Plant Molecular Biology8:327 336, (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

327

Characterization of the TrnD, TrnK, PsaA locus of Euglena gracilis chloroplast DNA Thianda Manzara, 1 Jian-Xian H u , 2 Carl A. Price 2 & Richard B. Hallick 3

1Department of Botany, University of California, Berkeley, CA 94720, USA; 2Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854, USA; 3Department of Biochemistry, University of Arizona, Tucson, A Z 85721, USA Received 10 December 1985; in revised form 5 January 1987; accepted 12 January 1987

Keywords: chlorophyll apoprotein gene, chloroplast DNA, Euglena, t R N A gene

Abstract

The EcoRI fragment Eco J ' of Euglena gracilis chloroplast D N A has previously been identified as a tRNA coding locus. The nucleotide sequence of a 2.3-kb region of the Eco J ' fragment known to contain the tRNA genes has been determined. This locus was found to contain two t R N A genes, trnD-GTC and trnK-TTT. Separated from the trnK locus by a 43-bp spacer is an open reading frame of 398 codons. The open reading frame is 73-75°70 homologous to the amino-terminal coding regions of the spinach and maize genes for the P700 chlorophyll a apoprotein of photosystem I. It has been identified as exon I of an intron-containing psaA gene. The exon is followed by an intron of at least 214 bp that has features characteristic of other Euglena chloroplast introns. Major chloroplast R N A transcripts of sizes 5.5, 4.7, and 2.3 kb hybridize to a psaAspecific probe. The gene for a second photosystem I P700 apoprotein, psaB, has been located on an adjacent EcoRI fragment, Eco C.

Introduction

We have been studying the t R N A coding loci of the Euglena gracilis chloroplast genome in order to determine the complete complement of tRNAs required for protein synthesis and to understand better the role of tRNAs in the regulation of chloroplast gene expression. The Euglena chloroplast tRNA loci have been mapped (4, 9, 20, 27), and 21 genes have been sequenced (8, 10, 11, 15, 28, 29). These t R N A genes are in general organized in clusters within the ribosomal RNA operons, with other t R N A genes, or with m R N A coding loci (10). This has led to the speculation that most tRNAs are part of polycistronic transcription units (10). The 3.8-kbp Euglena chloroplast fragment Eco

J ' was first identified as a tRNA coding region by Orozco et al. (27). The location of the t R N A genes within Eco J ' was more precisely defined by Southern blot analysis of cloned Eco J ' , using as a probe a t R N A gene-specific synthetic tetradecanucleotide (25). In this report, sequence data from the EcoR1 fragment J ' (Eco J ') of Euglena chloroplast DNA is presented. We have identified two tRNA genes and the first exon of a gene for a P700 chlorophyll a apoprotein. Materials and m e t h o d s

Generation of subclones of Eco J' The 3.8-kbp Eco J ' fragment of Euglena gracilis,

328 Pringsheim strain Z, chloroplast DNA was ligated to EcoRI-treated pMB9 and cloned as a recombinant plasmid designated pPG689. Fragments of Eco J ' were subcloned into phage M13 vectors for DNA sequence analysis. DNA fragments for the subcloning of Eco J ' were generated by one of the following methods: (a) restriction endonuclease digestion of pPG689 plasmid DNA for shotgun cloning; (b) gel purification of specific restriction fragments from pPG689 DNA; (c) digestion of the restriction endonuclease-cut pPG689 DNA with exonuclease III for varying lengths of time, digestion with S1 nuclease, followed by digestion with EcoRI, in order to generate a set of serially deleted DNA fragments (22); (d) partial digestion of pPG689 with DNase I followed by digestion with EcoRI as described by Hong et aL (14). All of the subfragments of Eco J ' were cloned into either M13 mp8, or mp9 (23).

DNA sequencing and data analysis DNA sequence analysis was accomplished by the dideoxy chain termination method developed by Sanger et al. (31) and modified by Biggin et al. (1). Analysis of DNA sequence data was performed on an IBM PC-XT computer using versions 3.2 and 3.9 of the DNA and Protein Analysis programs of Mount and Conrad, provided by D. W. Mount, University of Arizona. Protein open reading frames were aligned with known polypeptide sequences using the FASTP search algorithm described by Lipman and Pearson (21).

Hybridization probe An MI3 mp8 subclone of a portion of psaA exon 1 was used as a template for in vitro synthesis of a psaA probe for mRNA hybridization. Labeling of the probe was accomplished using the same reactions used for DNA sequencing, except that the dideoxynucleotide was omitted. The BamHI fragment Bam 20 from the spinach chloroplast psaB locus was provided by Dr R. Herrmann. This spinach DNA fragment was radiolabeled via the nicktranslation activity of E. coli DNA polymerase I.

Southern and northern hybridizations Isolation of Euglena chloroplast RNA and preparation of membrane filter blots of electrophoretically separated RNAs was performed as previously described (12). Northern hybridization of a homologous probe to Euglena chloroplast RNA was in 50% formamide, 0.02 M sodium phosphate buffer (pH 6.8), 1 x Denhardt's solution (2), 0.75 M NaCI, 0.075 M sodium citrate, and 0.1 mg/ml sheared, denatured herring sperm DNA for 16 h at 37 °C. The filters were washed 3 times for 45 min each at 37 °C in 25 ml of the reaction mixture lacking the hybridization probe. Isolation of Euglena chloroplast DNA and preparation of Southern blots was accomplished as previously described (12). Southern hybridization of the heterologous spinach probe to Euglena chloroplast DNA was in 2007o formamide, 0.075 M sodium phosphate buffer (pH 6.8), 1 x Denhardt's solution, 0.75 M NaCI, 0.075 M sodium citrate, and 0.2 mg/ml herring sperm DNA for 16 h at 37°C. The filter was washed 3 times for 45 min each at 37 °C in 25 ml of hybridization solution without the radiolabeled probe.

Results and discussion

DNA sequence of the trnD-trnK-psaA locus of Eco J' The 3.8-kbp EcoRI fragment Eco J ' of the Euglena chloroplast genome has previously been shown to encode tRNA(s) (25, 27). Based on hybridization of in vitro labeled tRNAs and synthetic oligonucleotide probes to Southern blots of Eco J ' restriction fragments, the tRNA coding locus could be localized to a region of approximately 1 kbp defined by 0.8- and 0.2-kbp Sau3A fragments (data not shown). Restriction maps of the tRNA coding region of Eco J ' as well as the sequencing strategies used to determine the structure of the tRNA genes are shown in Fig. 1. The DNA sequence of 2299 bp of the approximately 3.8-kbp Eco J ' fragment is shown in Fig. 2. Contained within the sequenced region are two t R N A genes, identified as trnD and trnK (described below). In addition, following the

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Fig. 1. Gene map, restriction map, and sequencing strategy. A. Gene m a p of a 2.5-kb region of Eco J ' . B - D. Restriction endonuclease maps. E - G . Sequencing strategies: E, clones generated following restriction endonuclease digestion of pPG689 DNA; F, clones generated following treatment of pPG689 D N A with exonuclease III and SI nuclease; G, clones generated following treatment with DNase I.

trnK locus is an open reading frame of 398 codons that has been identified as exon 1 of an introncontaining gene designated p s a A coding for a P700 chlorophyll a apoprotein of photosystem I (described below). All three genes are of the same polarity and are of the opposite polarity from the ribosomal R N A genes. The organization of the three genes is trnD -- 602-bp spacer -- trnK -44-bp spacer -- p s a A .

A s p a r t a t e a n d lysine t R N A genes

The cloverleaf structures for trnD and trnK are shown in Fig. 3. The identification of the trnD gene was based on the aspartyl anticodon sequence

of 5 ' - G T C and the very high homology with other chloroplast trnD genes. The Euglena trnD locus has 92% homology with the corresponding spinach chloroplast gene (13), 93% homology with the pea chloroplast gene (30), 92% homology with the tobacco gene (26), and 75% homology with the E. coli gene (6, 33, 37). The identification of trnK is based both on the lysyl anticodon sequence of 5'-TTT, and the earlier report by Kuntz et al. (20) that a Euglena chloroplast t R N A Lys, identified by aminoacylation, hybridized to Eco J ' . The trnK locus has 81% homology to the exons of the split trnK locus of tobacco chloroplast DNA (35) and 75% homology to the E. coli lysyl t R N A gene (36). The trnK and trnD loci are characteristic of Euglena chloroplast t R N A genes in that neither gene

330

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ACC GAT GTT GCA CAT CAT CAT TTG GCT TTA GCT Thr Asp Val Ala His His His Leu Ala Leu Ala 1850 CAT ATG TAT AAA ACA AAT TGG AAA ATT GGA CAT His Met Tyr Lys Thr Asn Trp Lys Ile Gly His 1900 TCT CAT ACT GGA CCT TTT ACA GGT CAA GGC CAT Ser His Thr Gly Pro Phe Thr Gly Gin Gly His 1950 ACT AAT TCT TGG CAT GCT CAA CTT AGT CTT AAC Thr Asn Ser Trp His Ala Gin Leu Ser Leu Asn 2000 TCC ATA ATA GTA GCA CAA CAT ATG TAT TCT ATG Ser lie lie Val Ala Gin His Met Tyr Ser Met 2050 ATA GAT TAT GGT ACA GAG TTA TCG TTA TTT ACA lle Asp Tyr Gl~InT~nr~ vv Glu Leu Ser Leu Phe Thr

GTT TTG TTT ATT TTG GCA GGT Val Leu Phe lle Leu Ala Gly GAT ATA AAA GGT TTA TTA GAA Asp Ile Lys Gly Leu Leu Glu AAA GGA CTT TAT GAA ATT TTT Lys Gly Leu Tyr Glu lie Phe CTT GCT ATG ATG GGC TCT CTT Leu Ala Met Met Gly Ser Leu CCT CCT TAT CCT TAC ATT GCA Pro Pro Tyr Pro Tyr lie Ala CAT CAT TAT TGG ATT GGA His His Tyr Trp lle Gly

GTTGCG AGCGTTATTT TTAAGAATTT TTAAATTGGA TCCCCTAATT ATTAATTATT 2150 2200 TTTAGTTTTT TGAAATTAAA ATAAA TTTA TTGAAAAATA AAAATTATTA AGCTAAAAAG 2250 GGCTGTGCTT GCTTTTAATT ATTAGTTAAA AAATATTAAA AATTTTACAA AATTAGAATT 2299 ATTTTCTCAA ATTCCTTTAA TTTATCTTTT TGAAAGATC

Fig. 2. The DNA sequence of the RNA-like strand of Eco J' containing the trnD, trnK, and psaA genes. The coding regions are as follows: trnD, 98-171; trnK, 774-846; psaA exon 1, 891-2085.

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encodes the 3' terminal C C A nor contains introns. It has previously been reported that the synthetic tetradecanucleotide of sequence 5 '-CTACCAACTGAGCT, designed as a probe to a highly conserved region within the D-stem and loop o f chloroplast t R N A genes, hybridized to Eco J' (11). This sequence is found within the Euglena chloroplast trnA, trnV, trnG, and trnF loci, and is also present in trnK of Eco J'.

The P700 apoprotein gene for photosystem I

(psaA) Fish et al. (5) reported that the maize chloroplast genome contains two genes in tandem that code for two similar proteins o f the PSI reaction center. In the nomenclature employed here, psaA denotes the first and psaB the second o f these genes. The two genes have been sequenced in maize (5), spinach

332 maize psaA, which is an addition with respect to Euglena and spinach, the three sequences can be

(17), pea (20a), and tobacco (34). The identification of the open reading frame of Eco J ' in Euglena chloroplast D N A as coding for a photosystem I polypeptide was accomplished by computerassisted comparison of the derived amino acid sequence of the open reading frame with a library of amino acid sequences from known chloroplast proteins. Using the FASTP amino acid homology search algorithm developed by Lipman and Pearson (21), the homologies between the Euglena open reading frame and the amino-terminal 400 codons derived from the spinach and maize D N A sequences were evident. The Euglena amino acid sequence shows 73°7o and 75°70 identity, respectively, with the spinach and maize sequences. The two plant sequences are 94% homologous to each other. The Euglena and higher plant amino acid sequences ~ire essentially colinear. Except for a short region within the first 15 codons and codon 40 in

aligned without assuming any insertions or deletions of amino acids. The alignment of the Euglena-derived amino acid sequence with that of maize is shown in Fig. 4. The region of amino acid homology of the Euglena polypeptide with both higher plant sequences ends at the same amino acid, corresponding to positions 394 and 396, respectively, in the spinach and maize polypeptides. No additional homology between the remaining Eco J ' sequence and the plant psaA loci could be identified. We interpret the 398 codon open reading frame Eco J ' to be the first exon of a split psaA gene for the following reasons: (a) The high degree of homology between the Euglena amino acid sequence and the higher plant sequences would argue against the possibility of the Euglena gene being a pseudogene. (b) The length of the proposed exon

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335 (Fig. 5). Because the 5.5-kb transcript did not hybridize to a sense strand-specific probe generated from the 812 base pair Sau3A fragment located less than 100 bp upstream from psaA, we expect that the 5' end of this transcript maps close to the putative ATG initiator codon. The ends ofpsaA and psaB within Eco C cannot be precisely located from the hybridization data, nor can the polarity of psaB be determined. It is likely, however, that psaA and psaB are located relatively close to one another. The psaA a n d p s a B genes have been sequenced in maize (5), spinach (17), pea (20a), and tobacco (34). In both cases, the psaA gene is upstream from the psaB gene and is separated from it by a short spacer. The two genes are of the same polarity and are transcribed as a polycistronic message in both higher plants. It is unclear whether psaA and psaB are transcribed as a polycistronic message in Euglena. No major transcripts of sufficient size to represent an unspliced, polycistronic psaA-psaB p r e - m R N A could be detected via a northern blot of Euglena R N A (Fig. 5). The largest major transcript which hybridized to the psaA-specific probe was approximately 5.5 kb, which would place the 3' end of the transcript approximately 1.5 kb into Hind 13. The spinach psaB probe hybridized with Hind 30, which would require at least an 8.5-kb transcript ifpsaA and psaB were transcribed together. A large primary transcript which is rapidly processed cannot be ruled out. In fact, at least two large minor transcripts were observed upon prolonged exposure of the northern blot. Additional experiments are required to determine if the arrangement of psaA and psaB in Euglena is similar to that of higher plant chloroplast.

Acknowledgements We wish to thank Dr R. H e r r m a n n for the gift of the spinach Bam 20 fragment. We are also grateful to Dr U. Johanningmeier for preparation of the northern blot, Dr J. Narita for preparation of the Euglena chloroplast DNA, and Dr C. Passavant for construction of some of the M13 clones. This work was supported by N I H Grants GM35665 and 35625

to R.B.H., and grants from the USDA and the Charles and Johanna Busch Memorial Fund to C.A.P.

References 1. Biggin MD, Gibson T J, Hong GF: Buffer gradient gels and 3sS label as an aid to rapid D N A sequence determination. Proc Natl Acad Sci USA 80:3963-3965, 1983. 2. Denhardt D: A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res C o m m u n 23:641-645, 1966. 3. Dix KP, Rawson JYR: In vivo transcriptional products of the chloroplast D N A of Euglena gracilis. Curr Genet 7:265-272, 1983. 4. E1-Gewely MR, Lomax MI, Lau ET, Helling RB, Farmerie W, Barnett WE: A m a p of specific cleavage sites and tRNA genes in the chloroplast genome of Euglena gracilis bacillaris. Mol Gen Genet 181:296-305, 1981. 5. Fish LE, Kuck U, Bogorad L: Two partially homologous adjacent light-inducible maize chloroplast genes encoding polypeptides of the P700 chlorophyll a-protein complex of photosystem I. J Biol Chem 260:1413-1421, 1985. 6. Folk WR, Hofstetter H, Birnstiel ML: Some bacterial t R N A genes are transcribed by eukaryotic R N A polymerase II1. Nucleic Acids Res 10:7153-7162, 1982. 7. Gingrich JC, Hallick RB: Euglena gracilis chloroplast ribulose-l,5-bisphosphate carboxylase gene I. Complete D N A sequence and analysis of the nine intervening sequences. J Biol Chem 260:16156-16161, 1985. 8. Graf L, K6ssel H, Stutz E: Sequencing of 16S-23S spacer in a ribosomal R N A operon of Euglena gracilis chloroplast D N A reveals two tRNA genes. Nature 286:908-910, 1980. 9. Hallick RB, Rushlow KE, Orozco EM, Jr, Stiegler GL, Gray PW: Chloroplast D N A of Euglena gracilis. Gene mapping and selective in vitro transcription of the ribosomal R N A region. I C N - U C L A Symp Mol Cell (Exchromosomal DNA) 15:127-141, 1979. 10. Hallick RB, Hollingsworth M J, Nickoloff JA: Transfer R N A genes of Euglena gracilis chloroplast DNA. Plant Mol Biol 3:169-175, 1984. 11. Hollingsworth M J, Hallick RB: Euglena gracilis chloroplast transfer R N A transcription units. 111. Nucleotide sequence analysis of a t RNATyr-t RNAHis-t RNAMe~-tRNATrptRNAGIu-tRNAGly gene cluster. J Biol Chem 257: 12795-12799, 1982. 12. Hollingsworth M J, Johanningmeier U, Karabin GD, Stiegler CL, and Hallick RB: Detection of multiple, unspliced precursor m R N A transcripts for the M r 32000 thylakoid membrane protein from Euglena gracilis chloroplasts. Nucleic Acids Res 12:2001-2017, 1984. 13. Holschuh K, Bottomley W, Whitfield PR: Sequence of the genes for tRNA Cys and t R N A Asp from spinach chloroplasts. Nucleic Acids Res 11:8547-8554, 1983.

336 14. H o n g GF: A systematic D N A sequencing strategy. J Mol Biol 158:539-549, 1982. 15. Karabin GD, Hallick RB: Euglena gracUis chloroplast transfer R N A transcription units. IV. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGIn gene cluster. J Biol Chem 258:5512-5518, 1983. 16. Karabin GD, Farley M, Hallick RB: Chloroplast gene for M r 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences. Nucleic Acids Res 12:5801-5812, 1984. 17. Kirsch W, Seyer P, H e r r m a n n RG: Nucleotide sequence of the clustered genes for two P700 chlorophyll a apoproteins of the photosystem I reaction center and the ribosomal protein S14 of the spinach plastid chromosome. Curr Genet 10:843- 855, 1986. 18. Koller B, Delius H: Intervening sequences in chloroplast genomes. Cell 36:613-622, 1984. 19. Koller B, Gingrich JC, Stiegler GL, Farley MA, Delius H, Hallick RB: Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-l,5bisphosphate carboxylase gene. Cell 36:545- 553, 1984. 20. Kuntz M, Keller M, Crouse E J, Burkhard G, Weil JH: Fractionation and identification of Euglena gracilis chloroplastic tRNAs and mapping of t R N A genes on chloroplast DNA. Curr Genet 6 : 6 3 - 6 9 , 1982. 20a. Lehmbeck K, Rasmussen OF, Bookjans GB, Jepsen BR, S t u m m a n n BM, Henningsen KW: Sequence of two genes in pea chloroplast D N A coding for 84 and 32 kD polypeptides of the photosystem I complex. Plant Mol Biol 7:3-10, 1986. 21. Lipman DJ, Pearson WR: Rapid and sensitive protein similarity searches. Science 227:1435-1441, 1985. 22. Manzara T, Hallick RB: manuscript in preparation. 23. Messing J: New M13 vectors for cloning. In: Wu R, Grossm a n L, Moldave K (eds) Methods in Enzymology, Volume 101 (part C). Academic Press, New York, pp 2 0 - 7 8 . 24. M o n t a d o n PE, Stutz E: Nucleotide sequence of a Euglena gracilis chloroplast genome region for the elongation factor Tu; evidence for a spliced m R N A . Nucleic Acids Res 11:5877-5892, 1983. 25. Nickloff JA, Hallick RB: Synthetic deoxynucleotides as general probes for chloroplast tRNA genes. Nucleic Acids Res 10:8191-8210, 1982. 26. Ohme M, Kamogashira T, Shinozaki K, Sugiura M: Structure and cotranscription of tobacco chloroplast genes for tRNAGIu(UUC), tRNATyr(GUA), and tRNAAsp(GUC). Nu-

cleic Acids Res 13:1045-1056, 1985. 27. Orozco EM, Jr, Hallick RB: Euglena gracilis chloroplast transfer R N A transcription units. I. Physical m a p of the transfer R N A gene loci. J Biol Chem 257:3258-3264, 1982. 28. Orozco EM, Jr, Hallick RB: Euglena gracilis chloroplast transfer R N A transcription units. II. Nucleotide sequence analysis of a tRNAVal-tRNAAsn-tRNAArg-tRNALeu gene cluster. J Biol Chem 257:3265-3275, 1982. 29. Orozco EM, Jr, Rushlow KE, Dodd JR, Hallick RB: Euglena gracilis chloroplast ribosomal R N A transcription units. II. Nucleotide sequence homology between the 16S-23S ribosomal R N A spacer and the 16S ribosomal R N A leader regions. J Biol C h e m 255:10997-11003, 1980. 30. Rasmussen OF, S t u m m a n n BM, Henningsen KW: Nucleotide sequence of a 1.1 kb fragment of the pea chloroplast gen o m e containing three tRNA genes, one of which is located within an open reading frame of 91 codons. Nucleic Acids Res 12:9143-9153, 1984. 31. Sanger F, Nicklen S, Coulson AR: D N A sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467, 1977. 32. Schantz R: Mapping of the chloroplast genes coding for the chlorophyll a-binding proteins in Euglena gracilis. Plant Sci Lett 40:43-49, 1985. 33. Sekiga T, Mori M, Takashi N, Nishimura S: Sequence of the distal tRNAAsp gene and the transcription termination signal in the Escherichia coli ribosomal R N A operon rrn r (or G). Nucleic Acids Res 8:3809-3827, 1980. 34. Sbinozaki K, O h m e M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M: The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5:2043-2049, 1986. 35. Sugita M, Shinozaki K, Sugiura. M: Tobacco chloroplast t R N A L y s ( u u u ) gene contains a 2.5-kilobase-pair intron: an open reading frame and a conserved boundary sequence in the intron. Proc Natl Acad Sci USA 82:3557-3561, 1985. 36. Yoshimura M, Kimura M, O h n o M, Inokuchi H, Ozeki H: Identification of transfer R N A suppressors in Escherichia coli. III. Ochre suppressors of lysine rRNA. J Mol Biol 177:609-625, 1984. 37. Young RA: Transcription termination in the Escherichia coli ribosomal RNA. J Biol C h e m 254:12725-12731, 1979.

Characterization of the TrnD, TrnK, PsaA locus of Euglena gracilis chloroplast DNA.

The EcoRI fragment Eco J' of Euglena gracilis chloroplast DNA has previously been identified as a tRNA coding locus. The nucleotide sequence of a 2.3-...
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