Plant Molecular Biology6: 1-12, 1986 © 1986 Martinus NijhoffPublishers, Dordrecht - Printed in the Netherlands

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA Daniel R. Shapiro & Krishna K. Tewari

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717, U.S.A.

Keywords: chloroplasts, gene localization, pea, sequences, tRNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNAVaI(GAC), tRNAASn(GUU), tRNAArg(ACG), tRNALeu(CAA), tRNATyr(GUA), tRNAGIu(uuc), tRNAHis(GUG), and tRNAArg(ucu) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition, two other tRNA genes, tRNA Gly (UCC) and tRNAne(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90-100°70 homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5' flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNAGIy(ucc), and the t R N A ° u ( u u c ) contains GATTC in its T-loop.

Introduction Chloroplast (ct) DNA from higher plants codes for its own tRNAs that are unique to organelles. Detailed studies on the hybridization of individually labelled aminoacyl tRNAs by Meeker and Tewari (28) have shown that genes for at least 17 tRNAs are present in pea ctDNA. The tRNA genes for amino acids cysteine, glutamic acid, and glutamine were not detected, but probably from a lack of adequate charging of tRNAs rather than the absence of genes in the ctDNA. Similarly, corn has been shown to contain 20-26 tRNA genes (16). The presence of tRNAs in chloroplasts has also been shown by two dimensional gel electrophoresis of ct tRNAs followed by hybridization to ctDNA fragments. Using this technique, 35 tRNA spots were separated and 21 tRNA genes mapped in spinach (11), 33 spots separated and 37 tRNA genes mapped in corn (44), 38 spots separated and 31 tRNA genes mapped in bean (31), 51 spots separated and 34 genes mapped

in tobacco (1), and recently 33 spots separated and 31 tRNA genes mapped in wheat (30). Our recent studies on pea ct tRNA separated 36 tRNA spots (6). These tRNAs were eluted from the acrylamide gels and each hybridized to a series of recombinant pea ctDNA clones that had been found to contain tRNA genes by Southern hybridization. These experiments mapped 30 tRNA genes in pea chloroplast DNA. A detailed sequence analysis of tRNA genes in ctDNA have identified 8 tRNA genes in spinach (4, 18, 19, 20, 21, 52), 12 tRNA genes in corn ctDNA (24, 25, 42, 43, 47, 48), 18 tRNA genes in tobacco ctDNA (8, 9, 10, 22, 32, 33, 49, 50), 6 tRNA genes in broad bean ctDNA (2, 3, 26), 3 tRNA genes in Spirodela ctDNA (23), 8 tRNA genes in wheat ctDNA (39, 40), 7 tRNA genes in barley ctDNA (35, 53), and 1 in mustard ctDNA (38). Detailed studies on ctDNA of E. gracilis have identified 21 tRNA genes (17). In this paper, we describe the complete nucleotide sequences of eight pea ct tRNA genes and

the partial sequences of two other tRNA genes. Our data on pea ctDNA are compared with the data obtained from five other higher plants: tobacco, spinach, corn, broad bean, and Spirodela. If all the data from different plants, obtained by hybridizing the aminoacyl tRNAs to ctDNA and its restriction digests, separating the tRNAs in two dimensional gel electrophoresis and hybridizing the separated tRNAs to the restriction digests, and, finally, by sequencing the ctDNA fragments known to contain tRNA genes, are summarized, it is apparent that ctDNA from higher plants contains tRNA genes for practically all of the twenty amino acids necessary for the autonomous synthesis of proteins in chloroplasts.

Materials and methods

Isolation of nucleic acids Chloroplast DNA was obtained by phenol extraction of Triton X100 disrupted chloroplasts followed by purification in CsC1/EtBr density gradients as described before (7). Plasmid DNA was obtained from the bacterial lysates by CsC1/EtBr density gradient centrifugation (34). Isolation of tRNAs from chloroplasts was carried out essentially by the method of Chu et al. (6).

Endonuclease digestion of DNA Restriction endonucleases were obtained from commercial suppliers and used under the recommended optimal conditions. Electrophoresis of DNA fragments was carried out in 0.8% to 1.5°70 agarose gels. DNA fragments were electrophoretically eluted from the gel onto DEAE cellulose paper

(5). Hybridization All hybridizations were carried out in 40% formamide, 1 × SSC, 10 mM Hepes, pH 7.5, 0.5% SDS, and 4 × Denhardt's solution @ 37 °C for 12-16 hr

(5). DNA sequencing The chemical degradation method of Maxam &

Gilbert was used to sequence the DNA fragments (27). DNA fragments were labelled at the 5' end using T 4 polynucleotide kinase and 3' 32p ATE The labelled DNA fragments were either strandseparated on a 5% polyacrylamide strand gel or redigested with the appropriate restriction endonuclease and separated on a 5 % polyacrylamide fragment gel before chemical degradati6ns were carried out.

Results

We have reported the localization of tRNA genes in the restriction endonuclease map of pea ctDNA by hybridizing the 3 'end-labelled tRNAs with the appropriate restriction digests (6). These studies have identified eight different regions of ctDNA that contain tRNA genes. For the sake of clarity, the location of these regions is presented in Fig. 1 showing the recombinant DNA which contain tRNA genes. We will describe our results starting clockwise from the 5' flanking region of the 16S rRNA gene. The recombinant pCB2-8 encodes the 5' flanking region of the 16S rRNA gene, the 16S rRNA gene, and most of the spacer region between the 16S and 23S rRNA genes, pCB2-8 (Figs. 1, 2) was digested with restriction endonuclease XbaI, end-labelled with 7 32p ATP, followed by digestion with PstL This DNA mixture was separated in a 5% polyacrylamide gel, and the 1.7 kbp DNA fragment was eluted and sequenced. These sequences were analyzed for the presence of tRNA genes or possible tRNA structures both visually and by using the Staden DNA analysis program (46). Our criterion for possible tRNA genes is to identify the conserved GTTC sequence in the T-loop of the tRNAs and identify them by their anticodon sequence as well as by comparison with published ct tRNA sequences from other plants. Nucleotide sequence analysis has identified the presence of a tRNA vai(GAc) gene located 257 bases from the start of the 16S rRNA gene. The gene contains the usual GTTC sequence, although the preceding base is A instead of the usual G. This tRNA gene is transcribed from the same strand as the rRNA genes. A tRNA TM gene has also been found to be present at the 5' end of the 16S rRNA gene in spinach, tobacco, corn, and mustard ctDNA (4, 38, 43, 50). The pea tRNA TM coding region is 72 bases long and differs by two nucleotides

pCB 2 - 8

pCP 1 2 - 7

BpC s-7

pSH 10 Pea Chloroplast

DNA

Sal 1

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tRNAs

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p~CBgl I1-1

Fig. 1. Restriction map of pea ctDNA showing recombinants containing ,XRNA gengs. The relative number of tRNA genes found by hybridization experiments (8) is shown by the size of the arrows. 1 - 2 ( ~ ), 2 - 4 ( ~ ) , 4 - 6 ( t ) . A A-T G-C G-¢ G-C G-T T-A A-T ~ "1"--"

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tRNA" ° L |o 20 ~o 40 50 60 70 so 90 too t Io tzo L~GGG~TITAA CTCAGCGGTA OAGTGTCACC TTGACGTGGT GGAAGTCATC AGTTCGAACC TGATTATCCC T~ACCCAAT ATCAATTTTT CCATTTGAGG ACGAGATCCA ATCTGAGTAG TCCCCATATT GAGTCGCCAT CTCACAGTGG AACTGCACCA CCTTCAGTAG TCAAGCTTGG ACTAATAGGG ATTTGGGTTA TAGTTAAAAA GGTAAACTCC TGCTCTAGGT TAGACTCATC 130 1• o 1s o i 60 170 1~0 19o 200 210 22o 230 240 ATAMCAGGAT AGGGAGTTGA CACAAGOGGG COTAAO~CCA TATAMrATTT ATC,6GAC~CA ACTCC~GCC~ AArA~TAA~C CCATGOATAC AAarCAA~Tr AT~TCTTCTC AGTTCAaTMA T*TTCTCCTA TCCCTCAACT GTGTTCCCCC CCATTCCGGT ATATTATAAA TACCCTCCGT

250

260

270

2go

290

TGAGGCCCGC

300

3to

TTATCATTCG GGTACCTATG TTCAGTTCAA TACAGAAGAG TCAAGTCATT

320

330

16S 3QO

rRNA 350

3~o

CTGAAATCAA ATTTAAGTTC AGTAAATGAA ATCAAATTCC GAATCAGCTT TGTCTAGAAA CAAGGAAGCT ATAAGTAATG CA/~CTA~[~AA GCTCATGGAG AGTTTG&TCT GGCTCAGGAT GACTTTAGTT TAAATTCAAG TCATTTACTT TAGTTTAAGG CTTAGTCGAA AL'AGATCTTT GTTCCTTCGA TATTCATTAC GTTGATCCTT CGAGTACCTC TCA~CTAGA CCGAGTCCTA ~70 ~eO 390 *00 • tO ~20 ~30 ~0 ~50 ~60 *70 ~eO GAACGETGGC GGCATGCTTT ACACATGCAA GTCGGACGGG AAGTGCTGTT TCCAGTGGCG AACGGGTGAG TAACGCGTAf* GAACCTGCCC TTGGGAGGGG GACAACAGCT GGAAACGGC'r CTTGCGACCG CCGTACGAAA TGTGTACGTT CAGCCTGCCC TTCACCACAA AGGTCACCGC TTGCCCACTC ATTGCGCATT CTTCGACGGG AACCCTCCCC CTGTTGTCGA CCTTTGCCGA

• 90

500

5to

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GCTAATACCC CGT&GGCI"G& GGAGCGAAAG GAGGAATCCG CCC&ACC=¢;G GGCTCCCGTC TGATT CGATTATGGG GCATCCGACT CCTCGC'FTTC CTCCTTAGGC GGGTTCCTCC CCGAGCGCAG ACTAA

Fig. 2. Sequencing strategy of the D N A fragment from the p C B 2 - 8 clone and the tRNA gene sequences. The location of the tRNA w'tGAc) gene (left) and the 5' end of the 16S rRNA gene (right) is shown by the bold lines on the restriction map. Their gene sequences are boxed in the D N A sequence printout. The cloverleaf structure of the tRNA is shown as deduced from the D N A sequence.

from the tRNA TM in mustard, tobacco, and corn. The tRNA TM between pea and spinach differs by only one nucleotide. Our recent studies (6) and other's previously (51) have shown the presence of two tRNA genes in the spacer region between the 16S and 23S rRNA genes. The EcoRI site is located at 170 nucleotides prior to the 3' end of the 16S rRNA, and, therefore, this site and the BamHI site 3' downstream were used to end-label the DNA. The labelled DNA was strand-separated and sequenced. Analysis of the nucleotide sequences identified half of a tRNA Ile(GgU) gene starting 225 nucleotides away

c

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from the BamHI site. We have not completely sequenced thespacer region; but our analysis of the available data on tRNA ne shows 100% homology with the equivalent genes in corn, spinach, and tobacco (24, 49). Hybridization with 3' end-labelled tRNAs and restriction digests of the recombinant pCP12-7 had shown that tRNA genes were localized immediately following the 5S rRNA gene. Further mapping of the tRNA genes indicated that a BgllI DNA fragment of 670 bases contained tRNA genes (Figs. 1, 3). This DNA fragment was isolated, labelled with 32p at the 5' ends, strand-separated, and then se-

. _

TT,m

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lOObp tRNA*'m to 20 30 ~0 50 ~0 70 60 90 tOO 110 t20 GATCTAGTGG AGACAGGGTG ~GGCTCGTAG CTCAGAGGAT TAGAGCAt'aC GGCTACGAAC CACGGTGTCG GGGGTTCGAA TCCCTCCTCG CCCAICAACCG GCTAAAAGGG GGAAGGACCT CTAGATCACC TCTGTCCCAC CCCGAGCATC GAGTCTCCTA ATCTCGTGCG CCGATGCTTG GTGCCACAGC CCCCAAGCTT AGGGAGGAGC GGGTGTTGGC CGATTTTCCC CCTTCCTGG*

t30

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CGGGGATA*G AAAATAAACT ATGATTGAGG TTGAAACAAA ATAGTCTTAC ATTTTTTATT TCTTATAAAT ACTATATTTA TATATAGTAT AAATAATAGA ATAGCCCCAG ACCGTAGAAC GCCCCTATTC TTTTATTTGA TACTAACTCC AACTTTGTTT TATCAGAATG TAAAAAATAA AGAATATTTA TGATATAAAT ATATATCATA TTTATTATCT TATCGGGGTC TGGCATCTTG

250 260 2;'O 280 2~0 300 310 320 330 aaO a50 360 AAAGGAATAT CAATAATCTA CACATAGGTA TTGACCGGGA GTCCCCAATA TAGTAGAATA GTATaCGATG AGATAGAATG TAATGAAACA AACTTTACAC TTCCTTTTGA TTCCAAATTC TTTCCTTATA GTTATTAGAT GTGTATCCAT AACTGGCCCT CAGGGGTTAT ATCATCTTAT CATATGCTAC TCTATCTTAC ATTACTTTGT TTGAAATGTG AAGGAAAACT AAGGTTTAAG tRNA A'" 410 370 300 3~0 400 4ZO ~ao 44o ~5o ~6o 47o aao TTTTAATTGG AAAATGAATA AAATCTCCCC AAATAGGATT TGAACCTACG ACCAATCGGT TAACAGCCGA CCGCTCTACC ACTGAGCTAC TGGGGAACAA CGGCAAATTC TATCCCATCT AAAA'rTAACC TTTTACTTAT TTTAItIAGGGG TTTATCCTAA ACTTGGATGC TGGTTAGCCA ATTGTCGGCT GGCGAGATGG TGACTCGATG ACCCCITrGTT GCCGTTTAAG ATAGGGTAGA

,+gO 500 510 520 530 5~0 550 560 570 ~qO 590 600 ATATTATCAT A T A A ~ A G A T ATCTAATAA T ~T~TTATACT AT~TATTTGT AATATATTCC ~TAAGATAGA TATCATATTC AT~GAATATG ATTGATI~CCT TGGTGGTGAA ATGGTGGTAG TATAATAG'rA TATTATTCTA TAGATTAT'TA TAGAATATGA TATATAAACA TTATATAAGG TAT~CTATCT ATAGTATAA'~ TATCTTATAC TAACTACGGA ACCACCACTT TACCACCATC tRNA L *. ~so 560 670 610 620 630 640 ACACGCGAGA CTCAAAATCT CGTGCTAAAC AGCGAGGAGG TTCGAGTCCT CTTCAAGGCA TAATATTGAG ATC TGTGCGCTCT GAGTTTTAGA GCACGATTTG TCGCTCCTCC AAGCTCAGGA GAAGTTCCGT ATTATAACTC TAG

A G-C G-C G-C C-G TC CT G-C T A G A

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AG C

tRNA L''

Fig. 3. Sequencing strategy of the D N A fragment from the p C B 1 2 - 7 clone and the t R N A gene sequences. The t R N A genes are marked by bold lines on the restriction map and their sequences are boxed in the D N A sequence printout. The cloverleaf structures of tgNAArgtAC% t R N A AsntGtm), and t R N A t+u(cAA) are shown as deduced from the D N A sequences. The palindrome between t R N A As" and tl~NA t~u is underlined.

tRNA Arg(ACG) gene differs from Spirodela tRNA Arg(ACG) by 3 bases (33). The tRNA Leu(CAA), deduced from the gene sequence, is 84 bases long and is 96'070 homologous to the corn (48), 95070 homologous to the soybean (37), and 96°7o to the bean tRNAs (36). Similarly, tRNA gsn(GUU), deduced from the gene sequence, is 95.°7o homologous to the Spirodela tRNA (23) and 96°7o homologous to the tobacco tRNA (22). Since the sequencing

quenced. The nucleotide sequences were analyzed both by visual observation and by computer programs. This DNA fragment is found to contain genes for tRNA Arg(AcG), tRNA Asn(Guu), and tRNA Leu(cAA). The tRNA Arg gene coding region is 74 bases long and is separated by 290 bases from the tRNA Asn gene. The tRNA Ash gene coding region is 72 bases long and is separated by 118 bases from the tRNA Leu gene. The coding region of the B

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100bp 10 20 30 ~0 50 60 70 ao 90 *00 Ii0 120 GATCTATTTT TTGTACCAAC TCATTAATGA ACAAAAAATT GGATTGACGA GTATAGTGAA AAAAGAATTT ATTCATATTG GATTTGATAA ATATCACTGG AATTCAATTT TTCAAAACCG CTAGATAAAA AACATGGTTG AGTAATTACT TGTTTTTTAA CCTAACTGCT CATATCACTT TTTTCTTAAA TAAGTATAAC CTAAACTATT TATAGTGACC TTAAGTTAAA AAGTTTTGGC

L3O t4o 15o 160 170 160 190 200 210 220 230 240 ATTCGAATTG AAGTCATCTT CATCATAACA CGAAATTAAT TTCATTGATA CATGTACCCC TAATTCAAAT ATTTCTGAAT CTTTGATAAA TGGATTCTAT CCTGTCCCTC AACTGAATTC TAAGCTTAAC TTCAGTAGAA GTAGTATTGT GCTTTAATTA AAGTAACTAT GTACATGGGG ATTAAGTTTA TAAAGACTTA GAAACTATTT ACCTAAGATA GGACAGGGAG TTGACTTAAG 2S0 260 270 260 290 300 310 320 330 340 350 360 TCATCCTTTT TTACTCAATT T C C A G A T T C A T T C T C G T T T T TTTATTTCCC GACTTAGTGT GAAATAGAAA TATACCTAAT TCAATCTTCT TAACACTGAA TGAAAGTGAA A A T A A C T A G A AGTAGGAAAA AATGAGTTAA AGGTCTAAGT AAGAGCAAAA AAATAAAGGG CTGAATCACA CTTTATCTTT ATATGGATTA AGTTAGAAGA ATTGTGACTT ACTTTCACTT

TTATTGATCT

370 300 390 400 4tO *20 ~30 440 ~50 ~60 470 400 AGAAATAAGG TGTTAATGCC CCCCTGTTCC AGCAAATCAA TCATTTCCAG AAAGGATTCT ATGTTTATTT TGGTTTGTTT CGCATTACTT CGTTTTATTT TGTATTTTGT TG&ATTATAG TCTTTATTCC ACAATTACGG GGGGACAAGG TCGTTTAGTT AGTAAAGGTC T T T C C T A A G A TACAAATAAA ACCAAACAAA GCGTAATGAA GCAAAATAAA ACATAAAACA ACTTAATATC 490

500

510

520

530

540 550 560 570 560 590 600 AATCATTATA TTGACAATTT CAAAAAACTG AACATTCCTT AAGACTTTCT ACTCTTCTAG GAAGACTAGC TTAGTAATAT AACTGTTAAA GTTTTTTGAC

A T T G G T G A T T GGAATGAACA A T T T C G A A A T CGAAAGGATT AATCAAAAAA TTGT&AGGAA TTCTGAAAGA TGAGAAGATC CTTCTGATCG

TAACCACTAA CCTTACTTGT

TAAAGCTTTA GCTTTCCTAA TTAGTTTTTT

tRNAm~.

610 620 630 640 650 660 670 6so §SO 700 710 720 TTCATACTAT GAACATAGTA GAATGGAGGT CGGGGAAGGA 11GCCCCCATC GTCTAGCGGT TTAGGACATC TCTCTTTCAA GGAGGCAACG GGGATTCGAC TTCCCCTGGG G~T,~GGGTAC AAGTATGATA CTTGTATCAT CTTACCTCCA GCCCCTTCCT ACGGGGGTAG CAGATCGCCA AATCCTGTAG AGAGAAAGTT CCTCCGTTGC CCCTAAGCTG AAGGGGACCC CCATCCCATG '"

tRNA'

730 740 750 760 ??O 780 790 ~00 alO e20 ca0 a40 TACGAAAGGA AATGGATCAG GGATTATCAA TAAAGACTAA ATTGGATTCT TCC~IGGGTCG ATGCCCGAGC GGTTAATGGG GACGGACTGT AAATTCGTTG GCAATATGTC TACGCTGGTT ATGCTTTCCT TTACCTAGTC C C T A A T A G T T ATTTCTGATT TAACCTAAGA AGGACCCAGC TACGGGCTCC CCAATTACCC CTGCCTGACA TTTAAGCAAC CGTTATACAG ATGCGACCAA

8SO 860 870 eao a90 goo 9|u 920 ~30 940 950 CAAATCCAGC TCGGCCCA]~A AATATTTCTG GATCCACCAT GAAATGATAG AACCCATTTG GTGTTCTCTT AAAATCACCA ATGGGTTCGG ATACAGAAGA TTAGAATCTC GA GTTTAGGTCG AGCCGGGTTT TTATAAAGAC CTAGGTGGTA CTTTACTATC TTGGGTAAAC CACAAGAGAA TTTTAGTGGT TACCCAAGCC TATGTCTTCT AATCTTAGAG CT

A G-T C-G C-G C-G C-G C-G A-T T C

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Fig. 4. Sequencing strategy o f the D N A fragment from the recombinant p C P 5 - 7 and the t R N A gene sequences. The t R N A genes are indicated by bold lines on the restriction map and by boxed lines in the D N A sequence printout. The cloverleaf structures of t R N A vyr(cUA)and t R N A G]"tuUc) are shown as deduced from the D N A sequences.

was carried out using a BglIID N A fragment, we are not able to orient the transcription of these genes in relation to the rRNA genes. The data do show that tRNA Ash is transcribed from the opposite strand in relation to tRNA Leu and tRNA Arg. However, in other higher plants the order has been established that the tRNA Arg gene is closest to and is on the same strand as the rRNA genes. Since the tRNA Leu(cAA) and tRNA Asn(°Uu) genes are oriented on opposite strands with their 5' flanks in between them, it is tempting to speculate that the region between the two genes contains sequences necessary for their transcription. It is interesting to note ,that the intergenic sequences between the tRNA Leu(cAA) and tRNA Asn(°uu) genes in pea ctDNA contain an eighty-six base palindrome (Fig. 3). The recombinant pCP5-7 has been found to contain tRNA gene sequences that are mostly confined to a BglII/XhoI D N A fragment of 950 bp (Figs. l, 4). This D N A fragment was sequenced by labelling at the 5' ends, followed by strand-separation. The

sequence analysis was also carried out by first restricting this D N A fragment with EcoRI, 5' endlabelling followed by strand-separation. Similar experiments were carried out by first digesting with HinfI. Two tRNA genes have been identified in this fragment: t R N A ~'r(oUA)and t R N A olu(uuc) (Fig. 4). i The two tRNAs are transcribedfrom the same strand as the r R N A genes starting from the t R N A G]u(UUc) gene. There are 60 nucleotides separating the two t R N A genes. It appears that these genes might be co-transcribed since,as in broad bean (26),there are no apparent termination sequences 3' to the t R N A Gmufuuc) gene, nor is there any initiation sequence apparent 5' to the t R N A "t~(GUA)gene. It has recently been demonstrated in tobacco that t R N A °u, t R N A ~:r,and t R N A Asp are cotranscribed (32). The t R N A °ufuuc), deduced from the gene sequence, is 73 nucleotides long and contains the sequence G A T T C in the T-loop. Spinach, tobacco, and broad bean t R N A GtufUUc) all also contain G A T T C in the T-loop (19, 26, 32). The t R N A °lutuUc) of pea

C G-C C-G. G-C G*T A-T T+G

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psbA 17~0 17~0 1740 1750 1760 1770 1760 1790 1800 z~O 17oo 17 o ~TGGTATGG~ AGTTATGCAT GAACGTAA~G CTCATAAYTT CCCTCTAGAC CTAGCTGC~ TTGAGGCTCC ~T~TATAA~T GGATAAITATT TTGGTTTAAA GATACGAATY TTTGTAAAGG GAGTATTAAA GGGAGATCTG GATCGACGCC AACTCCGAGG TAGATATTTA CCT&TT&TAA AACCAAATTT CTATGCTTAA AAACATTYC¢ AACCATACCT TCAAT&CGTA CTTGCATTAC 1840 taSO I060 ta?O laa0 189o 1900 1910 1920 18lo t820 te30 AGTAATATCA ACATTGTGGA TATTACTCCC TTACTTTTTG TTAGTATTCT TTTTCTGTAT ACATATACAG AAATAAATGT TTTCTTATTT TTTAATATTT TAGAAGAAAA GAATAATGAA TCATTATAGT TGTAACACCT ATAATGAGGG AATGAAAAAC AATCATAAGA AAAAGACATA TGTATATGTC TTTATTTACA AAAGAATAAA AAATTATAAA ATCTTCTTTT CTTATTACTT tRNA"'" 1960 1970 1980 1990 2000 2010 2020 2030 2040 1930 1~40 1950 AAGGTATAAA AAGTTATGTA ATTTAGACAT AGTTAGAGCE CGGATGTAGC CAAGTGGAT~ AAGGCA~TGG ATTGTGAATC CACCATGCGC GGGTTCAATT CCCGTCGTTC GCQICGCCCAT TTCCATATTT TTCAATACAT TAAATCTGTA TCAATCTCCC GCCTACATCG GTTCACCTAG TTCCGTCACC TAACACTTAG GTGGTACGCG CCCAAGTT&A GGGCAGCAAG CGGGCGGGTA 2080 2090 2100 21lO 2120 2130 2140 2150 2:80 2050 2060 2070 GAATCTCTTC AAATTCAAAC AAAAAAGAGA AAATAATTTA T~CTCCTGTT GCAGCTGCTA C~GCAGCTTT CGTGATTTAC CEGACGCTTT TGAGATGAGA CATTCATAAA CAACTCTACC CTTAGAGAAG TTTAAGTTTG TTTTTTCTCT TTTATTAAAT ATGAGGACAA CGTCGACGAT GCCGTCGAAA GCACTAAATG fiGCTGCGAAA ACTCTACTCT GTAAGTATTT GTTGAGATGG 2200 2210 2220 2230 22~0 2250 2280 2270 2280 2170 2180 2190 AT&ATTCTTA ~CGGATAACC CCAATTTTGG TTGAATACTA TAGTACAGTC GATTTTATTA TGTTTCATAA TTTTATTATA TAAATATAGA AAGAAATATA TAATAACAAA TTACTAAAAA TATTAAGAAT CGCCTATTGG GGTTAAAACC AACTTATGAT ATCATGTCAG CTAAAATAAT ACAAAGTATT AAAATAATAT ATTTAT~TCT TTCTTTATAT ATTATTGTTT AATGATTTTT 2320 2330 23~0 2350 2360 23?0 2330 . 2290 2300 2310 GATAAATACA AAAAAAAAAA TATACGAAGA AATTCGCCCC ACTCCCACAT ATTTGATAGC CTCICCTATA AAAAAACTGG AAATACCAAC TCCATTTGGA ATTC CTATTTATGT fTTTTTTTTT ATATGCTTCT TTAAGCGGGG TGA~GGTGTA TAAACTATCG GAGAGGATAT TTTTTTGACC TTTATGGTTG AGGTAAACCT TAAG

Fig. 5. Sequencing strategy of the D N A fragment from the recombinant pCBglII-1 and the t R N A gene sequences. The t R N A gene is marked by a bold line on the restriction map and is boxed in the D N A sequence printout. The sequences of the 3' end of the psbA gene are underlined. The cloverleaf structure of tRNAHis(tuG) is shown as deduced from the D N A sequence.

and broad bean are totally homologous and differ from spinach by only two nucleotides and from tobacco by three nucleotides. The tRNA Tyr(GOA), deduced from the gene sequence, is 82 bases long and contains the sequence GGTTC in the T-loop. The tRNA Tyr genes in pea, broad bean, tobacco, and spinach are completely homologous (19, 26, 32). While this manuscript was in preparation, Rasmussen et al. published the sequences of the pea ct tRNA Glu and tRNA Tyr genes, and our sequences confirm theirs (41). In addition, they found a tRNA Asp gene 454 bp 3' of the tRNA~r gene. Our earlier studies showed the presence of six tRNAs on the clone pCBglII-1, two in the 5' flanking region and four in the 3' flanking region of the psbA gene. The location and sequencing strategy of this fragment is shown in Figs. 1 and 5 along with the sequences. Unexpectedly, nacleotide sequence

analysis has revealed only one identifiable tRNA gene: tRNA His(GUG). The tRNA His(GUG) gene is 192 bases downstream from the stop codon of psbA and is transcribed from the same strand, The pea tRNA His(GUG), deduced from the gene sequence, is 100070 homologous to the corn (42) and tobacco (52) tRNA His(°UG) and 96°70 homologous to the spinach tRNA His(GUG)(52). It is 74 bases long and contains the GGTTC sequence in the T-loop. We have identified several possible tRNA genes which are atypical and do not resemble any previously known tRNAs. Further study is in process to determine if these are tRNA genes coding for active ct tRNAs, tRNA genes for IIe(AUA), IIe(AUU), and Lys(AAA) have not yet been identified in ctDNA and may be encoded in these atypical sequences. The recombinant pSHI0 was found to contain two tRNA genes from the dot hybridization experiA

G-C

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C-G G-C T-A

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AA T

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50

60

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~.CGA_C_TA_TAA.CCCTTAG~,~rTTCCAAGCTAA TGATGCGGGT TCGATTCCCG CTACCCGC'I~ ATATTCGATT TCTCTATATA TATTCTATTT TTTAGTAAAT TTC'rTTATTT AGTTATGAAT AGCTGATATT

GGGAATCGGA AGGTTCGATT AC'rACGCCCA AGCTAAGGGC GATGGGCGAA TATAAGCTAA AGAGATATAT ATAAGATAAA &AATCATT'rA AAGAAATAAA TCAATACTTA

zao 240 ! 30 140 150 180 170 ) so ! go 200 2 I0 220 ATTCAATTTG AATTATTTAG TCTTATTCTA GTTAGAATAA TTATTATTTC GATMTTCGGA TAGATAGAGT ATTTTATAGA ATTTTTTTTT TTCTTTCCCT TGAAATTTTC GTGAAAAGGG TAAGTTAAAC TTAATAAATC AGAATAAGAT CAATCTTATT AATAATAAAG CT&TAAGCCT ATCTATCTCA TAAAATATCT TAAA&AAAAA AAGAAAGGGA ACTTTAAAAG CACTTTTCCC tRNA* " * 350 36O 250 260 ;~70 280 290 300 3 LO 320 330 340 AAAGAAAAAA AGAAA~;~I~T C(~ATTGTCTA ATGGATAGGA CAGAGGTCTT CTA&ACCTTT GGTATAGGTT CAAATCCTAT TGGACGCIktA TTTATTACAT ATTGAATTTA TTGCATATAT TTTCTTTTTT TCTTTTCGCA GGTAACAGAT TACCTATCCT GTCTCCAGAA GATTTGGAAA CCATA'rCCAA GTTTAGGATA ACCTGCGTTT AAATAATG'rA TAACTTAAAT AACGTATATA

470 4eo 370 380 390 *00 410 420 • 30 440 *SO *60 ATTAC'rTGAA TTCGTATATT TCTAAAACTA TATTGTAAAT TTCTTATCTT GATTATTCTT ATTTAAATTT TAAATATTGA AACAGACTAA TTATCTAAAT TATAAAGGTA TGAAATTTGG TAA~'GAACTT AAGCATATAA AGATTTTGAT ATAACATTTA AAGAATAGAA CTAATAAGAA TAAATTTAAA ATTTATAACT TTGTCTGATT AATAGATTTA ATATTTCCAT ACTTTAAACC 590 60O 490 500 5 I0 S20 530 5*0 550 560 ST0 SBO AATCCATTTT TTTTCTACTT GTTCCTGAAG TCAAAAGAGT TCCATCTGTT CCTGAA'rAGc TTCCTTCAAA ATGGCTTCTG CTTCCCCAGT GAATGTC'rTG GTAGAAGATA TGATTTCGTT TTAGGTAAAA AAAAGATGAA CAAGGACTTC AGTTTTCTCA AGGTAGACAA GGACTTATCG AAGGAAGTTT TACCGAAGAC GAAGGGGI'CA CTTACAGAAC CATCTTCTAT ACTAAAGCAA 710 720 6 LO 620 630 640 650 e60 670 680 S90 700 GAATTTAGGT TTCTTCGTTT TTAAATAAGC CCGTAACTCA ACGATAAATT TCCTT&CCTG TGCAATTTCT ACTGAATCAA GATAAC'rAT'r CGTTCCAGTA TAAATAGTTA TTATCTGTTC CTTAAATCCA AAGAAGCAAA AATTTATTCG GGCATTGAGT TGCTATTTAA AGGAATGGAC ACGTTAAAGA TGACTTAGTT CTATTGATAA GCAAGGTCAT ATTTATCAAT AATAG&CAAG 730 7~0 750 760 770 780 790 ~o0 810 820 e30 TTCCACAGTA AGAGGAGCTG ATTGGGATTG TTTAAGCAAC TCGCGCAATC GTTGACCTCT TGCCAATTGA TTT'rGCGTAG CT'rTATCGAG ATCGGAAGAG AATTGTGCAA AACCT A&GGTGTCAT TCTCCTCGAC TAACCCTAAC AAATTCGTTG AGCGCGTTAG CAAC'rGGAGA ACGGTTAACT AAAACGCATC GAAATAGCTC TAGCCI"TCTC TTAACACGTT TTCGA

Fig. 6. Sequencing strategy of the recombinant pSH10 and the tRNA gene sequences. The tRNA genes are marked by bold lines on

the restriction map and are boxed in the DNA sequence printout. The cloverleaf structure of tRNAA'8(uc°)is shown as deduced from the DNA sequences.

ments. Sequencing of the tRNA region was carried out by digesting the recombinant with Sal! and Hind111, isolating the 835 bp DNA fragment, endlabelling the DNA fragment, and separating the strands (Figs. 1, 6). Further sequencing of the internal bases was carried out by 5' end-labelling o f EcoRI and Hinflsites. Nucleotide sequence analysis has identified tRNA Arg(ucU) and tRNA Gly(Ucc) genes in this clone. The two tRNA genes are separated by 195 bases and are transcribed in the opposite direction from the rRNA genes. The tRNA Arg(UcU), deduced from the gene sequence, is 72 bases long, contains the GGTTC sequences in the T-loop and differs from the Spirodela tRNA Arg by only one nucleotide (23). Our nucleotide sequences show only 48 nucleotides of the tRNA C[yfccc) gene at the 5' end o f the tRNA Arg(ucu) gene. These sequences differ by two nucleotides from the tobacco tRNA Gly(UCc) gene (10). In the tobacco and wheat tRNA Gly(UCc) genes there is an unusual intron in the D-loop of 691 nucleotides that, if present in pea ct, would explain the missing 3' end of the tRNA Gly(UCC) gene (10, 40).

Discussion The distribution of tRNA genes in pea ctDNA was analyzed by hybridizing 3' end-labelled tRNAs to the restriction digest of cloned recombinants (6). The nucleotide sequence analysis of those regions that hybridized with tRNAs has confirmed the location o f tRNA genes and identified the specific tRNAs. With the present identification of the tRNA ° u gene, a total of 18 amino acid tRNA genes have been found in pea ctDNA. Even though tRNA genes are widely distributed in the ctDNA genomes o f higher plants, some interesting patterns in the location of tRNA genes are apparent. There are two tRNA genes, tRNA ne and tRNA Ala, in the spacer region of the 16S and 23S rRNA genes. At the 3' end of the 5S rRNA gene we have identified three tRNA genes: tRNA Leu, tRNA gsn, and tRNA Arg. Similarly, in corn and tobacco ctDNA, this region contains genes for tRNA Leu, tRNA Ash, and tRNA Arg (1, 44). In addition, three other tRNA genes (tRNA Ash, tRNA Gly, and tRNA TM) have also been identified in this region (44). At the 5' end of the 16S rRNA gene, there is a tRNA TM gene in pea ctDNA. A tRNA TM gene

is found in the same region o f ctDNA from mustard (38), spinach (4), corn (52), and tobacco (50). The rDNA region of corn ctDNA, which has been extensively mapped for tRNA genes, contains a total o f about 11 tRNA genes (44). In pea, dot blot hybridizations with tRNAs separated by two dimensional gel electrophoresis had shown that this DNA hybridized to about 14 tRNA spots. One has to keep in mind, however, that not all tRNA spots may represent individual tRNAs. Some spots might result from degradation of tRNAs. Further sequencing of the rDNA region containing tRNA genes is necessary to definitely establish an accurate number of tRNA genes and their identity. tRNA genes appear to be located at the end of protein genes as well. For example, psbA is located in the pCBgllI-1 clone. About 150 bases from the 3' end of psbA is a tRNA His gene. Both psbA and the tRNA His gene are transcribed in the same direction. Similarly, tobacco ctDNA contains the tRNA Arg and tRNA GIygenes at the 3' end of the gene coding for the ot subunit of ATPase (atpA) (10). However, these two tRNA genes are transcribed in the opposite direction from atpA. In pea ctDNA, we have also located the tRNA Arg and tRNA GIy genes at the 3' end o f atpA. The significance of a tRNA gene immediately following the protein gene is not yet understood. Table 1 lists the tRNA genes that have been sequenced, tRNA genes of ctDNA of higher plants are extremely homologous. Competition hybridization experiments between heterologous ctDNA and tRNAs has shown homology between the total tRNAs of various higher plants (29). Nucleotide sequence analysis has confirmed such homology. For example, the tRNA His gene has been sequenced in ctDNA from pea, spinach, corn, and tobacco. There is not a single nucleotide difference in this tRNA gene in pea, corn, and tobacco. This gene differs from spinach tRNA His by only one nucleotide. Similarly, nucleotide sequences of the tRNA val gene differ by only two nucleotides in corn, tobacco, and pea. The other pea ct tRNA genes whose sequences are described in this paper exhibit similar homology. Nucleotide sequence analysis of pea ct tRNA genes, in agreement with findings of other workers, has shown that the 3' terminus common to all tRNAs is not encoded in ctDNA {unlike most bacterial tRNA genes) and is added posttranscriptional-

Table 1. Sequenced chloroplast transfer RNA genes I. tRNA

Plant Pea

Ala (UGC) Arg (ACG) Arg (UCU) Asn (GUU) Asp (GUC) Cys (GCA) Gln (UUG) Glu (UUC) Gly (UCC) Gly (GCC) His (GUG) Ile (GAU) Ile (CAU) Leu (UAA) Leu (CAA) Met (CAU) fMet (CAU) Phe (GAA) Pro (UGG) Ser (GCU) Ser (UGA) Ser (GGA) Thr (GGU) Thr (UGU) Trp (CCA) Tyr (GUA) Val (UAC) Val (GAC)

Broad bean

+ + + +

Spinach

Tobacco

Corn

+

+

Spirodela

Mustard

Barley

Wheat

+ + +

+ + + +

+ + +

+ +2

+

+ +2

+ +

+ + + +

+ +

+ + +

+ +

+

+ +

+ +

+ + + + +

+ + + +

+ + + + +

+

+

+

+

+ + +

+

+

+ +

+ + +

+ +

+ +

References are given in the text. 2 Partially sequenced.

ly. I n a d d i t i o n , s o m e t R N A genes in c t D N A c o n t a i n i n t r o n s ( a g a i n differing f r o m b a c t e r i a l t R N A genes). N u c l e o t i d e sequences o f ct t R N A s are m o r e h o m o l o gous to p r o k a r y o t i c t R N A s t h a n to e u k a r y o t i c t R N A s . However, a close analysis o f ct t R N A s (Table 2) shows t h a t the i n t e r n a l e u k a r y o t i c Pol I I I consensus sequences f o u n d by Galli et al. (12) a n d by S h a r p et al. (45) are present in p e a a n d o t h e r ct t R N A genes. I n t e r n a l e u k a r y o t i c Pol I I I consensus sequences o f T G G C N N A G T G G ( A sequences) a n d G G T T C G A N N C C (B sequences) are present in all o f o u r t R N A gene sequences. F o r example, the t R N A His, tRNAArg, tRNAAsp, tRNAGIy, tRNAArg, t R N A Leu, a n d t R N A ~yr genes have 90070 o r m o r e h o m o l o g y t o the i n t e r n a l e u k a r y o t i c Pol I I I B consensus sequences. Pea ct t R N A genes show 70-90070 h o m o l o g y to the A t y p e o f i n t e r n a l e u k a r y o t i c

Pol I I I consensus sequences. We c a r r i e d o u t similar analysis o f the t R N A gene sequences f o u n d in corn, spinach, a n d t o b a c c o c t D N A . T h e d a t a f r o m 17 t R N A genes a n a l y z e d showed h o m o l o g y o f a b o u t 80-100070. G r u i s s e m et al. have shown t h a t Euglena c h l o r o p l a s t t R N A genes possessing these sequences are t r a n s c r i b e d in H e l a extracts (15), however, it is n o t yet k n o w n w h a t role they m a y p l a y in vivo. T h e n u c l e o t i d e sequences o f the 5 ' f l a n k i n g regions o f p e a ct t R N A genes are shown in Table 3. Steinmetz et al. (48) have c a r r i e d o u t a d e t a i l e d analysis o f 5 ' n u c l e o t i d e s o f c o r n t R N A genes a n d c o m p a r e d t h e m with the n u c l e o t i d e sequences o f t R N A genes f r o m E. coli a n d o t h e r higher plants. T h e 5 ' end o f each c o r n c h l o r o p l a s t gene was f o u n d to cont a i n s h o r t c o n s e r v e d n u c l e o t i d e sequences t h a t are h o m o l o g o u s to varying degrees with the - 1 0 a n d

10 Table 2. Internal Pol III-like consensus sequences in chloroplast t R N A genes ~ TGGCNNAGTGG pea pea pea pea pea pea pea pea

LRNA tRNA tRNA LRNA tRNA LRNA tRNA tRNA

Ari(ACG) Ar|(UCU) Asn(GUU) Glu(UUC) HIs(GUG) Leu(CAA) Tyr(GUA) VaI(GAC)

tobacco tobacco tobacco tobacco tobacco tobacco tobacco tobacco tobacco

tRN^ LRNA tRNA tRNA tRNA tRNA tRNA tRNA tRNA

GIn(UUG) Gly(UCC) Gly(GCC) fHeL(CAU) Hmt(CAU) Pro(UGG) Ser(GCU) Trp(CCA) VaI(UAC)

mp|nach spinach spinach sp|nach

LRNA LRNA tRNA tRNA

Asp(GUC) Cys(GCA) Ile(XAU) Thr(GGU)

corn corn corn corn

L R N A Leu(UAA) L R N A Phe(GAA) LRNA Ser(UGA) LRNA Ser(GGA)

GGTTCGANNCC

GGGCTCGTAGCTCAGAGGATTAGAGCACGCGGCTACGAACCACGGT GCGTCCATTGTCTAATGGATAGGACAGAGGTCTTCTAAACCTTTGG

TCCCCAGTAG£TCAG__.TGG..TAGAGCGGTCGGCTGTTAACCGATTGGT GCCCCCATCGTCTAGCGGTTTAGGACATCTCTCTTTCAAGGAGGCA GCGGATGTAG¢CAAGTGGATCAAGGCAGTGGATTGTGAATCCACCA GCCTTGGTGGTGAAATGGTAGACACGCGAGACTCAAAATCTCGTGC GGGTCGATGCCCGAGCGGTTAATGGGGACGGACTGTAAATTCGTTG AGGGGTATAACTCAGC_GGTAGAGTGTCACCTTGACGTGGTGGAAGT

GTCGGGGGTT~GAA_TCC__CTCCTCGCCCA

TATAGGTT~kA__A~C~TATTGGACGC^ CGTAGGT_TCAAATCCTATTTGGGGAG ACGGG~T~G~TT_~CCCCTGGGGGTA TGCGCGGGTTCAAT___TCCCGTCGTTCGCC TAAACAGCGAGGAGGTTC_GAGTCC._TCTTCAAGGCA GCAATATGTCTACGCTGGTTCAA_ATCC_AGCTCGGCCCA

TGGGGCGTGGCCAAGTG_qTAAGGCAACGGGTTTTGGTCCCGCTATT

GCGGGTATAGTTTAGTGGTAAAACCCTAGCCTTCCAAGCTAACGAT GCGGATATGGTCGAAATGGTAAAATTTCTCTTTGCCAAGGAGAAGAT

CATCAGTTCGAACCTGATTATCCCTA CGGAGGT!CGAATCCTTCCGTCCCAG GCGGGT___TCGA__TTCCCGCTACCCGCT GCGGGTTCGATTCCCGCTATCCGCC

CGCGGGGTAGAGCAGTTTGGTAGCTCGCAAGGCTCATAACCTTGAG

GTCACGGGTTCAAATCCTGTCTCCGCAA

ACCTACTTAACT~AGTG_G_TTAGAGTACTGCTTTCATACGGCGGGAG AGGGATGTG_~GGGCAGGTT_GGTAGCGCGTTTGTTTTGGGTACAAAAT

TCATTGGTTC_AAATCCAATAGTAGGTA GTCACAGGTTGAA.8~CC_TGTCATCCCTA

GGAGAGATGGCTGAGTGGACTAAAGCGGCGGATTGCTAAT~CGTTGTA~GAGTTAAT~GTACCGAGGG-~TTCGAAT-~CC--CTCTCTTT~CG GCGCTCTTAGTTCAGTTCGGTAGAACGTGGGTCTCCAAAACCCGAT GTCGTAGGTTCAAATCGTACAGAGCGTG AGGGCTATAGCTCAGTTTG_GTAGAGCAACTCGTTTACACCGAGAAGG TCTACGGTTCGAGTCCGTATAGCCCTA GGGATTGTAqTTCA6TTGGTTAGAGCACCGCCCTGTCAAGGCGGAA GGCGGCATGGCCGAGT_G_GTAAGGCGGAGGACTGCAAATCCTTGTTC

GCATCCATG_GGCTGAA.TGGTTAAAGCGCCCAACTG^TAATTGGCGAA GCCCCTTTAAGTC.AGTGGTAGAGTAACGCCATGGTAAGGCGTAAGT

GCTGCGGGTTCGAGC_CCCGTCAGTCCCG CCCAGTT.C~ASA~C~TGGGTGTCGCCT TTCGTAGGTTCAATTCCTACTGGATGC^ CATCGGTTCAAAT__C.CGATAAGGGGCT

GGGGATATGGCGAAATCG_GTAGACGCTACGGACTTAAAAGCCGTCG ACTTTATAAGTCGTGAGGGTTCAAGTCCCTCTATCCCCA GTCAGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTG TCACCAGTTCAAATCTGGTTCCTGGCA GGAGAGATGGCTGAGT-G.GTTGATAGCTCCGGTCTTGAAAACCGGTATAGTTCTAGGAACTATCGAGGG--TT-~CG-AA-TCCCTCTCTCT~CT GGAGAGATGGC¢G_SGCGGTTCAAGGCGTAGCATTGGAACTGCTATGTAGACTTTTGTTTACCGAGGGTTCGAATTC_C_CTCTCTTTCCG

References are given in the text.

- 35 regions o f bacterial promoters. The consensus at - 1 0 from their data was found to be TAAGAT and at about 20 to 25 nucleotides away from the - 10 sequences was another consensus sequence o f ATTG A N A . In addition, Hallick and coworkers have analyzed the t R N A genes o f Euglena and spinach

c t D N A for 5' flanking consensus sequences (13, 14). They found the sequence G T A N T A A A A at about 14 bp upstream from t R N A genes. Our analysis o f pea ct t R N A genes (Table 3) shows that practically all o f our t R N A genes have both the consensus sequences found by Steinmetz et al. and Hallick et al.

Table 3. 5' flanking regions of pea chloroplast t R N A genes. tRNA Arg(ACG) -20 -10 GATCTA~TG~ ~A~AGGGTG tRNA Tyr(GUA) -60 -50 -40 -30 -20 -10 GGGT_ACTA~G_AAAGGAAATG GATCAGGGAT TATC~ATAAA GACTAAATTG GATTCTTCCT tRNA Glu(UUC) -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 TCTGAAAGAT GAGAAGATCC TTCTGATCGA ATCATTATAT TGACAATTTC AAAAAACTGT TCATACTATG AACATAGTAG AATGGAGGTCGGGGAAGGAT tRNA HII(GUG) -110 -100 -90 -00 -70 -60 -50 -40 -30 -20 -10 TTTTCTGTAT ACAATATACA GAAATAAATG TTTTCTTATT TTTTAAT~T.T_T_T~Ct~GAAA AGAATAATGA AAAG~T~Z/~_~/~tTJ~CJ_~TTT~GACA TAGTTAGAGG

LRNA Leu(CAA) -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 ATTCTATCCC ATCTATATTA TCATATAATA AGATATCTAA TAATATCTTA TACTATATAT TTGTAATATA TTCCATAAGA TAGATATCAT ATTCATAGAA TATGATTGAT tRNA AIn(GUU) -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 ATTCTATGAA TATGATATCT ATCTTATGGA ATATATTACA AATATATAGT ATAAGATATT&TJ~JLTJ~TC TTATTATATG ATAATATAGA T G G G ~ T T G C C G T T G tRNA Ari(UCU) -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 TCTAGTTAGA ATAATTATTA TTTCGATATT CGGATAGATA GAGTATTTTA TAGAATTTTT TTTTTTCTTT CCCTTGAAAT TTTCGTGAAA AGGGAAAGAA AAAAAGAAAA

(

) TAAGAT-Iike ( - 10) sequences; ( ~ )

(Hallick et al. 1984)

ATTGA-like ( - 35-) sequences; ( - - - ) GT(A/T)NTA(A/T) (A/T)A sequences

ll The significance of these sequences remains as yet unclear. We are carrying out in vitro transcription of cloned tRNA genes with pea chloroplast RNA polymerase to find out which of the consensus sequences o f tRNA genes in pea ctDNA have interaction with RNA polymerase. There is yet another level of homology in the 5' flanking sequences of tRNA genes in ctDNA of higher plants. For example, there is a stretch of 45 nucleotides that is identical in the tRNA Clu gene between pea, spinach, and broad bean. Similar conservation of sequences exists for other tRNA genes.

11.

12.

13.

14.

Acknowledgements 15.

We would like to thank Dr Ken Draper for his technical advice and Jim Massey for his support. This research was supported by PHS grant GM 31492-02.

References 1. Bergmann P, Seyer P, Burkard G, Weil JH: Mapping of transfer RNA genes on tobacco chloroplast DNA. Plant Mol Biol 3:29- 36, 1984. 2. Bonnard G, Michel F, Weil JH, Steinmetz A: Nucleotide sequence of the split tRNA Leu(UAA) gene from Viciafaba chloroplasts: evidence for structural homologies of the chloroplast tRNA Leu intron with the intron from the autosplicahie Tetrahymena ribosomal RNA precursor. Mol Gen(3enet 194:330- 336, 1984. 3. Bonnard G, Weil JH, Steinmetz A: The intergenic region between the Vicia faba chloroplast tRNALeu(CAA) and tRNALeu(UAA) genes contains a partial copy of the split tRNALeu(UAA) gene. Curr Genet 9:417-422, 1985. 4. Briat JF, Dron M, Loiseaux S, Mache R: Structure and transcription of the spinach rDNA leader region. Nucleic Acids Res 10:6865- 6878, 1982. 5. Chu NM, Oishi KK, Tewari KK: Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes. Plasmid 6:279- 292, 1981. 6. Chu NM, Shapiro DR, Oishi KK, Tewari KK: Distribution of transfer RNA genes in the Pisum Sativum chloroplast DNA. Plant Mol Biol 4:65- 79, 1985. 7. Chu NM, Tewari KK: Arrangement of the ribosomal RNA genes in chloroplast DNA of leguminosae. Mol Gen Genet 186:23 - 32, 1982. 8. Deno H, Kato A, Shinozaki K, Sugiura M: Nucleotide sequence of tobacco chloroplast genes for elongator tRNA Met and tRNA Val(UAC): the tRNA VaI(UAC) gene contains a long intron. Nucleic Acids Res 10:7511 - 7520, 1982. 9. Deno H, Sugiura M: The nucleotide sequences of tRNA Ser(GCU) and tRNA Gln(UUG) genes from tobacco chloroplasts. Nucleic Acids Res 11:8407- 8414, 1983. 10. Deno H, Sugiura M: Chloroplast tRNA Gly gene contains

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

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Received 8 July 1985; in revised form 30 September 1985; accepted 7 October 1985.