Nucleic Acids Research

Volume 1 1 Number 4 1983 Nucleic Acids Research

Isolation and nudeotide sequence of a plant tRNA gene: petunia asparagine tRNA

Nurit Bawnik, Jacques S.Beckmann*, Sara Sarid + and Violet Daniel

Departments of Biochemistry and + Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel, and *Institute of Field and Garden Crops, The Volcani Center, Bet Dagan 50200, Israel

Received 29 October 1982; Revised and Accepted 18 January 1983

ABSTRACT A 14.3 kb petunia genomic DNA fragment was isolated and found to contain a single tRNA gene coding for asparagine tRNA. The nucleotide sequence of the asparagine tRNA gene and its flanking regions has been determined. This gene does not contain intervening sequences nor the 3'-end CCA sequence of the mature tRNA and presents a similar overall sequence homology (70%) to both E. coli and mammalian asparagine tRNA. As in other eukaryotic tRNA genes the 5'-flanking region does not seem to contain any special sequence that could function as a regulatory element and the 3'-end is followed by a short cluster of T that may function as the transcription termination site.

INTRODUCTION In contrast to the large amount of information that has been accumulated on the arrangement and structure of tRNA genes from a variety of eukaryotes such as yeast, Neurospora, Bombyx, Xenopus, Drosophila, rat and man (1-13), nothing is known about plant nuclear tRNA genes. To study genes for tRNA in plant cells, we have constructed a petunia genomic library and screened it with an unfractionated cytoplasmic petunia tRNA probe. Here we describe the detailed analysis of a tRNA gene-containing clone and the nucleotide se- Asn quence of a DNA fragment carrying a tRNAMKC(U) gene.

MATERIALS AND METHODS Restriction enzymes were obtained from Biolabs, New England, T4 RNA ligase and T4 DNA ligase were from PL Biochemicals, la32PI nucleoside triphosphates and cytidine 3'-5'- 15'- IP bisphosphate (pCp) were from Amersham. Petunia Genome Library. Petunia nuclear DNA was partially digested with Eco RI and %2Okb DNA fragments were isolated by sedimentation through a 10-40% linear sucrose gradient as described by Maniatis et al. (14). The 20 kb petunia DNA fragments were ligated with a 2 fold molar excess of XCharon 4A purified arms (12 and 19 kb) using T4 DNA ligase (14) and the recombinant DNA was packaged in vitro into phage particles following the method of Hohn

© I R L Press Umited, Oxford, England. 1117 Nucleic Acids Research S~~~~~~~~~~~~~~~~~~~~~~~~~ and Murray (15). Approximately 105 independent clones were obtained. The library was amplified about 105 fold by low density plating on E. coli DP50supF (14). Petunia tRNA clones. The petunia genomic library was screened by the method of Benton and Davis (16) using as probe an unfractionated mixture of petunia cytoplasmic tRNAs labeled at the 3'- end with 15'-32P pCp. About 50 tRNA- hybridization positive clones were obtained for a petunia library of 10 clones. The final isolation of tRNA gene-containing clones was carried out by plaque purification (16). End labeling of tRNA. For 3' end labeling, unfractionated cytoplasmic petunia tRNA was first incubated for 120 min at 370 in 200 mM Tris-HC1 pH 8.3, 200 mM KCl and 10 mM MgCl2 to remove any 3'-end bound amino acid, then ethanol pre- cipitated. 15'-32 P pCp was then ligated to the tRNA using T4 RNA ligase following the procedure of England et al. (17). The 13' _32P - tRNA prepara- tion was further purified by electrophoresis on 10% acrylamide gel containing 7 M urea followed by elution with 500 mM NaCl, 100 mM Tris-HCl pH 7.4. DNA sequence analysis. 5' and 3'-end labeling of DNA fragments and sequence analysis were performed according to the procedures of Maxam and Gilbert (18). In vitro transcription. Transcription of petunia tRNA gene was carried out in Xenopus germinal vesicle extracts as described by Schmidt et al. (19). 0.5 1 g plasmid DNA were transcribed with 20 p1 of Xenopus germinal vesicle extracts (diluted 1:1 by volume with buffer containing 70 mM KC1, 7 mM MgCl2, 0.1 mM EDTA, 10 mM Tris-HCl pH 7.4, 2.5 mM dithiothreitol) by incubation at 23-240C for 90 min with IG-32PIUTP (0.01 mM, 400 Ci/mmole) and 0.1 mM each of ATP, GTP and CTP. The synthesized RNA was extracted and fractionated by two-dimensional gel electrophoresis in 10% acrylamide - 7 M urea followed by a 20% acrylmide - 7 M urea gel (20).

RESULTS AND DISCUSSION Isolation of tRNA gene-clones from a petunia genom-ic library A petunia genomic library was screened for tRNA gene-containing clones by the Benton and Davis plaque hybridization method (16), using as probe an unfractionated mixture of petunia cytoplasmic tRNAs labeled at the 3'-end with 15'-15321'pp.Te32PlpCp. The P-labeled tRNA probe was previously purified by electrophoresis on 10% polyacrylamide gel and the hybridization was performed in the presence of a large excess of tRNA-free petunia ribosomal RNA. Working with a collection of 105 phage recombinants we have detected 50 clones carrying tRNA genes. A clone, XCh4-pNl, containing 14.3 kb of petunia

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Figure 1. Restriction endonuclease diges- tion of recombinant DNA from bacteriophage XCh4-pNI. DNA digests with Eco RI (A) and Hind III (B) were electrophoresed through 1% agarose slab gels and stained with ethi- dium bromide. In parallel with fluores- cence of DNA fragments is presented an auto- radiogram of a Southern blot of the same gel hybridized with 3'-32P-labeled, unfractionated petunia tRNA. An Eco RI digest of XDNA is presented in the separate lane at right.

DNA was isolated, purified and recombinant DNA analyzed by restriction with Eco RI and Hind III endonucleases. Fig. 1 shows the pattern of restriction and Southern blots obtained by transfer of the DNA fragments to nitrocellu- lose and hybridization with I3I_32PI-labeled tRNA. The tRNA genes appear to be located within a Hind III fragment of 1.0 kb. This fragment was in- serted into the Hind III site of plasmid pBR322 and the recombinant plasmid used to transform_E. coli HB101. The recombinant plasmid pBR322-petunia DNA (pN1) was subsequently used for the study of petunia tRNA gene sequence and transcription. Restriction enzyme mapping and nucleotide sequence The subcloned 1.0 kb petunia DNA fragment was cleaved with several restriction endonucleases, thereby reducing the size of tRNA gene-containing region to a 200 bp fragment (Alu I-Bam HI). The DNA sequence presented in Fig. 2 shows that the 1.0 kb Hind III DNA fragment contains a tRNA gene Asn Asnn coding for tRNAA MC(U)C Since the 200 bp fragment containing the tRNA is the only fragment hybridizing with ~~~32P-labeled tRNA, we conclude that the 14.3 kb petunia DNA insert of clone ACh4-pNl probably contains a single tRNA gene. The 5'-flanking region (shown in Fig. 2) does not present any sequence that could be identified as a regulatory element for transcription. The 3'- flanking region contains a sequence TTTTT in the nontranscribed strand, one nucleotide from the coding region, followed by the sequence CTTTTC, 16 nucleo-

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2 .~ _

I a- cEx = IM < m I m *f I, I , zL±4 4,,1. I , 100 300 500 700 900

-50 -40 -30 -20 -10 -1 ATAGGTTGGCCAAGAACGAGAGTCGACTTCCrAATTAAGTCCGTTGT

1 10 20 30 40 50 LCCTCAGTAGCTCAGTGGTAGAGCGGTCGGCTGTrAACCGATTGGTCGTA

60 70 +1 10 +20 GGTTCGAAT~CrAcTTGGGGAGTTrGAGTFATCGC=TCTGACCTA

+30 +40 +50 +60 .70 GCGCGACCCCTGTCCTTCTTCTAGl-lCTAAACTAGCAGAATCGTGGACA

+80 +90 +100 110 +120 TCMAAGCGGTAGITGATTGTrGGITIATTCCTCACTCTCGTATAGG

Figure 2. Restriction map of the 1.0 kb Hind III fragment of petunia DNA from XCh4-pN, and the nucleotide sequence of the tRNA-gene containing region. The non-coding strand is shown and the asparagine tRNA corresponding sequence is underlined. tides downstream. These short T clusters must be the sites for transcription termination since all RNA polymerase III transcripts that have been reported (21,22) terminate within 4 or more consecutive T residues in the nontranscribed strand. The DNA sequence of the tRNAAsn gene can be arranged in a clover leaf secondary structure as shown in Fig. 3. The sequence shows that this gene does not contain an intervening DNA sequence nor the sequence corresponding to the 3'-end CCA found in mature tRNAs. A comparison of the nucleotide sequence of petunia tRNAAsn gene with the sequences (23) of E. coli and mammalian tRNAAsn reveals a 70% homology with each. It should be observed that there is a 60% homology between tRNAAsn of prokaryote and eukaryote origin (24). There is an identity of Asn sequences among the mammalian tRNA (human, bovine, rat) (24) and a near Asn identity of those with the sequence of a Drosophila tRNA gene (9). This indicates a conservation of primary structure of cytoplasmic tRNAs during the evolution of these eukaryotes of the animal kingdom. In vitro transcription of tRNAAsn gene Asn The RNA sequence of tRNA , as that of most tRNA species in plants, is Asn unknown and it cannot presently be determined if the tRNA gene described

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G TA CG CG TG CG AT GT T A A T G A AT C A TCC G CT C G GT AG G C G GAG C T GT A GT T G T-A G C G G C G C C A T A G T T

Figure 3. Petunia tRNAAc(U) gene in clover leaf arrangement. is expressed in the plant cell. In order to test whether petunia tRNA sn gene is recognized by RNA polymerase III and transcription factors, we have transcribed the pNl plasmid DNA in a nuclear extract of Xenopus oocytes with labeled nucleoside triphosphates and separated the products by two- dimensional acrylamide gel electrophoresis. Fig. 4 shows the synthesis of a main tRNA-size molecule (spot 5) and several minor RNA species (spots 1-4) which probably represent precursor intermediates. These minor spots indeed disappear upon an additional incubation of 2 hrs in the presence of unlabeled nucleoside triphosphates (data not shown). The in vitro synthesized tRNA-size

Figure 4. Two-dimensional polyacrylamide gel electrophoresis of in vitro transcription products of pN1 DNA.

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molecule (spot 5) was extracted from the gel and used as a hybridization probe with the restriction fragments of the 1 kb petunia DNA insert of pNl plasmid (Fig. 2). It was found to hybridize exclusively with the 200 bp Alu I-Bam HI fragment proving unequivocally the origin of the transcript. Although the petunia tRNA 8 gene transcription in a heterologous Xenopus system is not a definitive proof of its expression in the plant cell, the primary DNA sequence and secondary structure derived from it seem to contain all the characteristics required for a tRNA gene to be expressed. This paper is the first description of the fine structure of a tRNA gene from a plant nuclear genome. The features of the structure of this plant tRNA gene indicate that the coding sequence is as close to that of other eukaryotes of animal origin as it is to that of bacteria while the flanking regions seem to be essentially the same as those found in other eukaryotes.

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