ABSTRACT
In the ciliated protozoa Tetrahymena thermophila introns have been detected in rRNA and mRNAs until now. We have isolated and sequenced seven tRNATyr genes from the T.thermophila nuclear genome. All of these genes contain introns of identical length and sequence. The 11 bp long intervening sequences are located 1 nt 3' to the anticodon as found in other eukaryotic nuclear tRNA genes. Tetrahymena tRNATyr genes are efficiently transcribed in HeLa cell nuclear extract. Moreover, processing and splicing occurred in HeLa as well as in wheat germ extracts, supporting the notion that Tetrahymena tRNATyr introns can be classified as authentic tRNA introns. We have also isolated cytoplasmic tRNATyr from Tetrahymena cells. This tRNATyr isoacceptor has a Q[Psi]A anticodon and is not a UAG suppressor as shown in in vitro translation studies. Since UAG and UAA codons are used as glutamine codons in Tetrahymena macronuclear DNA, the presence of a strong natural UAG suppressor such as tRNATyr with G[Psi]A anticodon should cause misreading of the glutamine as tyrosine codons and the absence of the latter had thus been predicted. Furthermore we have studied the organization of tRNATyr genes in the genome of T.thermophila and have found two types of tRNATyr gene arrangement. A minimum of 12 tRNATyr genes are present as single copies in genomic DNA HindIII restriction fragments ranging in size from 0.6 to 7 kb. Additionally one cluster of tRNATyr genes consisting of six members has been detected in a 2.3 kb HindIII fragment.
Ciliated protozoa emerged as an evolutionary group more than 109 years ago before the appearance of fungi, plants and animals (1 ). Despite their great genetic diversity, ciliates have in common the presence of nuclear dimorphism: a germ-line micronucleus, which is transcriptionally inactive and a vegetative macronucleus which is responsible for the transcription. Some ciliates, such as Tetrahymena, Stylonichia and Paramecium use an altered genetic code. It was reported by several workers independently that a number of macronuclear genes in these ciliates contain internal TAA and TAG codons. A comparison of the derived amino acid sequences with other known homologous protein sequences revealed that these two codons which are stop codons in the universal genetic code appear to be used as glutamine codons (2 ,3 ). This assumption was confirmed when two unusual glutamine tRNA isoacceptors with CUA and UmUA anticodon, in addition to the normal tRNAGlnUmUG, were purified from Tetrahymena thermophila cells (4 ,5 ). Moreover it was demonstrated that tRNAGlnUmUA reads the UAA and UAG codons in vitro, whereas tRNAGlnCUA recognizes only UAG (5 ), indicating that T.thermophila uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon employed by this organism in the nuclear genome.
We have previously shown that tobacco and wheat cytoplasmic tRNATyr with G[Psi]A anticodon is a powerful natural UAG and to a lesser extent a UAA suppressor, whereas tRNATyr with Q[Psi]A or GUA anticodon is unable to interact with these two stop codons (6 -8 ). Since UAG and UAA are sense codons in Tetrahymena mRNAs, we postulated that a tRNATyr isoacceptor with G[Psi]A anticodon should not be present in the cytoplasm of Tetrahymena cells in order to avoid misreading of the glutamine codons as tyrosine. Therefore, we have isolated and sequenced tRNAsTyr from total tRNA of T.thermophila and have identified a single cytoplasmic tRNATyr isoacceptor with Q[Psi]A anticodon.
The synthesis of pseudouridine in the centre of the tRNATyr anticodon, i.e., [Psi]35 has been shown to be dependent on the presence of an intron in the corresponding tRNA precursors in yeast (9 ), animals (10 ,11 ) and plants (8 ). Until now, intron-containing tRNA genes have not been found in the nuclear genome of ciliated protozoa. For that reason it seemed to be of general interest to learn if tRNATyr genes from Tetrahymena accommodate intervening sequences. We first used tRNATyr- specific sequences in order to amplify tRNATyr genes present in the Tetrahymena nuclear genome and subsequently isolated seven full-length tRNATyr genes and a single truncated tRNATyr gene from a genomic library. All of the seven tRNATyr genes contain identical 11 bp long intervening sequences. Furthermore, we show here that Tetrahymena tRNATyr genes are transcribed in HeLa cell nuclear extract and that intron-containing tRNATyr precursors are processed and spliced in HeLa as well as in wheat germ extracts.
Restriction endonucleases, nuclease P1, calf intestinal alkaline phosphatase, T4 RNA ligase, T4 DNA ligase and T4 polynucleotide kinase were from Boehringer, Mannheim. [3H]Tyrosine with a specific activity of 1 Tbq/mmol was obtained from Amersham Buchler (Braunschweig) and all other radiochemicals were from Hartmann Analytic, Braunschweig. The Sequenase kit (7-deaza-dGTP version) from USB was used for sequencing reactions. Untreated wheat germ was a gift from SynPharma GmbH, Augsburg.
Escherichia coli JM109 was used as a host for propagation of plasmid DNA. The recombinant plasmid pAtY3II carries an Arabidopsis thaliana tRNATyr gene on a 1.4 kb RsaI fragment cloned into pUC19 DNA (12 ). Escherichia coli DH5[alpha]F' was purchased from New England Biolabs, Schwalbach.
Unfractionated tRNA and aminoacyl-tRNA synthetase from Tetrahymena were isolated from the cells of T.thermophila, mating type IV essentially as described by Kuchino et al. (4 ). Fractionation of tRNAs by BD-Cellulose column chromatography, purification of tRNAs by successive polyacrylamide gel elctrophoresis and sequencing of tRNA by post-labelling techniques was carried out according to Beier et al. (6 ) and Zerfaß and Beier (8 ).
About 108 cells from a fast growing culture of T.thermophila, mating type IV were harvested by centrifugation, washed with cold 10 mM Tris-HCl, pH 7.4 and suspended in 1 ml of the same buffer. The cell suspension was mixed with 10 ml lysis buffer (10 mM Tris-HCl, pH 7.4, 0.1 M Na2EDTA, 1% SDS) and 6 ml phenol and incubated for 10 min at 60oC, then twice extracted with chloroform/isoamylalcohol (24:1). The aqueous phase was recovered, dialyzed against SSC and subsequently treated with 100 µg RNase A/ml and 100 µg proteinase K/ml.
Oligonucleotides were synthesized with the Gene Assembler Plus from Pharmacia LKB. They were end-labelled with [[gamma]-32P]ATP using T4 polynucleotide kinase and purified by using Nucleobond AX 5 columns (Macherey-Nagel, Düren). Labelled DNA fragments of high specific activity were prepared by using the random primed DNA labelling kit from Boehringer, Mannheim.
The amplification reaction was carried out in a total volume of 100 µl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% gelatin, 200 µM each of dGTP, dATP, dTTP and dCTP, 1 µM of each primer: (Tyr 1: 5'-CCGAACTTAGCTCAGTTGG-3', identical to nucleotides 1-19 and Tyr 2: 5'-TCCGTTCTCCCGGGTTCG-3', complementary to nucleotides 56-73 of Tetrahymena tRNATyr shown in Fig. 1 B), 100 ng genomic DNA from T.thermophila and 2.5 U AmpliTaqTM Taq DNA polymerase (Perkin Elmer Cetus). The mixture was subjected first to a 3 min denaturation at 95oC, followed by 30 cycles of 60 s, 95oC, 60 s, 55oC and 60 s, 72oC in a Perkin Elmer Cetus Thermocycler.
Approximately 100 µg of genomic DNA from T.thermophila was digested with HindIII and the fragments were separated on a preparative 0.8% agarose gel employing Seakem GTG agarose from Biozym. HindIII fragments of 2.0-2.3 kb were isolated by electroelution and ligated into pUC19 DNA which had been cleaved with HindIII and treated with alkaline phosphatase. The DNA mixture was used to transform the E.coli strain DH5[alpha]. Colony screening was carried out with 32P-labelled oligonucleotides as probes. Prehybridization of the nitrocellulose filter was in 6* SSC, 10* Denhardts, 0.01 M phosphate buffer, pH 6.8, 1 mM ATP, 0,05% SDS, 10 mM Na2EDTA, 0.1 mg/ml herring sperm DNA at 42oC for 4 h. Hybridization was at 42oC for 18 h in the same buffer and 3 × 106 c.p.m. 32P-labelled probe/ml. After hybridization, the filter was washed two times with 6* SSC, 0.05% SDS at room temperature, once at 42oC for 30 min and once at the stringent temperature (50-53oC) for 7 min.
Direct sequencing of plasmid DNAs was performed according to Hattori and Sakaki (13 ), employing the method of `primer walking'. The clone pTetY3 was partially sequenced by MediGene AG DNA-Analytics, München.
Nuclear extract was prepared from HeLa S3 cells essentially as described by Dignam et al. (14 ). Transcription assays and the elution of precursors from preparative gels were performed as described by Stange and Beier (15 ).
Cell-free wheat germ S23 extract was prepared from wheat embryos according to Stange and Beier (15 ). In vitro processing and splicing of tRNA precursors was performed in a total volume of 30 µl, containing 6 µl S23 extract (25 mg/ml), 20 mM Tris-HCl, pH 7.4, 100 mM potassium acetate, 6 mM magnesium acetate, 80 µM spermine, 10 mM creatine phosphate, 0.8% Triton X-100 (Surfact-Amps X-100 from Pierce), 1 mM ATP, 0.1 mM CTP and 104 c.p.m. precursor tRNA.
Total tRNA from T.thermophila was fractionated by BD-cellulose chromatography. A linear gradient of 0.35-1 M NaCl (0.01 M MgCl2, 0.02 M NaOAc, pH 4.5) was first applied and the hydrophobic tRNAs were then eluted with 2 M NaCl/15% ethanol. Aliquots of appropriate fractions were selected for aminoacylation assays. The presence of tRNATyr was determined by using [3H]tyrosine and a crude synthetase preparation from T.thermophila cells. All tRNATyr isoacceptors eluted late from the BD-cellulose column (i.e., fraction IV) and were found also in the ethanol eluate (i.e., fraction V). The tRNAsTyr in each fraction were further purified by successive gel electrophoresis in a native 10% polyacrylamide gel, pH 8.3, containing 10% glycerol, a 10% denaturing polyacrylamide gel, pH 3.5 and then in a 12.5% denaturing polyacrylamide gel, pH 8.3. The nucleotide sequences of the purified tRNAsTyr were determined according to Stanley and Vassilenko (16 ).
Two mitochondrial tRNATyr isoacceptors with GUA and QUA anticodon (Fig. 1 A) were present in fraction V in about equal amounts whereas a single cytoplasmic tRNATyr isoacceptor with Q[Psi]A anticodon was observed in fractions IV and V (Fig. 1 B). Since we had used a crude synthetase preparation for the detection of tRNAsTyr it was no surprise that we had identified the mitochondrial isoacceptors in addition to the desired cytoplasmic tRNATyr. Comparison of the mitochondrial tRNATyr sequence with the known Tetrahymena mitochondrial tRNATyr gene (17 ) revealed one difference in the long extra arm, i.e., an extra cytidine at position 47:F (Fig. 1 A).
The sequence of cytoplasmic tRNATyr is the first one for this isoacceptor characterized in any ciliate organism until now and shows the typical features of eukaryotic as compared to prokaryotic tRNATyr, i.e., a short extra arm of 5 nt and two cytidines at positions 1 and 2 (18 ). The modified nucleoside in the first anticodon position of mitochondrial and cytoplasmic tRNAsTyr is the known queuosine found also in bacterial and plant tRNAsTyr as deduced from its chromatographic migration pattern in two different solvents (19 ).
Suppressor activity of Tetrahymena mitochondrial and cytoplasmic tRNAsTyr was examined in a messenger-dependent reticulocyte lysate to which tobacco mosaic virus (TMV) RNA and the corresponding tRNA were added. Tobacco tRNATyr with G[Psi]A anticodon stimulates readthrough over the leaky UAG termination codon at the end of the 126K cistron of TMV RNA to a great extent (Fig. 2 , lane d) as has been previously demonstrated (6 ), whereas the two Tetrahymena tRNATyr isoacceptors exhibit no UAG suppressor activity in this assay (Fig. 2 , lanes b and c).
We used the nucleotide sequence established for Tetrahymena cytoplasmic tRNATyr (Fig. 1 B) to design primers hybridizing to the 5' and 3' ends of the putative gene(s) for the amplification reaction. The PCR products were separated on a 2% agarose gel. A number of products of variable size were detected in ethidium bromide stained gels. The products were blotted onto a nylon membrane and identification of tDNATyr-containing fragments was achieved by hybridization with a tRNATyr-specific probe (i.e., Tyr 3) identical to nucleotides 18-37 of cytoplasmic tRNATyr (Fig. 1 B). A strong hybridizing fragment of ~345 bp and two weakly hybridizing fragments of ~600 and ~780 bp were detected. The purified 346 bp PCR fragment was subsequently cloned into SmaI-cleaved pUC19 DNA. The nucleotide sequence of this DNA fragment is shown in Figure 3 . The amplified genomic DNA fragment contains two tRNATyr genes separated by a spacer region of 179 bp. Both tRNATyr genes have intervening sequences of identical length (i.e., 11 bp) and sequence. The first tRNATyr gene codes for the sequenced tRNATyr shown in Figure 1 B, whereas the second gene differs in eight positions from the latter. The presence of amplified products of even higher size than 346 bp indicated that tRNATyr genes might be clustered at a single site in Tetrahymena macronuclear DNA.
In order to obtain more precise information about the tRNATyr gene organization in the Tetrahymena genome we used first the random-primed 346 bp long PCR fragment as a probe for hybridization to EcoRI-, HindIII- and XbaI-digested genomic DNA from T.thermophila. Southern blot analysis revealed the existence of at least 11 hybridizing fragments in each digest, ranging in size from 0.6 to >10 kb (Fig. 4 ). If we utilized 5'-labelled oligonucleotide Tyr 3 as a probe, essentially the same hybridization pattern was obtained (not shown). A remarkably strong hybridization fragment was seen in each digest which had the approximate length of 10, 2.3 or 1.6 kb upon cleavage with EcoRI, HindIII and XbaI, respectively (Fig. 4 ). We subsequently isolated HindIII fragments in the range from 2.0 to 2.3 kb from a preparative agarose gel for generating a mini genomic library assuming that the DNA section corresponding to the 346 bp fragment amplified by PCR (Fig. 3 ) was contained in one of them.
About 1.5 × 104 white colonies containing HindIII fragments of 2.0-2.3 kb from Tetrahymena genomic DNA ligated into the HindIII site of pUC19 were screened with 32P-labelled tRNATyr-specific probe Tyr 3 (Fig. 1 B). About 10 hybridizing colonies were thus identified and repeated rescreening with the same probe yielded eventually two positive clones which were called pTetY1 and pTetY2. Unexpectedly, preliminary sequence data revealed that neither clone accommodated the genomic equivalent to the 346 bp PCR fragment shown in Figure 3 . The remaining positive clones were therefore screened with a labelled oligonucleotide complementary to a sequence of the spacer region between the two tRNATyr genes located on the amplified PCR fragment as indicated in Figure 3 . This probe, i.e., IR-346, enabled us to identify unambiguously the desired clone, which was called pTetY3. All three clones contain HindIII fragments of ~2.2-2.3 kb as expected (Fig. 5 ). We have elucidated the complete nucleotide sequences of the inserts contained in pTetY2 and pTetY3 and part of the 2.3 kb insert of pTetY1. Cleavage of the latter clone with a variety of other restriction enzymes did not reveal the presence of more than one tRNATyr gene on this individual fragment. Likewise the clone pTetY2 contains only a single tRNATyr gene. However, the 2.3 kb HindIII fragment contained in pTetY3 harbours five tRNATyr genes and a pseudogene, consisting of a truncated 5' half of the coding region. Moreover, this fragment contains the 346 bp region amplified earlier by PCR as indicated in Figure 5 . The six tRNATyr genes of pTetY3 have the same orientation and are separated by spacer regions ranging from 166 to 179 bp. Five of the eight tRNATyr genes in clones pTetY1, pTetY2 and pTetY3 correspond exactly to the sequenced cytoplasmic tRNATyr (Figs 1 B and 6 ). The tRNATyr genes TetY4 and TetY7 vary in a number of positions from the major species as indicated in the secondary structure models shown in Figure 6 . Some of these mutations result in mismatches in the anticodon and/or T stem. Disruption of base pairs in either stem has been shown to generate processing-defective pre-tRNA molecules in HeLa and plant extracts (20 ), indicating that TetY4 and TetY7 may be pseudogenes. All seven full-length tRNATyr genes have 11 bp long introns of identical sequence (Figs 3 and 9 ).
The HeLa nuclear extract has been shown to support RNA polymerase III-dependent transcription of numerous heterologous tRNA genes originating from yeast, plants and animals (21 -24 ). Transcription is initiated generally upstream of the tRNA gene at a purine neighbouring a pyrimidine and is terminated at a stretch of at least five consecutive thymidine residues either immediately following the end of the tRNA gene or within ~20 bp downstream (25 ). The seven full-length Tetrahymena tRNATyr genes all contain putative initiation signals at positions -3 and/or -6 and a stretch of T residues immediately flanking the tRNA genes consisting of six (TetY3, TetY4, TetY7), eight (TetY6), nine (TetY8), 14 (TetY2) and 15 (TetY1) residues. Consequently they should be substrates for the HeLa RNA polymerase III.
We selected TetY2* for our studies, located on a 236 bp DraI fragment derived from the original 2162 bp HindIII insert of pTetY2 as indicated in Figure 5 . In vitro transcription of pTetY2* in HeLa cell nuclear extract in the presence of 4 mM MgCl2 yielded two major primary transcripts of 91 and 94 nt after 20 min and upon further incubation a processed intermediate product of 87 as well as the mature tRNA of 76 nt (Fig. 7 ). The two high molecular weight polynucleotides which were synthesized also at 1 mM MgCl2, i.e., in the absence of processing events (12 ), are probably transcripts which have originated by initiation of transcription at two different sites, since a very strong termination signal of 14 T residues is located immediately downstream of the tRNATyr gene. A similar pattern of products is generated upon in vitro transcription of pTetY1 in HeLa extract (not shown).
The two major primary transcripts of 91 and 94 nt synthesized preparatively in HeLa nuclear extract at 1 mM MgCl2 were subsequently used as substrates for studying processing and splicing in a cell-free S23 extract from wheat germ. We had previously demonstrated that homologous and heterologous tRNA precursors are faithfully processed, that is removal of 5' and 3' flanking sequences was equally efficient with plant, animal and yeast pre-tRNAs. However, intron excision by the wheat germ splicing endonuclease appeared to request exclusively homologous substrates for its action (26 ). Figure 8 shows the processing pattern of Tetrahymena intron-containing pre-tRNATyr (i.e., P 94) and for comparison, the maturation of an Arabidopsis pre-tRNATyr (i.e., P 113) derived from transcription of pAtY3II (12 ). The latter is first converted to intron-containing pre-tRNA with processed ends (i.e., I 88) followed by intron excision and ligation of the resulting tRNA halves to produce mature tRNA (M 76). Maturation of either of the two Tetrahymena primary transcripts (i.e., P 91 and P 94) proceeds essentially by the same pattern albeit with a lower efficiency.
Natural suppressor tRNAs encoded by nuclear DNA have been characterized in a number of eukaryotic organisms (27 ,28 ). With the exception of selenocysteine tRNA(U*CA) all of them are normal cytoplasmic tRNAs. Accordingly, misreading of either stop codon involves unconventional base pairing. And in return unorthodox codon reading depends on the nucleotides surrounding the suppressed stop codon as well as on properties of the tRNA itself. Thus, we have previously shown that base modifications in the first and second position of the G[Psi]A anticodon in tRNATyr have strong inhibitory or stimulating effects on UAG suppression: tRNATyrG[psi]A is a very powerful UAG suppressor whereas tRNATyrQ[psi]A and tRNATyrGUA are not suppressors (7 ,8 ).
A single tRNATyr isoacceptor with Q[Psi]A anticodon was identified in the cytoplasm of T.thermophila cells (Fig. 1 B). In vitro translation experiments clearly demonstrated that this tRNA is not a UAG suppressor in vitro (Fig. 2 , lane b), confirming earlier results with tRNATyrQ[psi]Afrom Drosophila (29 ) and wheat (7 ). Due to an altered genetic code employed by Tetrahymena, UAA and UAG are glutamine codons in the nuclear genome of this organism (5 ,30 ). It is conceivable that the release factor in Tetrahymena has become specific for UGA and thus should be unable to recognize UAA and UAG codons any longer. Consequently, competition between release factor and any putative suppressor tRNA at these sites is abolished. This implies that the presence of a tRNATyr isoacceptor with G[Psi]A anticodon in the cytoplasm of Tetrahymena cells should be more deleterious than in other organisms and, as shown here, is in fact avoided.
We are grateful to Dr H.-M.Seyfert (Gießen) for a culture of T.thermophila cells and for advice on the preparation of genomic DNA. We thank H.-D.Sickinger for capable technical assistance. This work was supported by a grant from theDeutsche Forschungsgemeinschaft to H.B.
*To whom correspondence should be addressed. Tel: +49 931 888 40 31; Fax: +49 931 888 40 28
Present addresses: +MPI für Züchtungsforschung, Carl-von-Linné Weg 10, D-50829 Köln, Germany and §Boehringer-Mannheim GmbH, Nonnerwald 2, D-82377 Penzberg, Germany
REFERENCES