Plant Molecular Biology 49: 161–169, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

161

Identification of a tRNA isopentenyltransferase gene from Arabidopsisthaliana

Anna Golovko, Folke Sitbon, Elisabeth Tillberg and Björn Nicander∗Department of Plant Biology, P.O. Box 7080, Swedish University of Agricultural Sciences, Uppsala, 750 07 Sweden(∗author for correspondence; e-mail Bjorn.Nicander@vbiol.slu.se)

Received 19 February 2001; accepted in revised form 17 October 2001

Key words: cytokinins, Arabidopsis thaliana, tRNA isopentenyltransferase, tRNA modification, yeast complemen-tation

Abstract

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, theyhave been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli andSaccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning andfunctional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiaeshowed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsictRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in thetRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.

Introduction

The cytokinins are a group of modified adenines foundin the tRNA of plants, animals and most micro-organisms, but not Archeaebacteria. Situated at po-sition 37 next to the anticodon of certain tRNAsthat bind to codons starting with U, cytokinins havebeen shown to influence translational fidelity and effi-ciency in micro-organisms (reviewed by Persson et al.,1994). For instance, studies using mutant strains ofEscherichia coli and Salmonella typhimurium lackingcytokinin indicate that the moieties influence transla-tional proof-reading by decreasing or increasing mis-reading of the first and third position, respectively. Thefar fewer studies done with eucaryotes indicate that inhigher organisms i6A is important in translation. Theloss of i6A biosynthesis in a Saccharomyces cerevisiaesuppressor line gave decreased i6A levels in tRNA,including the SUP7 (tRNA(UAA)tyr), and loss of sup-pression (Laten et al., 1978). Studies on mammalian

The nucleotide sequence data reported will appear in the EMBLand GenBank Nucleotide Sequence Databases under the accessionnumber AF109376.

selenocysteine tRNA indicated that the presence ofi6A, the sole cytokinin in animal tRNAs, is importantfor the efficient decoding of the cognate codons bythis tRNA (Warner et al., 2000). i6A is present in anatural UGA suppressor tRNAcys in tobacco (Urbanand Beier, 1995).

The name ‘cytokinin’ stems from the strong bi-ological effects that many of these compounds, inthe tRNA-free form, exert on the cell cycle inplants (Skoog and Armstrong 1970). In plants theyare classed as hormones, influencing developmentalprocesses like cell division, cell expansion, shoot mor-phogenesis, leaf greening, and sink activity (Mok,1994).

Cytokinins are synthesized by enzymes that trans-fer an isopentenyl group from �2-dimethylallylpyro-phosphate to position N6 on a conjugated adenine.These isopentenyltransferases can be divided intothree subclasses, depending on which conjugated ade-nine they utilize. The tRNA isopentenyltransferases(EC. 2.5.1.8) catalyses the transfer to adenine at posi-tion 37 of certain tRNAs. The tRNA isopentenyltrans-ferases of E. coli, S. typhimurium and S. cerevisiae

162

are probably the best studied of all tRNA-modifyingenzymes (Björk, 1995; Winkler, 1998). Certain plantpathogenic bacteria have cytokinin synthases thatcatalyse the same transfer but use AMP as the accep-tor (AMP isopentenyltransferase, EC 2.5.1.27). Re-cently, a family of genes from Arabidopsis coding forcytokinin biosynthesis enzymes have been identified(Kakimoto, 2001; Takei et al., 2001). At least one ofthese code for an isopentenyltransferase that uses ATPor ADP as an acceptor (Kakimoto, 2001), forming athird subclass. We report here the cloning of a plantcDNA with homology to known tRNA isopentenyl-transferases (IPPTs), and show that the gene productcan function as an IPPT when expressed in yeast.

Materials and methods

Strains and growth conditions

E. coli strains DH5α and TOP10 (Invitrogen) wereused for propagation of the recombinant DNA. S. cere-visiae strains used were MT-8 (MATa SUP7 ura3-1his5-2 leu2-3,112 ade2-1 trp1 lys1-1 lys2-1 can1-100mod5::TRP1; Gillman et al., 1991) and H57 (MATaMOD5 SUP7 can1-100 ade2-1 his5-2 lys1-1 ura3-1)(gifts from Dr Anita Hopper, Pennsylvania State Uni-versity College of Medicine). The yeast lines weremaintained on YPD medium (Gillman et al., 1991).For all experiments, the cultures were grown in syn-thetic complete medium (Gillman et al., 1991) lackinguracil. For complementation experiments, the mediumwas modified by exclusion of either adenine or lysine,or by the addition of 60 µg/ml canavanine (Sigma).

Cloning of an Arabidopsis cDNA

A. thaliana plants (ecotype Columbia) were grownunder sterile conditions under a 16 h light/8 h darkregime. Total RNA was extracted from seedlings 3weeks old. RT-PCR was performed in 40 µl con-taining 0.5 µM of each primer, 50 µM dNTP,1.3 mM MgCl2, 1xII AmpliTaq Gold buffer, and0.15 units AmpliTaq Gold (PE Biosystems, Fos-ter City, CA). Oligonucleotide primers used were5′-CGGTTAAAATTGATAAATCGGTG-3′ (forward)and 5′-TCATGAGCTATTTGAATTCAAATG-3′ (re-verse). PCR cycling parameters were: 95 ◦C for12 min, then 42 cycles of 94 ◦C for 15 s, 52 ◦C for10 s, and 72 ◦C for 90 s. The product was subclonedinto pBS+ (Clontech) and sequenced.

mRNA 5′- and 3′- end analysis

The 5′ and 3′ ends of the Arabidopsis mRNAwere determined by rapid amplification of cDNAends (RACE) with a kit from Gibco-BRL, fol-lowing the supplied protocol. For the 5′ end,the Arabidopsis cDNA-specific antisense primerRACE1, 5′-TGGTCAACCACTGAAGCAAC-3′, wasused for first-strand cDNA synthesis, and a sec-ond cDNA-specific antisense primer RACE2, 5′-CCTCTGCTGCTGCATCGTCAAGAA-3′, for thefirst round of PCR. Amplified specific DNA productswere diluted 100-fold and used for a second PCRalong with a second sense primer from the kit andthe RACE2 primer. To characterize the 3′ ends, thetranscript was reverse-transcribed with Superscript IIreverse transcriptase and an oligo-dT adapter primerprovided by the kit. Specific cDNAs were then am-plified by two rounds of PCR using cDNA-specificnested primers 5′-GGAGACATGTGCTGGTCATTT-3′ and 5′-GGGTGAAGTTAAATACCAGAA-3′ andanother adapter primer from the kit. SpecificcDNAs were subcloned into the TOPOpCR 2.1 vector(Invitrogen) and sequenced.

Southern blot analysis

Arabidopsis genomic DNA (8 µg) was digested withEcoRI, HindIII and BglII, separated on a 1% agarosegel and blotted onto Hybond-N membranes (Amers-ham Pharmacia Biotech). Hybridization with 32P-labelled Arabidopsis IPPT cDNA was for 18 h eitherat 65 ◦C (high stringency) or at 52 ◦C (low strin-gency) in 5× SSC, 0.5% SDS, 5× Denhardt’s solutionand 500 µg/ml denatured salmon sperm DNA. Highstringency washes were done at 65 ◦C in 0.1× SSCcontaining 0.5% SDS, and lower-stringency washeswere done at 52 ◦C in 1× SSC containing 0.5% SDS.

Northern blot analysis

Total RNA was prepared as described (Logemannet al., 1987). The poly(A)+ fractions were iso-lated with Dynabeads (Dynal) following the manu-facturer’s instructions. Poly(A)+ RNA (0.75 µg perlane) was electrophoretically separated on denaturingformaldehyde-agarose gels, blotted onto Hybond-Nmembranes (Amersham) and fixed in a UV crosslinker(Amersham). Hybridization with a 32P-labelled Ara-bidopsis IPTT cDNA was done overnight at 68 ◦Cin 5× SSC, 0.5% SDS, 5× Denhardt’s solution and500 µg/ml denaturated salmon sperm DNA. The

163

membranes were washed twice in 2× SSC, 0.5% SDSand twice in 0.1× SSC, 0.5% SDS at 68 ◦C followedby exposure to X-ray film (Kodak or Fuji).

DNA sequencing and analysis

DNA sequencing was done with the dRhodamine ter-minator chemistry kit (PE Biosystems). DNA and pro-tein sequence analysis was performed with MacVectorsequence analysis software (Oxford Molecular Group,Oxford, UK). Multiple sequence alignments wereobtained with a CLUSTAL W algorithm (Thomp-son et al., 1994). The BLAST search program wasused for sequence comparisons in AtDB, GenBank,PROSITE and ProDom databases. Prediction of in-tron splice sites in the Arabidopsis IPPT genomicsequence and prediction of sorting signals in proteinswere done with NetPlantGene and PSORT programs,respectively. Phylogeny inferences were drawn usingMacVector (version 7.0) and PAUP (version 4.0).

Yeast transformation

The Arabidopsis cDNA was blunt-ligated into theblunted NotI site of the vector pFL61 (Minet et al.,1992). Yeast cells were transformed with a lithiumacetate-based method (Gietz and Schiestl, 1997).

tRNA analysis

Yeast cultures grown to late log phase (OD600 ca.1.0) were harvested by centrifugation and lysed bysonication with 0.2 mm glass beads in a buffer con-taining 0.15 M NaCl, 50 mM sodium acetate, 10 mMmagnesium acetate and 6% SDS, pH 4.5. The tRNAwas extracted by phenolization, LiCl precipitationand DEAE chromatography (Buck et al., 1983). ThetRNA fraction was degraded to nucleosides for HPLCanalysis by means of nuclease P1 (Roche MolecularBiochemicals) followed by Bacterial Alkaline Phos-phatase (Sigma) (Gehrke et al., 1984). The nucleaseP1 incubation time was extended to 14 h.

Cytokinin analysis

tRNA nucleosides were separated by HPLC (Gilson,Middleton, WI) on a 250 mm × 4 mm C8 RP-selectB column (Merck, Darmstadt, Germany). The gradi-ent was 0.25 M ammonium acetate at pH 6.0 to 40%acetonitrile (Rathburn, Walkerburn, UK) in water, at aflow rate of 1.5 ml/min (Buck et al., 1983). Column ef-fluents were scanned with an UV 3000 detector (TSP,

Figure 1. Schematic illustration of the Arabidopsis IPPT cDNA andmRNA. The predicted ORF is represented by an open box. Verticalarrowheads indicate positions of spliced introns. Thick arrows indi-cate positions of the primers used for RT-PCR. The positions of theprimers used in the 3′- and 5′-RACE analyses are indicated.

Figure 2. Southern blot analysis of the Arabidopsis IPPT gene.Arabidopsis genomic DNA was cleaved with EcoRI, HindIII andBglII and probed with 32P-labelled Arabidopsis IPPT cDNA. Thefilter was washed at high stringency.

Riviera Beach, FL) to determine the UV spectrum andλmax (PC1000 software, TSP).

Material from the HPLC peak eluting at the posi-tion of isopentenyladenosine (i6A) was evaporated, analiquot removed, mixed with 10 pmol of 2

6H-i6A (ApexOrganics, Honiton, UK) and analysed in a Q-TOFtandem mass spectrometer (Micromass, Manchester,UK). The amount of i6A was estimated in scanningmode from the ratio between the molecular massesof the isolated compounds and the authentic stan-dard (336.2 and 341.2, respectively). The putativei6A fragmented into three major ions, 136, 148 and204, identical to the pattern obtained from authentici6A. The deuturated analogue, as expected, gave thefragments 136, 148 and 210.

Results

Cloning of an Arabidopsis cDNA

An Arabidopsis thaliana genomic sequence (GenBankaccession number AC005824) showing homology to

164

Figure 3. Alignment of the deduced amino acid sequence of the Arabidopsis IPPT (GenBank accession number AF109376) with the IPPTsfrom Homo sapiens (AF074918), Saccharomyces cerevisiae (M15991), and Escherichia coli (M63655). Dark boxes indicate identical residues,shaded boxes indicate similar residues. Dashes indicate gaps added for the alignment.

the yeast MOD5 gene that codes for a tRNA isopen-tenyltransferase (IPPT) was found by searching theAtDB database. An intron-exon identifying programrecognized 9 introns of various sizes (ranging from69 to 394 bp) in the putative ORF (Figure 1). Acorresponding 1502 kb cDNA containing the entirepredicted ORF was cloned by RT-PCR on Arabidopsisseedling mRNA.

Nucleotide sequence

Figure 1 illustrates schematically the cDNA clone andthe mRNA. The clone contained three closely spacedpotential start codons around position 60, and a fourthabout 50 nucleotides further downstream (not shown).The longest ORF would encode a product of 466amino acids with a molecular mass of 53 094 Da. The

165

cDNA extended 43 bp beyond this. 3′-RACE showedthat the transcript extends 158 nucleotides (nt) fromthe termination codon, with a potential polyadeny-lation signal (AATAAT) starting 14 nt before thepoly(A) tail (not shown). 5′-RACE showed that a sin-gle transcript is made from the gene, with a 5′ endlocated 63 nt upstream of the first ATG codon, 4 ntlonger than the cDNA.

Southern analysis was performed to determine thecopy number of the gene in the Arabidopsis genome(Figure 2). Both low- and high-stringency hybridiza-tion conditions gave the same results, and the pat-terns correlated well with predictions based on therestriction enzymes cut sites in genomic sequence.This strongly indicates that the Arabidopsis IPPT isencoded by a single-copy gene.

Protein sequence

The deduced plant protein sequence displayed a highsimilarity to the IPPTs of Homo sapiens, S. cerevisiaeand E. coli (Figure 3). The identity and similarity is28% and 49%, respectively, for H. sapiens, 24% and44% for S. cerevisiae, and 20% and 33% for E. coli.All regions highly conserved among the IPPTs arepresent also in the plant protein (Figure 3). A hydropa-thy plot (not shown) indicated that the plant proteinis very similar to human IPPT, except for the first 15amino acids which are hydrophobic in the human en-zyme, and that both are soluble proteins. The predictedpI is 6.2.

Based on ClustalW alignments of a number of theIPPT sequences available in GenBank, a phylogenetictree was constructed (Figure 4). The three eukaryoticIPPTs, including the plant protein, are more related toeach other than to any of the prokaryotic IPPTs.

The Arabidopsis cDNA complements a yeast mod5mutant

To determine if the cDNA encodes an IPPT activity,it was cloned in the yeast expression vector pFL61(Minet et al., 1992) and subsequently transformed intothe S. cerevisiae mutant strain MT-8 (Gillman et al.,1991). This strain has three sets of genetic modifi-cations. First, there are a number of UAA nonsensemutations, i.e. ade2-1, in genes for key metabolic en-zymes. In order to grow the strain requires that thecatalytic products of these enzymes are supplied in themedia. Second, the strain contains a mutated tRNASUP7, which can insert a tyrosine at UAA, therebysuppressing the above point mutations and restoring

Figure 4. Rooted phylogenetic tree depicting the relationshipamong eukaryotic and prokaryotic IPPT enzymes. The tree wasrooted using bacterial sequences as an outgroup. Sequences used notlisted in Figure 3 can be found under the following accession num-bers in GenBank: Aquifex aeolicus, AE000721; Bacillus subtilis,Z99113; Agrobacterium tumefaciens, M83532; Treponema pal-lidum, AE001238; Pseudomonas putida, AF016312; Synechocystissp. PCC6803, D90911; Streptomyces coelicolor, AL022268.

metabolic independence. The SUP7 tRNA was shownto require the i6A modification to be able to suppressUAA (Laten et al., 1978). Third, the MOD5 gene,which codes for the single IPPT of this species, hasbeen disrupted by an insertion. This results in a lossof UAA suppression, and MT-8 is thus a suitablesystem to test the IPPT functionality of the clonedcDNA. Only the cDNA-carrying plasmid was able torestore both adenine independence (Figure 5A), lysineindependence (data not shown), and canavanine sus-ceptibility (Figure 5B). This strongly indicated that thecDNA-encoded protein functionally substitutes for theabsent MOD5 protein.

To see if the complementation of the phenotypewas accompanied by restored synthesis of i6A, thetRNA nucleoside composition was analysed by HPLC(Figure 6). The MOD5 line H57 contains a peak atthe position of i6A (Figure 6, top panel), while MT-8lacks this modification (Figure 6, middle panel). The

166

expression of the Arabidopsis cDNA in MT-8 resultsin the appearance of significant amounts of i6A (Fig-ure 6, bottom panel). The identity of the i6A peaks wasverified by UV and MS spectroscopy.

Expression in Arabidopsis

Spatial and temporal patterns of expression of the genewere analysed by northern blotting with poly(A)+RNA from different organs and developmental stages.A single transcript of about 1.8 kb was detected inall organs (Figure 7). The size of the transcript isconsistent with the one predicted from the 5′- and 3′-RACE. The signal levels were similar in all samples,indicating that the expression is constitutive.

Discussion

The functional complementation and the analyses ofthe tRNA nucleoside compositions clearly show thatthe cloned cDNA encodes a functional IPPT. TheIPPTs (EC 2.5.1.8) form a group of closely relatedenzymes, whose function has been studied in E. coliand S. cerevisiae (Leung et al., 1997; Tolerico et al.,1999). Recently, we cloned a human member of thefamily (Golovko et al., 2000). The alignment in Fig-ure 3 shows that key features of the microbial IPPTshave been preserved throughout evolution to the twohigher eukaryotes.

In contrast to the yeast and human homologues, theplant protein sequence does not appear to have a po-tential bipartite nuclear localization signal (NLS), butthere is a stretch of basic residues, HRKR (Figure 3,442–445), that might be involved in nuclear targeting.A C2H2 Zn-finger-like motif found in the yeast and hu-man amino acid sequences (Golovko et al., 2000), butnot in the sequences of prokaryotic origin, is presentalso in the plant IPPT (Figure 3, 419–442).

In addition to cytoplasmic tRNA, cytokinins arefound in the tRNA of plastids and mitochondria (Takeiet al., 2000). Plants could thus have IPPT activity alsoin these compartments. Seven additional genes withhigh homology to isopentenyltransferases are presentin the Arabidopsis genome, but none of these codefor an IPPT. Instead they all code for cytokinin syn-thases (Kakimoto, 2001; Takei et al., 2001), indicatingthat the IPPT gene studied here is the single IPPTof Arabidopsis, and supplies the tRNA modificationfunction also to the organelles. In yeast, the MOD5gene gives rise to two separate proteins, the longer

of which has an additional 11 amino acids at the N-terminus, harbouring a mitochondrial targeting signal(Gillman et al., 1991). However, even though the 5′end of the plant IPPT has four potential translationalstarts, no obvious properties typical of signal peptideswas found.

The level of i6A in yeast tRNA from cells withthe Arabidopsis IPPT is significantly lower than thatin tRNA from control cells (Figure 6). This indicatesthat the plant protein recognises the yeast tRNA sub-strates less efficiently than MOD5, and that a part ofthe tRNA population in the transgenic cells has beenleft unmodified. An analysis of the tRNAs could givevaluable insights into the substrate requirements of theplant IPPT.

The main form of cytokinin in plant cytoplasmictRNA is cis-zeatin [Taller, 1994), which differs fromi6A in that it has an hydroxyl group at carbon 4 on theisopentenyl moiety. No cis-zeatin was formed in thetransformed yeast cells (Figure 6). The nature of thehydroxylation reaction in plant tRNA is not known,but it could possibly be related to that catalysed bythe tRNA hydroxylating activity carried by the miaEgene from S. typhimurium (Persson and Björk, 1993).The miaE gene product did not recognize i6A-tRNAas a substrate, instead a further modification with amethylthiol moiety at carbon 2 of the purine ring wasrequired for the reaction to take place. Plants couldalso have an alternative substrate for the IPPT, forinstance an hydroxylated analogue of the dimethyl-allylpyrophosphate that could give cis-zeatin-tRNAdirectly. An analogous situation has been suggested tobe the case in the synthesis of the cytokinin hormonesof the trans-zeatin type (Åstot et al., 2000).

Many possible fashions in which the tRNA cy-tokinins could be related to the cytokinin plant hor-mones have been suggested over the years (Lethamand Palni, 1983; Prinsen et al., 1997). One possibilityis that tRNA is a source of the hormones, since tRNAturnover releases cytokinins. An example of a smallbut possibly not insignificant production of cytokininfrom tRNA has been shown in the plant pathogenAgrobacterium tumefaciens (Gray et al., 1996). Calcu-lations based on in vivo studies in plants indicate thattRNA turnover could account for only a fraction of thefree cytokinins (Letham and Palni, 1983). It has neverbeen shown that cytokinin production from tRNA hasphysiological consequences. Also, the recent identifi-cation of enzymes in a direct pathway to the cytokinins(Kakimoto, 2001; Takei et al., 2001), bypassingtRNA, suggests that the tRNA pathway is, at the most,

167

Figure 5. Growth of yeast cells from four cell lines on selective minimal media either lacking adenine (A) or supplemented with canavanine(B). Line H57 is a wild-type strain expressing the yeast IPPT gene. Line MT-8, which has an inactivated native IPPT gene, was transformedwith the plasmid pFL61 without insert, or containing the Arabidopsis IPPT cDNA.

Figure 6. HPLC analyses of yeast tRNA nucleosides. Top panel: 4 OD260 units from yeast line H57. Middle panel: 8 OD260 units of tRNAfrom yeast line MT-8. Bottom panel: 4 OD260 units from MT-8 transformed with the Arabidopsis IPPT cDNA. The arrows indicate the positionwhere authentic i6A elutes.

168

Figure 7. Northern blot analyses of Arabidopsis IPPT mRNA. Eachlane separates the poly(A)+-fraction from 75 mg of total RNA. A.tissues of plants harvested in week 5 (flowers) and week 4 (all oth-ers) B. seedlings harvested at day 8 (first lane), and week 2, 3, 4 and5 (lanes 2–5), respectively. Blots were probed with the 32P-labelledArabidopsis IPPT cDNA. Lower panels in both A and B show thesame blots probed with 32P-labelled EF1α cDNA, as an internalcontrol for RNA loading.

a minor contributor. The Arabidopsis IPPT appears tobe constitutively expressed (Figure 7), in line with thefunction as a tRNA modifying enzyme, but is unlikethe pattern that would be expected from a key enzymein the pathway to a hormone. Given the potent effectsof the cytokinin hormones, it is perhaps more likelythat there exists some mechanism in plants that pre-vents cytokinins arising from tRNA degradation frominterfering with the hormone-active compounds. Thefact that the biologically relatively inactive cis-zeatinis the most abundant cytokinin form in plant tRNAcould be such a mechanism (Kaminek, 1982). Whenmade available through tRNA degradation, cis-zeatinwould thus not affect total hormone activity signifi-cantly. cis-Zeatins are sometimes found in plants in thetRNA-free form (Watanabe et al., 1982; Nicander etal., 1995). One species, Cicer arietinum, even has cis-zeatins as the predominant form of cytokinin (Emeryet al., 1998). The biogenesis and role of this type ofcytokinin is not known.

The ATP/ADP and AMP isopentenyltransferases,which synthesize cytokinins by a pathway bypass-ing tRNA, have significant sequence similarities tothe tRNA isopentenyltransferases, and may thus haveevolved from the latter group. Such a process could

well have started with a shift in substrate specificityfrom tRNA to other adenine compounds.

Acknowledgements

This work was in part supported by grants from theTrygger Foundation and the Nilsson-Ehle Foundation.We thank Dr A. Hopper of the Dept of Chemistryand Molecular Biology, Pennsylvania State Univer-sity College of Medicine for her kind gift of the yeaststrains. Dr Göran Hjälm, Dept. of Medical Biochem-istry and Microbiology, Uppsala University, Sweden,is thanked for expert help with RT-PCR. We thank DrBo Ek (Dept of Plant Biology, SLU, Uppsala, Swe-den) for excellent mass spectrometry analyses. Dr UlfLagercranz is gratefully acknowledged for valuablehelp with phylogenetic analyses.

References

Åstot, C., Dolezal, K., Nordström, A., Wang, Q., Kunkel, T.,Moritz, T., Chua, N.H. and Sandberg, G. 2000. An alternativecytokinin biosynthesis pathway. Proc. Natl. Acad. Sci. USA 97:14778–14783.

Björk, G.R. 1995. Biosynthesis and function of modified nucleo-sides. In: D. Söll and U. RajBhandary (Eds.) tRNA: Structure,Biosynthesis, and Function, ASM Press, Washington, DC, pp.165–205.

Buck, M., Connick, M. and Ames, B.N. 1983. Complete analy-sis of tRNA-modified nucleosides by high-performance liquidchromatography: the 29 modified nucleosides of Salmonella ty-phimurium and Escherichia coli tRNA. Anal. Biochem. 129:1–13.

Emery, R.J.N., Leport, L., Barton, J.E., Turner, N.C. and Atkins,C.A 1998. cis-Isomers of cytokinins predominate in chickpeaseeds throughout their development. Plant Physiol. 117: 1515–1523.

Gehrke, C.W., Kuo, K.C., McCune, R.A. and Gerhardt, K.O. 1982.Quantitative enzymatic hydrolysis of tRNAs. Reversed-phasehigh performance liquid chromatography of tRNA nucleosides.J. Chromatog. 230: 297–308.

Gietz, R.D.and Schiestl, R.H. 1997. Transforming yeast with DNA.Meth. Mol. Cell. Biol. 5: 255–269.

Gillman, E.C., Slusher, L.B., Martin, N.C. and Hopper,A.K. 1991. MOD5 translation initiation sites determine N-6-isopentenyladenosine modification of mitochondrial and cyto-plasmic transfer RNA. Mol. Cell. Biol. 11: 2382–2390.

Golovko, A., Hjälm, G., Sitbon, F., and Nicander, B. 2000. Cloningof a human tRNA isopentenyl transferase. Gene 258: 85–93.

Gray, J., Gelvin, S.B., Meilan, R. and Morris, R.O. 1996. TransferRNA is the source of extracellular isopentenyladenine in a Ti-plasmidless strain of Agrobacterium tumefaciens. Plant Physiol.110: 431–438.

Kakimoto, T. 2001. Identification of plant cytokinin biosyntheticenzymes as dimethylallyl diphosphate:ATP/ADP isopentenyl-transferases. Plant Cell Physiol. 42: 667–685.

169

Kaminek M., 1982. Mechanisms preventing the interference oftRNA cytokinins in hormonal regulation. In: P.F. Wareing (Ed.)Plant Growth Substances 1982, Academic Press, New York, pp.215–224.

Laten, H., Gorman, J. and Bock, R.M. 1978. Isopentenyladenosinedeficient tRNA from an antisuppressor mutant of Saccharomycescerevisiae. Nucl. Acids Res. 5: 4329–4342.

Letham, D.S. and Palni, L.M.S. 1983. The biosynthesis andmetabolism of cytokinins. Annu. Rev. Plant Physiol. 34: 163–197.

Leung, H.-C.E., Chen, Y. and Winkler, M.E. 1997. Regulation ofsubstrate recognition by the miaA tRNA prenyltransferase mod-ification enzyme of Escherichia coli K-12. J. Biol. Chem. 272:13073–13083.

Logemann, J., Schell, J. and Willmitzer, L. 1987. Improved methodfor the isolation of RNA from plant tissues. Anal. Biochem. 163:16–20.

Minet, M., Dufour, M.-E. and Lacroute, F. 1992. Complementationof Saccharomyces cerevisiae auxotrophic mutants by Arabidop-sis thaliana cDNAs. Plant J. 2: 417–422.

Mok, M.C. 1994. Cytokinins and plant development: an overview.In: D.W.S. Mok and M.C. Mok (Eds.) Cytokinins: Chemistry,Activity, and Function, CRC Press, Boca Raton, FL, pp. 155–166.

Nicander, B., Björkman, P.-O. and Tillberg, E. 1995. Identificationof an N-glucoside of cis-zeatin from potato tuber sprouts. PlantPhysiol. 109: 513–516.

Persson, B.C. and Björk, G.R. 1993. Isolation of the gene(miaE) encoding the hydroxylase involved in the synthesis of2-methylthio-cis-ribozeatin in tRNA of Salmonella typhimuriumand characterization of mutants. J. Bact. 175: 7776–7785.

Persson, B.C., Esberg, B., Ólafsen, Ò. and Björk, G.R. 1994. Syn-thesis and function of isopentenyl adenosine derivatives in tRNA.Biochimie 76: 1152–1160.

Prinsen, E., Kaminek, M. and van Onckelen, H.A. 1997. Cytokininbiosynthesis: a black box? Plant Growth Regul. 23: 3–15.

Skoog F. and Armstrong, D.J. 1970. Cytokinins. Annu. Rev. PlantPhysiol. 21: 359–384.

Takei, K., Sakakibara, H. and Sugiyama, T. 2001. Identificationof genes encoding adenylate isopentenyltransferase, a cytokininbiosynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem.276: 26405–26410.

Taller, B.J. 1994. Distribution, biosynthesis, and function of cy-tokinins in tRNA. In: D.W.S. Mok and M.C. Mok (Eds.) Cy-tokinins: Chemistry, Activity, and Function, CRC Press, BocaRaton, FL, pp. 101–112.

Thompson, J., Higgins, D. and Gibson, T. 1994. ClustalW: improv-ing the sensitivity of progressive multiple sequence alignmentthrough sequence weighing, position-specific gap penalties andweight matrix choice. Nucl. Acids Res. 22: 4673–4680.

Tolerico, L., Benko, A., Aris, J., Stanford, D., Martin, N. andHopper, A. 1999. Saccharomyces cerevisiae Mod5p-II con-tains sequences antagonistic for nuclear and cytosolic locations.Genetics 151: 57–75.

Urban, C. and Beier, H. 1995. Cysteine tRNAs of plant origin asnovel UGA suppressors. Nucl. Acids Res. 23: 4591–4597.

Warner, G.J., Berry, M.J., Moustafa, M.E., Carlson, B.A., Hatfield,D.L. and Faust, J.R. 2000. Inhibition of selenoprotein synthesisby selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine. J.Biol. Chem. 275: 28110–28119.

Watanabe, N., Yokota, T. and Takahashi, N. 1982. Transfer RNA,a possible supplier of free cytokinins, ribosyl-cis-zeatin andribosyl-2-metylthiozeatin: quantitative comparison between freeand transfer cytokinins in various tissues of the hop plant. PlantCell Physiol. 23: 479–488.

Winkler, M.E. 1998. Genetics and regulation of base modificationin the tRNA and rRNA of procaryotes and eucaryotes. In: H.Grosjean and R. Benne (Eds.) Modification and Editing of RNA,ASM Press, Washington, D.C., pp. 441–469.