, Yokohama, Kanagawa 227, Japan

Received 18 November 1992; accepted in revised form 11 February 1993

Key words: expression, genomic gene, protein synthesis, ribosome, RNA N-glycosidase, wheat germ

Abstract

A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe. The 5'-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression. The cloned DNA encoded tritin consists of 275 amino acids with no se- cretion signal sequence. The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrulamide gel electrophoresis and immunological analysis.

Abbreviations: MAP, Mirabilis antiviral protein; RIP, ribosome-inactivating protein; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ICs0, median of inhibitory concentration of L-[ 35S] methionine incorporation.

Many, and possibly all, plants contain ribosome- incativating proteins (RIPs) that have highly spe- cific RNA N-glycosidase activity. They cleave an N-glycosoidic bond at an essential adenine se- quence such a s A 4324 of rat liver 28S rRNA to inactivate the ribosome, resulting in the inhibition of protein synthesis [5, 6]. RIPs have an antivi- ral activity against animal and plant viruses [2]. Some RIPs such as pokeweed antiviral protein from Phytolacca americana and trichosanthin from Trichosanthes kirilowii have been reported to specifically inhibit the replication of human im- munodeficiency virus [20, 24]. Recently, a trans-

genic tobacco plant with increased fungal protec- tion was created by the introduction of a barley toxin gene, an RIP from Hordeum vulgare having antifungal properties [16, 17].

Analysis ofnucleotide sequences of several RIP genes indicates that an RIP from a dicot plant is produced in a precursor form with a secretion signal peptide [11, 13, 14]. It has been suggested that an RIP is secreted into some compartments such as vacuole or cell wall matrix so as not to inactivate ribosomes of the original plant [ 13, 21 ]. On the other hand, maize RIP was reported to be present in soluble cytoplasm [7]. Barley toxin and

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CATGTCTTTG

AAAATAGAGC

TTATTACTGT

AATCGAGACA

TCCAAGCTGA GAAGTG~GAT GAACTC~TCT

TGCCGTCTCC ATTCCAGAAC GATATTTCTG

TTCTTTGGAC TAGCTAGCTA GGAAAGCAAG

CGCGTGCATG TTATATAGAT CAATAGGGTT

CAAAGATAAG GATCAGCTCC AAAAACGCCT

AAGTCAAGCT ATACAAAGAT AAGGATCATC

TTACATATGA CACGTACACG TATACAAAGC

TTCGTTCATC CTCTTCCATA TCTCAACCCC

ATG GCG AAG AAC GTG GAC AAG CCG

M A K N V D K P

P49

TAT GTC ACC TTC ATC AAC GGC ATC

Y V T F I N G I

CCC GTG CTG CCG CCG ATC GAG CCC

P V L P P I E P

TCG CCG GCA AGC ACC GGG CTC ACG

S P A S T G L T

AGC AGC GAC GGC ACC TGG TGG GAG

S S D G T W W E

GGC GGC ACG TAT CGC GAC CTC CTC

G G T Y R D L L

CAG ATG GCG GAC GCG GTG ACC GCG

Q M A D A V T A

P42

TTTTTATTTC TCCCTACCCG

CGAGAGTGAT CCTCCTGTTT

GACTTTTGCG CCTT~,ATAA

TGACATCCGC AGGTTTCTTA

T TTCCGCGGGG ATGGTTGCCT GAACATGTCG 31

AGGCAGAACC TGCTACCTTT GCTCTGCCTG ATCATTGGCT TCATAACAGA 111

CTCTATGAAA GATCATGTTT ACAGCTCGAC ACAAGTACAA TCCTTCTGGA 191

TGTA/~CATAT ACTCAGAATT CCAAAGACAG AACTGATCCC TGCATCACTA 271

TTCAAGTTAG TGGTAGAAGG ATAGCGCTTA ]~ATGCTGCG /~CCGATTAAT 351

TCGGCATAAA TAAAGGGATC ATCCACTAGT TAATCCCCAG AAGA~CA 431

CGTAGCCGTG CAGTGCTGCA AATCAAACCA AATTGAATCT TGGCATCTCC 511

CAAGCATATG TAAAAGTGAA GGCGTGTAGG CGTATACATC TAGGTAAGGA 591

GCAATTGGCG GCAGGTTATT TAGTAGGACA GACCCCTAAA CCAAGCAATA 671

TCGTGTAAGC ATACGCACAC ACATCACGTA GTAGTATTAG CATTGACCTT 751

TCTGACAACC TGCCTTGTAT ACAACACACA AGCACAGCAT CACGTACAAG 831

CTATCATTTG ACAGCATGCA CCTGTCGATC GCCTATAAAT TCATCTCCAG 911

TCTCGCTTGA TAGTACGTCT CATCTAAGCC TCGTATCCAT ~CGGCAAAG 991

NcoI

CTC TTC ACG GCG ACG TTC AAC GTC CAG GCC AGC TCT GCC GAC 1057

L F T A T F N V Q A S S A D 22

CGC AAC AAG CTC CGC AAC CCG GGG CAC TCC TCC CAC AAC CGC 1123

R N K L R N P G H S S H N R 44

AAC GTC CCG CCG AGC AGG TGG TTC CAC ATC GTG CTC AAG ACA 1189

N V P P S R W F H I V L K T 66

CTC GCC ACC CGC GCC GAC AAC CTC TAC TGG GAG GGC TTC AAG 1255

L A T R A D N L Y W E G F K 88

CTC ACC CCC GGA CTC ATC CCC GGC GCC ACC CAC GTT GGG TTC 1321

L T P G L I P G A T H V G F 110

GGC GAC ACC GAC AAG CTG ACC AAC GTC GCT CTC GGC CGG CAG 1387

G D T D K L T N V A L G R Q 132

CTC TAC GGG CGC ACC AAG GCC GAC AAG ACC TCC GGC CCG AAG 1453

L Y G R T K A D K T S G P K 154

CAG CAG CAG GCG AGG GAG GCG GTG ACG ACG CTG CTC CTC ATG GTG CAC GAG GCC ACG CGG TTC CAG 1519

Q Q Q A R E A V T T L L L M V H E A T R F Q 176

P40a

ACC GTG ~CG GGG TTC GTG GCT GGA GTG CTG CAC CCC AAG GAG AAG AAG AGC GGG AAG ATC GGC AAT 1585

T V IS G F V A G V L H P K E K K S G K I G N 198

GAG ATG AAG GCC CAG GTG AAC GGA TGG CAG GAC CTG TCC GAA GCG CTG CTG AAG ACG GAC GCG AAC 1651

E M K A O V N G W O D L S E A L L K T D A N 220

P41,40b GCC CCG CCG GGA AAG GCG CCA GCG AAG TTC ACG CCG ATC GAG AAG ATG GGC GTG AGG ACG GCG GAG 1717

A P P G K A P A K F T P I E K M G V R T A E 242

CAG GCG GCT GCC ACC CTG GGG ATC CTG CTG TTC GTC CAG GTG CCC GGT GGG ATG ACG GTG GCC CAG 1783 Q A A A T L G I L L F V Q V P G G M T V A Q 264

GCG CTG GAG CTG TTT CAT AAG AGT GGG GGG AAA TAG 1819 A L E L F H K S G G K *** 275

GTAGTAGTTG TGCAGGTATA TCTGCATGGG TATTGTACAA GTCG~ATAAA CATGTCACAG AGTGCATGAA TGATATAAAT 1899

AAATAAATGT CACAGAGTCA GTGAATGATA TGAATAAATA AACATGTCTA GTTTATATTG GGCCCAACCA TAGAATTGCT 1979

ATCCAACATA TGTTTTGCAT ATGCACATAT TAGAAGCGCG AACTATGTTC ATCCTTTTAG ACTCCCAGGG GCTCATTTGG 2059

TTGTTGGGGT GAAAAAACCA TGGGCAGTAA ACTCCCCTAG GGGATTTCCC TGCTCATATG AGATCCTCCT CCTGCCATGT 2139

NcoI GAAATATCCC ATTGCCATTT GGCGACCAAG GGAGAGTGAA GAGAGTGAAG GGAGAAGAAC AGAAGAACAG AGCGCCAAGG 2219

ACCTCAGATC ACAAGAAAAG AGACAAGGGG A 2250

Fig. i. Nucleotide sequence of a genomic gene encoding tritin. The full-length barley toxin gene was obtained from nuclei of H. vulgare using polymerase-chain reaction as described previously [ 14]. The cDNA of tritin was obtained from a wheat cDNA li- brary using the barley toxin gene as a probe. High-molecular-weight DNA was isolated from purified nuclei of T. aestivum cv. Asakaze seedlings (ca. 10 cm) grown in a greenhouse. The genomic library was constructed with EMBL3 (Stratagene) and 9 to 23 kb wheat DNAs digested by Bam HI, positive clones were isolated using the 32p-labelled tritin cDNA, the insert was subcloned in the Sal 1 site of pBluescript (Stratagene), and its nucleotide sequence was determined as described previously [ 14]. Nucleotides and amino acid sequences were analyzed using a DANASIS system (Hitachi, version 6.0). Boxes indicate the sequences homol- ogous to the Opaque-2-binding site. The TATA boxes, polyadenylation signals, Nco 1 sites and amino acid sequences deduced by Edman degradation analysis are indicated by underlines. The obtained cDNA clones were downstream from the arrow.

maize RIP do not have any secretion signal pep- tide [16, 23]. These findings suggest that cereal plants have a different system of avoiding attack by endogenous RIPs.

Previously, the N-glycosidic bond of a specific adenine of wheat rRNA was reported to be cleaved by endogenous tritin [ 15]. To character- ize tritin further, it was purified and its genomic gene was cloned. Here we describe the nucleotide sequence of a genomic clone for tritin and its expression in Escherichia coli.

Tritin was purified to yield a single band on SDS-polyacrylamide gel electrophoresis from wheat germ. In order to discover its partial amino acid sequences, it was first cleaved by CNBr, and then the amino acid sequences of the following four peptide fragments as revealed by high- performance liquid chromatography (P40, P41, P42 and P49) were determined by Edman degra- dation analysis (Fig. 1). The results are: P40, (K/ V)V(Q/E)(V/A)(N/T)(G/A)(L/F), which appears to be composed of two totally different peptide fragments, a and b; P41, K(A/D)QVNGWQ; P42, AD(Q/A)V(T/N)AL(Y/Q)G; and P49, A K N V D K P L F T A ? F N (? indicates an unchar- acterized amino acid residue). The amino acid residue of the NH2 terminal of the native protein

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was methionine and the following sequence was the same as that of P49.

The amino acid sequences of the peptide frag- ments were highly homologous to that of barley toxin [1]. Therefore, the barley toxin gene was used as a probe for cloning of the tritin gene. The barley toxin gene was obtained using polymerase- chain reaction with barley genomic DNA and primers, 45mer each, complement to the DNA sequences coding the NH2 and COOH terminals [ 16]. The resulting full-length DNA sequence was almost identical to the gene sequence reported previously [ 16]. As the two cDNA clones of tritin obtained from a wheat cDNA library did not cover the full-length sequence, one of them was used as a probe for cloning of the genomic gene of tritin (Fig. 1).

The sequence of the genomic gene is shown in Fig. 1. It consists of a 5'-non-coding flanking re- gion, an open-reading frame, and a 3 '-non-coding flanking region composed of 991, 828 and 431 nucleotides, respectively. The 3'-non-coding flanking region has several polyadenylation sig- nals. The 5'-non-coding flanking region not only has several TATA boxes but also three sequences, GATAATGTAA, G A T G C T G C G A , and GAT- GAACTGA, homologous to the binding se-

i0 20 30 40 50 TR M3%KNAq9 KP LFTATFNVQAS SAD~'VTFI NGI P/qKLRNP - - GHS S H I ~ P I BT MAAKMAKNVD KP LFTATFNVQASSADTATFIAGI RNKLRNP - -AHFSHN~PV ZM ~ R I V P K F T E IFPVEDANYPYSAFIASVRKDVI KHCTDI~KGIFQP%~LPP -

60 70 80 90 I00 ii0 TR EPN~P SRWFHIVI2KTSPASTGLTLAT~IfWEGZ'KSSDGT~LTP GL I p ..... GATHVGT BT F, PNVP P SR~HVVIA~AS P T SAGLTIAI IA~I YLEGB'KS S D~LTP GL I p ..... GATYVGT ZM EK~ -ELN~'YTEIA~TR- -TS S I TLAIR~YLVGFRTPGGV~FGKDGDTHLLGDNPRWLG~

120 130 140 150 TR GGT~'R~TD KLTN~QQMADAVTALY GRTK ..................... ADKTSGPK BT GGTYR~LGDTDKLTNVAI~QQLADAVTALHGRTK ..................... ADKPSGPK ZM GGRYQ~a51 GN -KGLE TVTMGRAEMTRAVNDLAKKKZ~AT LEE E EVKMOMOMP EAAD LAAAAAAD p

160 170 180 190 200 210 TR QQQAREAVTTLLI~TRFQ~GFVAGVLHP K- - E KKS GK I GNEMKAQVNGNQD LSEALLKT BT QQQAREAVTTLLLM~T~Q'~ GFVAGL LHP KAVEKKS GK I GNEMKA~f~'NG~ DLSA~ LKT ZM ~ADTKS KLVKLVV~LRFNT~ RTVDAGFN .... S QHGVTLTVTQGKQVQKWDRI SKAAFEW

220 230 240 250 260 270 TR DANAPPGKAPAKFTP IEKMGVRTAEQ~AATLG I LLFVQVP GGMTVAQALE LFHKSGGK BT DVKPPPGKSPAKFAP ZEKMGVRTAVQ~%ANTLG I LLFVEVP GGLTVAKALE LFHASGGK ZM AD HP TAVI P DMOKLG I K D ~ IVAL%~KNOTTA~%ATAASADNDDD ~ A

Fig. 2. Comparison of amino acid sequences of cereal RIPs. TR, BT and ZM indicate RIPs from wheat (tritin), barley and maize. The amino acid residues conserved among the three RIPs are indicated by bold letters and those conserved in all RIPs by arrowheads [10]. The peptides removed during the processing of maize RIP are indicated by underlines [23].

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quence of the maize transcriptional activator pro- tein Opaque-2. Eight out of ten nucleotides are identical to the consensus Opaque-2-binding site, GATGAPyPuTGPu [18]. Opaque-2 was re- ported to have the leucine zipper motif identified in DNA binding proteins and is thought to acti- vate the expression of maize RIP gene [3, 12]. Therefore, a similar DNA-binding protein may also control the expression of the tritin gene.

The open-reading frame had no intron and en- coded an protein of 275 amino acid residues with a high pl of 10.13 as calculated from its sequence. The GC content was as high as 66.5 ~o, whereas that of the total sequence was 51.5~o. The de- duced amino acid sequence was in good agree- ment with the partial peptide sequences of tritin cleaved by CNBr (Fig. 1).

Some diversification of amino acid residues was found in peptides P41 and P42. The purified protein that yielded a single band on SDS- - PAGE produced three peaks in high-performance liquid chromatography, each of which had an in- hibitory effect on in vitro protein synthesis (data not shown). Together with our isolation of other genomic genes encoding tritin, which confirms the previously reported finding that wheat has three RIP molecules, these observations support the idea that wheat expresses several tritin genes [22].

The amino acid sequences of RIPs from cereal plants are aligned as shown in Fig. 2 [ 16, 23]. The sequence of tritin has 88 ~o homology with that of barley RIP. Three cereal RIPs conserve five amino acid residues of tritin, Tyr-83, Tyr-ll4, Glu-171, Arg-174 and Trp-207, that have been shown to play an important role in RNA N-glycosidase activity [9].

To express the tritin gene in Escherichia coli, the DNA fragment containing the open-reading frame was cleaved with Nco I and inserted into the Nco I site of the expression vector pTV119N [19]. The ATG sequence in the 5'-Nco I site of the genomic clone coincided to the translation frame of the tritin gene, and the ATG in the Nco I site of the plasmid coincided to the starting sig- nal for translation. The recombinant tritin puri- fied after expression in E. coli was analyzed by SDS-PAGE and sequentially by immunoblotting

Fig. 3. SDS-polyacrylamide gel electrophoresis of native and recombinant tritins. Tritin was extracted from wheat germ homogenized with glass beads and suspended in four-fold volumes (w/w) of 20 mM Tris-HCl pH 8.0, 20 mM KC1, 5 mM MgC12, 1 mM dithiothreitol. After removal of the precipitate formed upon the addition of solid ammonium sulfate to a final saturation of 50%, tritin was obtained from the precipitate formed upon the addition of ammonium sulfate to a satura- tion of 90%. This precipitate was suspended in 10 mM so- dium phosphate pH 6.0 and dialyzed against the buffer. Tritin was partially purified by means of carboxymethyl-Sepharose and Blue-Sepharose column chromatographies as described previously [9]. The tritin fractions obtained were dialyzed against 10 mM sodium phosphate pH 7.0, and applied to a monoS column. After the column was washed with the buffer, the protein was eluted with a 0-0.2 M linear gradient of NaC1. To purify the recombinant tritin, the E. coli (JM 109) transfor- mant was cultured at 37 °C in L broth (1% Bacto trypton, 0.5% Bacto yeast extract, 0.5% NaC1) containing 50/~g/ml of ampicillin. When the absorbance of the culture at 550 nm reached 0.5, isopropyl-fl-D-thiogalactopyranoside was added to a final concentration of 1 mM. After expression for 3 h at 37 °C, cells were collected and disrupted. Proteins were sus- pended in 20 mM Tris-HCl pH 8.0, 20mM KCI, 5 mM MgC12, 1 mM dithiothreitol. Tritin was purified by means of salting out, and carboxymethyl-Sepharose and Blue- Sepharose column chromatographies as described above. Lanes 1 and 2 indicate the native and recombinant tritins, respectively.

analysis. The recombinant tritin showed the same migration as the native one in SDS-polyacryla- mide gel (Fig. 3), and cross-reacted with antise- rum against native tritin (data not shown). The results indicate that the gene did in fact encode tritin.

Wheat ribosomes have previously been found to be susceptible to the RNA N-glycosidase ac- tivity of tritin. In fact, the purified tritin substan- tially inhibited the protein synthesis of wheat germ. At concentrations of 100 and 470 nM, it inhibited protein synthesis ca. 68 and 21~o, re- spectively, with an ICso of approximately 250 nM. The maize RIP was reported to be a soluble cy- toplasmic protein and no secretion signal se- quence was found in the maize RIP gene [7]. Therefore, maize may have a special system of avoiding the attack of endogenous RIPs different from the compartmentalization system found in dicot plants. Tritin may also be a cytoplasmic protein because its gene has no secretion signal sequence, and it is reported to be present at a high concentration of 2 ~o of the total soluble protein in wheat [4]. Therefore, wheat may have a spe- cial system as well.

Acknowledgments

We wish to thank Dr Kazuo Shishido, Tokyo Insitute of Technology, for his technical advice; Dr Koichiro Tsunewaki, Kyoto University, for providing the seeds ofH. vulgare and T. aestivum; and Mr. Y. Kanzaki, Nisshin Flour Milling Co., Ltd., for providing the wheat germ.

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