©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Accurate Transcription of the Trypanosoma brucei U2 Small Nuclear RNA Gene in a Homologous Extract (*)

(Received for publication, May 11, 1995)

Arthur Günzl (1)(§), Christian Tschudi (1), Valerian Nakaar (1), Elisabetta Ullu (1) (2)(¶)

From the  (1)Department of Internal Medicine and (2)Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8022

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element.


INTRODUCTION

Studies of transcription in the protozoan family Trypanosomatidae have revealed unusual features such as polycistronic transcription units and RNA polymerase I-mediated transcription of protein-encoding genes. However, compared to what we know about the transcriptional machinery in a number of different organisms, including yeast and higher eukaryotes, our understanding of promoter structures and trans-acting factors in trypanosomatid protozoa is extremely limited. This is in large part due to the fact that only a few functional promoters have been identified and that no in vitro transcription system is available. To date, RNA polymerase I and III promoters have been characterized in detail in Trypanosoma brucei. RNA polymerase I transcription units include the genes encoding the large ribosomal rRNAs(1, 2) , the variant surface glycoprotein gene expression sites(3, 4) , and the procyclic acidic repetitive protein genes(1, 5, 6) .

The analysis of RNA polymerase III-mediated transcription in T. brucei has led to a number of surprising findings. Based on RNA polymerase inhibitor studies with the drugs -amanitin and tagetitoxin and on the architecture of the promoters, the U2, U4, and U-snRNA()B (the U3 snRNA homolog) genes are transcribed by RNA polymerase III(7, 8) . This is in contrast to most other eukaryotic organisms studied to date, where these RNAs are synthesized by RNA polymerase II(9) . The only exception being the plant U3 snRNA gene which is also transcribed by RNA polymerase III (10) . In particular, studies with tagetitoxin, a specific inhibitor for RNA polymerase III, showed that transcription inhibition of the T. brucei U2, U4, and U-snRNA B genes closely followed that of typical RNA polymerase III genes, namely 5S, 7SL, U6 snRNA, and tRNAs, but not that of RNA polymerase I or II genes(7) .

The characterization of cis-acting elements required for expression of the U2 and U6 snRNA genes in T. brucei has been a major focus of our research. In vivo transcription of both genes is dependent upon the integrity of three regulatory elements (7, 8) .()Two upstream elements coincide with divergently oriented A and B boxes, which in the case of the U6 snRNA gene locus, are part of a functional threonine tRNA gene. In addition, expression of the U2 and U6 snRNA genes requires intragenic elements close to the 5` end of the coding region, which are most likely responsible for positioning the RNA polymerase at the correct transcription start site.

Although the above studies have unraveled a novel promoter architecture for the U2 and U6 snRNA genes, the experiments reported thus far do not provide insight into the underlying mechanism. Therefore, as a first step toward a detailed biochemical understanding of the transcriptional apparatus assembling on these U-snRNA genes, we developed a T. brucei DNA-dependent in vitro transcription system. Using the G-less cassette approach (11) and a modified Dignam extract(12) , RNA polymerase III-mediated transcription was shown to initiate at position +1 of the U2 snRNA gene, and transcription activity was dependent on the upstream B box element.


MATERIALS AND METHODS

Plasmid Construction

Plasmids pTbU2_GL-AAA, pTbU2_GL-AT-, pTbU2_GL-CAA, pTbU2_GL-CAT, pTbU2_GL-TAT, and pTbU2_GL-TCA were derived from construct pTU283 (7) by oligonucleotide-directed mutagenesis of single-stranded template(13) . The oligonucleotide used in this mutagenesis was U2-GL1, 5`-GCATATCTTCTC(ATC)(ATC)CTATTTA(ATC)CTAAGATC-3` (parentheses indicate degenerate positions), containing the sequences of the U2 snRNA gene from nucleotides -2 to +28.

The U2 G-less cassette has the following sequence (sequence of the nontranscribed strand, +1 indicates the U2 transcription start site):

The cassette was constructed by using two complementary oligonucleotides. Oligonucleotide U2-GL2 is 111 nt long and complementary to the G-less sequence listed above. It spans the whole cassette and has additional CG and CT dinucleotides at its 5` and 3` end, respectively. Oligonucleotide U2_GL3, 5`-CCACTACATATCTTCTCAAC-3`, was designed to generate an overhang characteristic for the restriction enzyme SalI at the 5` end of the sequence shown above. The oligonucleotides were hybridized to each other, and double-stranded DNA was prepared by the Klenow-mediated fill-in reaction. The G-less cassette was cloned into the SalI and NruI restriction sites of plasmid pTbU2_BS-12/-2b thereby replacing the U2 snRNA coding region. pTbU2_BS-12/-2b is a derivative from mutant BS-12/-2 (7) and was generated by removing a SalI site in the vector. Two U2 G-less cassette mutants were obtained. TbU2_GLa contains the G-less cassette sequence as shown above, whereas TbU2_GLb lacks three of the five 3`-terminal thymidine residues. Therefore, transcription of TbU2_GLb is expected to terminate 10 nt downstream at the putative endogenous termination signal. Standard transcription reactions were carried out with TbU2_GLb, because this template reproducibly gave rise to stronger transcription signals. The extra sequence in TbU2_GLb contains a guanosine residue. Thus, RNase T1 cleavage at this site will trim TBU2_GLb RNA to the same size as RNA obtained from TbU2_GLa (see Fig. 2B).


Figure 2: In vivo expression of the U2 G-less cassette gene. A, schematic outline of the U2 G-less cassette gene. Filled in and open boxes represent wild-type and G-less sequences, respectively. Transcripts expected from correct initiation (97 base pairs) and nonspecific readthrough transcription (106 base pairs) are indicated. GLESS_PE denotes the oligonucleotide complementary to the tag at the 3` end of the G-less cassette. B, 3`-terminal sequences of mutants TbU2_GLa and TbU2_GLb. C and D, total RNA was isolated from transfected T. brucei cells and subjected to Northern blot (C) and primer extension (D) analyses with oligonucleotide GLESS_PE. Cells transfected with vector DNA served as a control (lane 1). Lane 2, pTBU2_GLa; lane 3, pTbU2_GLb; M, MspI-digested pBR322 marker.



Mutant pTbU2_GL-B52 is a derivative of pTbU2_GLb carrying six point mutations in the upstream B box element(7) .

DNA Transfection and RNA Analysis

Transfections, RNA isolation, primer extension analysis, and Northern blot hybridizations were carried out as described(7, 8) . The following oligonucleotides were used in primer extension analysis and/or Northern blot hybridizations: U2k(14) , complementary to nucleotides 128 to 148 of the U2 snRNA; GLESS_PE, 5`-GAGTGAATGATGATAGATTTG-3`, complementary to nucleotides +75 to +94 of the U2 G-less cassette gene.

Preparation of Nuclear Extracts

A 2-liter culture of the procyclic forms of T. brucei rhodesiense strain YTaT 1.1 was grown as described previously (15) to a density of 1 10 cells per ml. Cells were harvested at room temperature, yielding a packed cell volume of 2 to 2.5 ml. The pellet was rinsed twice with 10 ml of wash solution (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM MgCl) and once with 10 ml of ice-cold transcription buffer (150 mM sucrose, 20 mM potassium L-glutamate (Sigma), 10 mM Hepes-KOH, pH 7.9, 2.5 mM MgCl, 1 mM dithiothreitol, 10 µg/ml leupeptin). The following steps were carried out at 4° C. Cells were finally resuspended in 1.5 times packed cell volume of transcription buffer, preincubated for 5 minutes, and broken in a 7-ml dounce with a type A pestle by applying rapid strokes continuously for about 10 min, until the vast majority of cells were broken. The resulting cell extract was spun in an Eppendorf centrifuge at 4,000 g for 10 min, the supernatant was removed, and the pellet was subjected to another spin for 2 min at 16,000 g. The final pellet was resuspended in an equal volume of transcription buffer. KCl was added to a final concentration of 400 mM, and the nuclear fraction was extracted by rotating the tube at 4° C for 30 min. The extract was spun in an Eppendorf centrifuge for 10 min at 16,000 g, and the supernatant was desalted using a Centricon-10 concentrator (Amicon) until the KCl concentration was around 40 mM. Final extracts ranged in protein concentration from 3 to 6 mg/ml and were stored at -70° C. No correlation was detected between protein concentrations within this range and extract activity.

In Vitro Transcription Reactions

A standard transcription reaction was carried out in a volume of 25 µl containing 10 µl of nuclear extract. The reaction further contained 1 µg of circular plasmid DNA (40 µg/ml final concentration), 20 mM potassium L-glutamate, 2.5 mM MgCl, 10 mM Hepes-KOH, pH 7.9, 2 mM ATP, 0.8 mM CTP, 0.8 mM 3`-O-methyl-GTP (3`-OMeGTP), 5 µM UTP, 0.26 µM [-P]UTP (20 µCi), 20 mM creatine phosphate, 0.48 mg/ml creatine kinase, 3% polyethylene glycol, 10 units of RNase T1 (Calbiochem), 1 mM dithiothreitol, and 10 µg/ml leupeptin. Unless otherwise stated, the reaction was conducted at 28° C for 90 min and stopped by the addition of 400 µl of HES buffer (5 mM Hepes-KOH, pH 7.9, 5 mM EDTA, 0.5% SDS, 0.3 M NaAc, 5 µg/ml tRNA). The RNA products were extracted with an equal volume of buffered phenol/chloroform (1:1), precipitated from the aqueous phase with ethanol, and analyzed on 6% polyacrylamide/50% urea gels. Following electrophoresis, the gels were dried and transcription signals were visualized by autoradiography and quantitated by phosphorimaging (Molecular Dynamics).

In transcription reactions without radioactive labeled UTP, the unlabeled UTP concentration was raised to 0.8 mM; in reactions without 3`-OMeGTP, GTP was added to a final concentration of 0.8 mM. For the time course experiments, the standard reaction was scaled up 10 times, and 25-µl aliquots were removed at specified time intervals.

Determination of the Transcription Start Site

A standard transcription reaction in the absence of radiolabeled nucleotides was carried out, and the RNA products were purified and analyzed by primer extension with radiolabeled oligonucleotide GLESS_PE. With the same oligonucleotide chain-terminating cycle, sequencing of the plasmid pTBU2_GLb was conducted using the Taq cycle sequencing kit (United States Biochemical Corp.) according to the manufacturer's protocol.


RESULTS AND DISCUSSION

The U2 G-less Cassette Gene

In the past, we repeatedly attempted to prepare cell-free T. brucei extracts active in transcription and capable of supporting correct transcription initiation of tRNA and 5 S rRNA genes. One of the major problems we encountered was the abundance of nonspecific labeling activities in our extracts which made it impossible to detect specific transcription signals. As an alternative strategy, we decided to apply the G-less cassette approach (11) to the RNA polymerase III-transcribed U2 snRNA gene, since this approach is very efficient in removing nonspecifically labeled RNAs and has been previously employed to establish an in vitro transcription system for the human U1 snRNA gene(11) . A G-less cassette gene lacks cytosine residues in the transcribed strand and thus gives rise to guanosine-less (G-less) RNAs. Transcription reactions are supplemented with the RNA chain-terminating nucleotide 3`-OMeGTP to suppress nonspecific transcription either from cryptic promoters in the template DNA or from genomic DNA contamination present in the extract and with RNase T1 to further degrade nonspecific transcripts and to trim RNAs generated by readthrough transcription (see Fig. 2for a more detailed description). Since the first 24 nucleotides of the U2 snRNA coding region are essential for in vivo expression(7) , we first determined whether the three guanosine residues in this element could be replaced without affecting transcription efficiency. We therefore randomly substituted the guanosine residues at position +11, +12, and +20 relative to the transcription start site with adenosine, thymidine, and cytidine residues and analyzed the effect of these mutations in vivo by transient transfection of trypanosome cells. As shown in Fig. 1, this analysis revealed that the six different combinations of G replacements tested did not significantly reduce the transcription efficiency of the U2 snRNA gene.


Figure 1: In vivo expression of mutant U2 genes containing G replacements in their intragenic control region. A, schematic outline of the tagged U2 snRNA gene. The upstream A and B box promoter elements and their orientation are indicated(7) . The mutated guanosine residues in the sequence of the intragenic control region (ICR) are printed in bold and marked by arrows (+1 marks the transcription start site). B, wild-type (WT) and mutated U2 genes were transiently transfected into T. brucei cells, and total RNA was analyzed by primer extension with oligonucleotide U2k which is complementary to the 3` end of U2 snRNA. The letters above each lane indicate the G replacements within the intragenic control region, i.e. TCA stands for the mutant in which the guanosine residues at positions +11, +12, and +20 are replaced by a thymidine, a cytidine, and an adenosine residue, respectively (- marks a deletion).



This result made it possible to construct the G-less U2 snRNA gene schematically outlined in Fig. 2A. In the U2 snRNA gene, all Gs were replaced by As from position +1 to +74 of the RNA sequence. In addition, to prevent possible premature termination by RNA polymerase III, we disrupted stretches of three Ts by one T to C transition. To the 3` end of the coding region we attached a gene-specific tag which included five 3`-terminal thymidine residues for efficient transcription termination by RNA polymerase III. Finally, the guanosine residues immediately upstream of the transcription start site up to position -9 were replaced by adenosine residues, since we have previously shown that these sequences are not essential for U2 snRNA gene expression in vivo(7) . These substitutions will allow us to distinguish between correct initiation at position +1, which should give rise to a transcript of 97 nt, and nonspecific readthrough transcription, which instead will produce a 106-nt-long transcript (Fig. 2A). The final U2 G-less cassette gene was then tested in vivo by transient DNA transfection. Northern blot analysis revealed that the transcript is indeed 97 nt long (Fig. 2C, lane U2_GLa). In the process of cloning the U2 G-less cassette, we isolated a second construct, TbU2_GLb, which differed only at the very 3` end (Fig. 2B) and produced an approximately 8-nt longer transcript (Fig. 2C, lane 3), most likely terminating at a stretch of Ts at positions +157 to +160 of the U2 snRNA gene(16) . Primer extension with an oligonucleotide complementary to the tag resulted in a product whose size is consistent with transcription initiation at position +1 (Fig. 2D). Thus, both U2 G-less cassette genes are expressed in vivo and initiate at the correct site.

A Cell-free Extract Active in RNA Polymerase III Transcription

Based on our experience with permeabilized trypanosome cells and on our previous attempts to prepare transcriptionally active extracts(17) ,()we introduced several modifications to the protocol of Dignam et al.(12) . Most notably, we replaced glycerol in all our buffers by sucrose. Extracts containing glycerol were not able to maintain a stable ATP pool even in the presence of an ATP regenerating machinery. Next, we broke trypanosome cells by douncing in an isotonic solution. Douncing of T. brucei cells in hypotonic conditions results in substantial leakage of nuclear contents into the cytoplasmic fraction (16, data not shown). Finally, potassium chloride was substituted with potassium glutamate in all buffers, because transcription in permeabilized cells is not inhibited by a wide range of salt concentrations when the transcription buffer contained potassium glutamate (data not shown, see also (18) ).

After varying extract preparation procedures, testing different extract fractions, and trying out different reaction conditions, we eventually were able to detect a weak signal of the correct size. In order to improve the transcription efficiency, we varied a number of components in the incubation mixture. Most dramatically was the addition of polyethylene glycol, a compound which reduces the reaction volume and, therefore, can increase transcription efficiencies. We found that polyethylene glycol at a final concentration of 3% resulted in an approximately 20-fold increase of the specific transcription signal (data not shown). In vitro transcription was sensitive to small changes of the magnesium chloride concentration, and maximal synthesis occurred at 2.5 mM MgCl (Fig. 3). Changing the potassium glutamate concentration from 10 to 75 mM did not significantly affect transcription of the U2 G-less cassette gene, whereas a further increase to 100 mM reduced the signal by 70%. However, in agreement with data obtained with the permeabilized cell system(17) , KCl concentrations above 30 mM reduced the transcription signal by more than 80% (data not shown). Specific transcription was dependent upon addition of exogenous DNA, and the strongest signal was obtained at a concentration of 40 µg/ml (data not shown). Finally, we determined that adding 5 µM unlabeled UTP enhanced the transcription signal by about one-third (data not shown). Taken together, these optimizations increased the transcription reaction efficiency more than a 100-fold.


Figure 3: Magnesium chloride titration of the in vitro transcription reaction. A, in vitro transcription reactions were carried out in the presence of 1.0, 2.0, 2.5, 3.0, or 4.0 mM MgCl, and the RNA was analyzed on a 6% polyacrylamide-50% urea gel. The appearance of a second band just above the specific product is most likely due to variable 3` ends of the U2 G-less cassette RNA since analysis of the 5` end by primer extension revealed only one signal of the correct size. M, MspI-digested pBR322 marker. B, histogram showing the relative strengths of the specific transcription signals. The strongest signal at 2.5 mM MgCl was set to 100%.



Fig. 4shows an array of reaction conditions that illustrate the effects of the addition of template, 3`-OMeGTP, and RNase T1. The control reaction lacking all three components gives rise to bands superimposed on a smear on the autoradiograph (lane 1). The generation of these RNA molecules may be due to end-labeling of RNAs or to endogenous transcription from chromatin present in the extract. Although the effect of template addition is hard to detect because of the high background, a faint band of 105 nt, which is not present in the control reaction, can be seen when TbU2_GLb was added (lane 2). RNase T1 removes the labeling background by degrading all but the G-less cassette RNAs (lane 3). The two remaining bands correspond to correctly initiated (97 nt) and readthrough transcripts (106 nt). The further addition of the chain terminator 3`-OMeGTP blocked nonspecific readthrough transcription and enhanced the specific product (lane 4). Finally, high magnesium concentrations prevent correct transcription initiation and enhance nonspecific readthrough transcription, so that the 106-nt product becomes detectable even in the presence of 3`-OMeGTP (lane 5).


Figure 4: Effects of template DNA, RNase T1, 3`-O-methyl-GTP, and MgCl on the in vitro transcription reaction. In vitro transcription reactions were carried out in the presence of -P-labeled UTP, and RNA products were analyzed on a 6% polyacrylamide-50% urea gel. DNA template, RNase T1, 3`-O-methyl-GTP, and MgCl were added as indicated above the lanes. M, MspI-digested pBR322.



Accurate Transcription Initiation

To determine the transcription start site, the in vitro transcription reaction was conducted in the absence of labeled UTP, and total RNA was subjected to primer extension analysis with an oligonucleotide complementary to the U2 G-less cassette tag. As shown in Fig. 5, this analysis gave rise to one major band corresponding to correct initiation at position +1(16) .


Figure 5: The 5` end of the U2 G-less cassette RNA coincides with the mapped initiation site of the U2 snRNA gene. In vitro transcription reactions were carried out in the absence of radiolabeled nucleotides, and RNA was analyzed by primer extension with 5`-end-labeled oligonucleotide GLESS_PE (lane pTbU2_GL). In a control reaction, the vector without the U2 gene was used as template (lane vector). For comparison, the U2 G-less cassette gene was sequenced by chain-terminating cycle sequencing with the same end-labeled oligonucleotide (lanes marked pcr seq.). On the right side, the double-stranded sequence surrounding the U2 transcription start site is shown. The sequence on the left is from the transcribed strand and corresponds to the sequencing ladder. The adenosine residue at position +1 (nontranscribed strand) is aligned with the sequencing ladder as indicated by the arrow. Marker, MspI-digested pBR322.



Kinetic of RNA Synthesis

Fig. 6shows a time course of the in vitro transcription reaction. There is a distinct lag phase of about 10 min after which a faint signal can be detected on a long exposure. The lag phase most likely reflects the time needed to assemble transcription initiation complexes. A linear increase then occurs between 20 and 90 min, when a plateau is reached.


Figure 6: Kinetic analysis of U2 G-less cassette gene transcription in vitro. A, after incubation times of 0, 5, 10, 20, 30, 40, 60, 90, and 120 min, aliquots were taken from a 250-µl in vitro transcription reaction, and the RNA was analyzed on a 6% polyacrylamide-50% urea gel. B, quantitation of the transcription signal. The strongest signal at 90 min was set to 100%.



RNA Polymerase III Transcription

Studies with permeabilized T. brucei cells showed that a concentration of 2 µg/ml -amanitin had only a moderate effect on transcription of the U2 snRNA gene by RNA polymerase III, whereas 100 µg/ml -amanitin inhibited U2 snRNA synthesis by more than 90%(7) . In the in vitro system, -amanitin concentration of 2 µg/ml did not significantly affect transcription of the U2 G-less cassette gene (an average decrease of 13%), but a concentration of 100 µg/ml reduced transcription to undetectable levels (Fig. 7, lanes 2-4). To verify this result, we used the RNA polymerase III-specific inhibitor tagetitoxin(19) . Similar to what was observed in permeabilized cells(7) , tagetitoxin concentrations of 15 µM and 60 µM inhibited in vitro transcription of the U2 snRNA gene by 52% and 75%, respectively (Fig. 7, lanes 5-7). Hence, we conclude that in vitro transcription of the U2 G-less cassette gene is mediated by RNA polymerase III.


Figure 7: In vitro transcription of the U2 G-less cassette gene is mediated by RNA polymerase III and depends on the upstream B box promoter element. A, in vitro transcription reactions with radiolabeled UTP were carried out in the presence of 0, 2, or 100 µg/ml -amanitin (lanes 2-4). In a second set of reactions, the RNA polymerase III-specific inhibitor tagetitoxin was added to final concentrations of 0, 15, and 60 µM (lanes 5-7). Lane 1, the vector without U2 gene sequences served as template. Lane 8, the U2 G-less cassette template was replaced by a mutant which carried six point mutations in the upstream B box promoter element. The RNAs were analyzed on a 6% polyacrylamide-50% urea gel. B, quantitation results of the transcription signals from three independent experiments with -amanitin. In each case, the signal of the control reaction was set to 100%. C, quantitation results of two experiments with tagetitoxin.



Effect of B Box Mutations

To further test the validity of the in vitro system, we examined the effect of mutations in a known regulatory element of the U2 snRNA gene. Similar to what was observed in vivo(7) , six point mutations within the upstream B box promoter element completely abolished U2 transcription in vitro (Fig. 7, lane 8), further indicating the close resemblance of the in vitro and in vivo systems.


CONCLUSIONS

In this study we report the development of the first in vitro transcription system for the protozoan family Trypanosomatidae. This goal was achieved by adapting the G-less cassette approach to the RNA polymerase III-transcribed U2 snRNA gene of T. brucei. With this in vitro transcription system we are now in a position to identify and characterize transcription factors essential for U2 snRNA gene transcription.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI28798 (to E. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by an EMBO long term fellowship.

To whom correspondence and reprint requests should be addressed. Tel.: 203-785-7332; Fax: 203-785-3864.

The abbreviations used are: snRNA, small nuclear RNA; nt, nucleotide(s); 3`-OMeGTP, 3`-O-methyl-GTP.

V. Nakaar, A. Günzl, E. Ullu, and C. Tschudi, manuscript in preparation.

A. Günzl, C. Tschudi, and E. Ullu, unpublished data.


ACKNOWLEDGEMENTS

We thank Tim Nilsen for valuable suggestions and Philippe Male for photography.


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