(D) 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 14 3773-3777

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs Kenji Oda, Katsuyuki Yamato, Eiji Ohta, Yasukazu Nakamura, Miho Takemura, Naoko Nozato, Kinya Akashi and Kanji Ohyama* Laboratory of Plant Molecular Biology, Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, 606-01, Japan Received January 27, 1992; Revised and Accepted May 26, 1992

ABSTRACT Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer. from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes. INTRODUCTION Transfer RNA genes encoded by large mitochondrial genomes of higher plants are reported to be fewer than those encoded by compact mammalian and yeast genomes and not enough to recognize all codons used in the mitochondrial genome of higher plants. This deficiency is overcome by the import of nuclearencoded tRNAs from the cytoplasm. Among 31 species of tRNAs in potato mitochondria, only 20 species are encoded by the mitochondrial genome and the rest by the nuclear genome (1). Wheat and maize mitochondrial genomes are estimated to contain *

To whom correspondence should be addressed

GenBank accession no. M68929

less than 20 tRNA (2), and 16 tRNA (3) genes, respectively. Bean mitochondria import at least 8 species of tRNAs from the cytoplasm (4). Surprisingly, some of the tRNAs encoded in the mitochondrial genome show very high sequence homology (90-100%) with their chloroplast counterparts. They are defined as 'chloroplast-like' tRNAs. For instance, the maize mitochondrial trnH gene is completely identical to that in the chloroplasts, indicating that the tRNA gene was inserted during evolution into mitochondrial DNA (5). 'Native' mitochondrial tRNAs decode exclusively codons for initiator methionine, proline, tyrosine, glutamine, lysine, aspartic acid, glutamic acid, and serine (AGY codon). Codons for elongator methionine, histidine, asparagine, tryptophan, and serine (UCY codon) are recognized by 'chloroplast-like' tRNAs. Mitochondrial tRNA genes for leucine, threonine, alanine, and arginine are encoded by the nuclear genome. There are two kinds of tRNA genes, 'native' trnS-UGA and 'chloroplast-like' trnS-GGA for the UCN codon box in wheat (6). The trnG and trnV genes are present on the mitochondrial genome in potato (1) and on the nuclear genome in maize (3). The trnC and trnF genes are classified into 'native' tRNA genes in potato (1) and into 'chloroplast-like' tRNA genes in maize (3). From the evolutionary point of view, it is very important to compare the mitochondrial tRNAs among various species of plants, especially between higher and lower plants. In order to obtain information on plant mitochondrial tRNAs and their origin, we have identified all of the tRNA genes on the liverwort mitochondrial genome. Here, we report that the liverwort mitochondrial genome has 27 species of tRNA genes, many more species than those found in mitochondrial genomes of higher plants. None are 'chloroplast-like' tRNA genes.

MATERIALS AND METHODS The complete nucleotide sequence (accession number M68929 in the GenBank Data Library) of mitochondrial DNA from a liverwort, Marchantia polymorpha, has been determined by dideoxy sequencing methods as described elsewhere (7, 8).

3774 Nucleic Acids Research, Vol. 20, No. 14

Computer aided analysis of the nucleotide sequence to identify tRNA genes was performed using a Hitachi DNASIS program on a NEC PC-9801VM computer and an IDEAS program on a FACOM M-780 computer (Data Processing Center, Kyoto

leaf secondary structure. Twenty-nine tRNA genes were identified in the sequence. The corresponding amino acids were determined from their unmodified anticodon sequences according to the universal codon table, since our sequence data indicated utilization of the universal genetic code in the liverwort mitochondrial genome. It has been reported that a potato mitochondrial tRNAIle initially having a CAU anticodon for methionine is capable of accepting isoleucine and presumably recognizing the isoleucine codon (AUA) upon post-transcriptional modification of the C as reported in eubacterial and chloroplast tRNAIe (9). Thus, liverwort tmnl-CAU gene was identified by high homology to the potato tmnI-CAU gene. Nucleotide sequences of 27 species of liverwort tRNA genes are given in Figure 1. Unlike highly clustered tRNA genes in fungal mitochondria (10), all the liverwort tRNA genes are located in the semicircle region of the genome except for the duplicated tRNA genes, tinY and trnR (Figure 2). Most liverwort mitochondrial tRNA genes are found in five regions. Gene arrangements in these regions are also different from those in the yeast tRNA clusters. Mammalian tRNA genes are not highly clustered, but rather are located at the termini of protein genes, and serve as punctuation signals for the processing of mRNA (11). Although most liverwort tRNA genes are far from adjacent genes, however, the liverwort trnG-UCC gene is 29 bp upstream from the rps4 gene, the trnP-UGG gene is 26 bp upstream from the orfl37, and the tmnS-GCU gene is 3 bp upstream from the trnA gene. Moreover, the liverwort tnW gene is located 23 bp upstream from the nad3 gene, and the trnF gene is 18 bp upstream from the rps2 gene, but both tRNA genes are on the opposite DNA strand from the coding strand of the respective protein genes. The liverwort mitochondrial genome has a single large inversion (orf62 to atp9) for protein and rRNA genes (Figure 2). Ten of the tRNA genes (tmF, tmQ, tmnH, trnL-CAA, tmnL-UAA, trnW, trnT, tmnY, trnRUCG, and tmnS-UGA) are located at the strand opposite to the DNA strand coding protein and rRNA genes in the respective

University).

RESULTS AND DISCUSSION Identification and genomic location of the tRNA genes Transfer RNA genes were deduced from the complete nucleotide sequence of the liverwort mitochondrial DNA using the conserved sequence of the TYC loop (GTTCRA) and the typical cloverTabl 1. Sequence homologies of the liverwon mitochondrial tRNA genes with the native mitochonda tRNA genes from higher plants and the liverwort chloroplast tRNA genes

Homology (%) with Uvervort mitochondial gencs trnA-UGC trnC-GCA trnD-GUC trnE-UUC trnF-GAA trn-GCC

trnG-UCC &WMH-CUG trnl-CAU trnK-UUU trnLUAA rnL-CAA trnL-UAG

trjM-CAU

'native' mitochondrial genes

liverwon chloroplast genes (14)

ref.

-

83.1% 87.8% 88.3% 91.9% 93.1% -

Tomato Wheat Soybean Oenothera Oenoehera

(20) (2) (21) (22) (22)

Potato Maize

(9) (23)

Otnothera

(22)

68.5% 59.2% 67.6% 63.0% 73.5% 69.9% 76.1% 68.9% 55.4% 67.6%

-

94.0% 93.2% 93.2% -

60.6% 58.8% 61.8% 72.1%

-

76.2% 76.2%

trnM-CAU trnN-GUU trnP-UGG trnQ-UUG trnR-ACG trnR-UCG trnR-UCU

81.9% 87.5%

trnS-UGA trnS-GCU

98.2% 90.9% 90.9%

IrnT-GGU trnV-UAC trnW-CCA irnY-GUA

-

Wheat Oenothera

(12)

(13) 503% 77.2% 73.0% 68.1% 62.2%

-

-

Wheat Wheat

(2) (24)

-

-

54.8% 75.6% 69.3%

-

Wheat Wheat Ocenohera

(2) (2) (22)

inversion.

67.6% 74.5% 62.6%

-

-

96.4% 95.8% 95.2%

Maize Wheat Oenothera

(23)

Structure of liverwort tRNA genes None of the liverwort mitochondrial tRNA genes has a CCA sequence at its 3' terminus, a situation similar to that in other mitochondrial and chloroplast tRNA genes with a few exceptions

69.1%

(2)

(22)

Homology is expressed as a percentage (%) of identical residues per total number of residues.

Genes

Aminoacyl stem

stem

D

loop

Anticodon stem

stem

trnA-UGC trnC-GCA trnD-GUC trnE-UUC trnF-GAA trnG-GCC trnG-UCC trnli-GUG trnl-CAU trnK-WU trnL-UAA trnL-CAA trnL-UAG trnf?-CAU trns-CAU trnN-GUU trnP-UGG trnQ-UUG trnR-ACG trnR-UCG trnR-UCU trnS-UGA

loop

stem

Extra arm

GCTC AATTGGTA GAGC G TATGT TTTGCAA GCATA AAGCT GGGGACG TA TA ACAT AAGGGTA TCCTA TAAA GGCTAAG ATGT A TTGGA GGGGAAA TA GCTT AGTGGTTTA TAGC G CTGGT TTGCAAA CTGTCAA GCCAG AAGTC TC GTCT AGGGGTATA GGAC A TCGTC TTTTCAT GTCGA AAAC GTCCCTT TA GCTC AGGTGGTTA GAGC A AAGGA CTGAAAA TCCTT GTGTC GTTTAGA GCGGAAA TA GCTT AATGGTA GAGT A TAGCC TTGCCAA GGCTA AGGTT TA GATT AAAGGTA AATT A TCTGC CTTCCAA GCAGA GGAT GCGGATA TA T GCAGA TTGTGGC TCTGA AAAC GGCGGATA TA ACTT AGGGGTTA AAGT GGGCTTA GTTT AATTGGTTC AAAC AG CACCG CTCATAA CGGTG ATATT GGGTGTA TA GCTC AGTTGGTA ACAC GAGC ATAGG CTTTTAA CTTAA AGGTC GCGCATT TG GTGA AAAAGGTAA G ACGGA TTTAAAA TCCGT TCCTATTGGTT ACTCGCT TG GTGG AACGGCAA ACAC G GCAGA CTCAAAA TCTGT TTCTAATGGAAT G CCAGG TTTAGGT TCTGG TGACCATAATGTTCGT GCGGATA TA TG ATGG AATTGGTA AATTGGTAG ACAT ACTC G TCAGG CTCATAA TCTGA ATGTT CGCGGGA TA GTTT GAGT GTCGGAA AGTAGGGTA GAAC A GCGGG ATCATAA TTCGC ACAC TCTTTAG TA GCTC AGCGGTTA GAGC A AATGG CTGTTAA CTATT GGGTC CAAGGTG TA GCGC AATCTGGTA GCGC G TCTGC CTTGGGC GCAGA AAGTT TA GCCA AGTGGTA A TCGGC CTTTGAT GCCGA GAAAC TGGAGTA TA AGGC GTGCTTG GCTC AATTGGATA GAGC A CCAAA CTACGGA TTTGG GGGTT GCATTCT TA GCTC AGTTGGATA GAGC A ACAAC GTTGA TGGTC GCATTCT TA GCTC AGTTGGATA GAGC A ACAAC CTTCGAA CTTCTAA GTTGA AGGTC GGATGGA TG TCTG AGCGGTTGA AAGA G TCGGT CTTGAAA ACCGA AGTATTGAGAATACC trnS-GCU GGAGGTA TG AGTGGTTGA AAGC A TTGGT TTGCTAA ATCAA AATACAACAATATTGTATC trn2-GGU GCCCGCG TA GCTG GCTC AGATGGTA GAGC A TTCCC ATGGTAA GGGAA AGGCC GAGT CCTCG TTTACAC CGAGA GAGTC trnV-UAC GGGTAAT TA GCTC AGTTGGTA trnP-CCA AGGAGAA TA GTTC CAATGGTA GAAC AG ATGGT CTCCAAA ACCAA AGGTT trnY-GUA GGGAGAG TG GCCG AGTGGTTAA AAGC G ACAGA CTGTAAA TCTGC TGAAGGTTTTCTAC

Figure 1. Nucleotide sequences of 27 species of tRNA genes in the liverwort tmS-GCU gene.

TYC stem

loop

stem

Aminoacyl stem

GTCGG TTCAAAT CCGAT CGTCTCC GATGG TTCGAAT CCGTC CTTAGCC GCGGG TTCAAAT CCCGT TTTTCCC ACGGG TTCAAAT CCCGT AAGGGAT AGTGG TTCGAAT CCACT TCTAAAC GAGGG TTCAAGT CCCTT TTTCCGC

ATGGG TTCGATT ACGGG TTCGAAT GTAGG TTCGAGT GCAGG TTCAAGT ATTGG TTCAAGT ATCGG TTCGAAT GGGGG TTCGAGT GTGGG TTCGAAT GGGGG TTCAAAT GTTGG TTCGAAT ACAGG TTCAAAT

CCCGT TATCCGC

CCCGT TATTCGC CCTAC TAAGCCT CCTGC TATACCC

CCAAT AATGCGC

CCGAT AGCGAGT CCCTC TATCCGT CCGAC TCCCGCC CCCTC TTCCGAT CCAAC CTAGAGA

CCTGT CACCTTG AAAGG TTCGAAT CCTTT TACTCCA

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

GAGAG TTCAAAT CTTTC CAAGCAT G CCTGT AGGATGC G CCTGT AGAATGC G CCCTC TCCATCC G

ACAGG TTCAAAT ACAGG TTCAAAT GGGGG TTCGAAT ATGGG TTCAAAT TCCGG TTCAAGT AGCGG TTCAAGT AAGGG TTCGAAT GTAGG TTCGAAT

CCCAT TTCCTCC G CCGGT CGTAGGC T CCGTT ATTACCC A CCCTT TTCTCCT G CCTGC CTCTCCC A

mitochondrial genome. The arrowhead indicates the splicing site of the intron in the

.~ ~

such as the potato trnI-L*AU gene that encodes the CCA triplet at the 3' terminus (9). So far, thirty-two introns have been found in the 17 identified genes of the liverwort mitochondrial genome.

Nucleic Acids Research, Vol. 20, No. 14 3775

However, only the liverwort mitochondrial trnS-GCU gene has

a group II intron in the anticodon stem. None of the tRNA genes in mitochondrial genomes of higher plants is known to have an intron. Liverwort tRNA genes for phenylalanine, isoleucine (CAU anticodon), initiator methionine, serine (GCU and UGA

anticodons), proline, tyrosine, glutamine, lysine, aspartic acid, glutamic acid, cysteine, and glycine (GCC anticodon) show very high sequence similarities to the corresponding 'native' tRNA genes in the mitochondrial genomes of higher plants (Table 1), although the liverwort trnjM gene exhibits less homology to those

of wheat and Oenothera (12, 13). By contrast, the similarities of liverwort mitochondrial tRNA sequences with liverwort chloroplast counterparts are moderate ranging from 50.3% for trnM to 77.2% for trnN-GUU (14). This led to the conclusion that the liverwort mitochondrial genome contains no 'chloroplastlike' tRNA gene. Acquisition of tRNA genes from the chloroplast genome and loss of corresponding 'native' genes might have occurred only in the mitochondrial genome of higher plants after the divergence from bryophytes.

Gene

duplications Twenty-five species of tRNA genes are present as a single copy, but trnjM and trnY genes are duplicated in the liverwort

Figure 2. Location of 29 tRNA genes in the liverwort mitochondrial genome. The tRNA genes are indicated by the one-letter amino acid code with their anticodons given in parentheses. Mitochondrial genes (rrn18, rrn26, rps2, rps4, orfl37, nad3, orf62 and atp9) are also shown with their relative location. Genes shown outside the map are transcribed counterclockwise, and those inside are transcribed clockwise. The asterisk indicates the tmS-GCU gene having an intron.

(A)

mitochondrial genome. The duplicated trnjM genes are encoded upstream and downstream from the 26S rRNA gene on the same DNA strand. Both have a completely identical nucleotide sequence. The homology of the repeated sequences extends to the 5' and 3' flanking regions including a few base substitutions (Figure 3A). It is uncertain whether both are transcribed in the liverwort mitochondria. The trnYgenes also exist in the regions of repeated sequences (about 800 bp long) that are located more

145607 GATAAATAATGATGAATCCTCATTAATAAGGAATTATCATAAATAAAGAATGATGAATCATCATAGATAAATAAAAGGCGATAAACAAAAATATATTTCTTTTTTTGTTGGCT 151133 TTGCGTAGCCTGCCTTTAGCCAAAGCCCGAACGA iAiTATiAAAG;AATGAiTA OTCAGCATAGATAAATAAAAGGCGATAAACAAAA.TATATTTCTTTTTTTGTTGGCT

GT7CWGGaWd6

lftltttttftftkti>ttftftttt#tftika"T

GGTGATAAAACGGGTGAGAAAACAAAAATGCAAAAAGG

AACAACCCACCCACAAAACCAAAAACTGTCCGACAAAACAAAGGTTTGATTATGTCTCCCCTCTCGGGCATGCAAATGATAATGGGTCAGGTACACAAAACTGTAGGACGA 14 52 62 AACAACCCACCCACAAAACACGAAGTCCGGTTCTAGGTATAACTTTCTGAr=TTTccCCCGCCTGGGGCTTAGcccGTATTTTCAAAAAATCTATAGAAGTCGCTTATAT 150o7 88 (3) 14 8 36 CGCACTCTGCGCCQCGCCCCGGGGCCGTCTTAGGCTTCTGAGAACCAATAACACGTAAACTATGTAAAACTAAAGTAAATCAATATACAAAAAACACGAATTATAATTTCC TC C 7 9 962 GTAAGAAAGGGTTAGCTAATAAAAAAATGAAcTcTTcTGAGAAccAATAAcAcGTAAAccATGTAAAAcTAAAGCAAATCAATATCCAAAAAACACGAATTCGAACTTCCTCC AA-AATAGTTTTTAATACAAAATTCATTATATTTCGTCGTGTGTTGAACCAGATCA AAACCAAGAAAACGCAAAGCA_AAAATATATATATTTTTTT-TCGTTTTATTCTATTGGA

AAAATAGTTTTTAATACAAAATTCATTATATTTCGTCGTGTGTTGAACCTGATCAAAACTAAGAAAACGCAAAGCAAAAAAAAATATATATATTTTTwTGTTCGTTTATTCTATTTGA C ~TCTTTCQGCCTTCATTCGCGATGCGAATAAAGCGGGAGAAGAGAAGCAAGCTTCT CCGGATTCACTCTTTTTTGCGAAGTGGTTAGTAATGTACTC .: ..... .

.. . .. . . . ....................................... ......

CCCTTCTTTTTCAGCCTTCATTCGCGATGCGAATAAAGCGGGGAGAAGAGAAGCTTGCTTCTTTCTTCTCTGGAGTTCACTCTTTTTTTGCGAAGTGGTTAGTGiiATG6t=CEe

CTTAG;CTCAG

TGTAGAATGC

.... ... ... ... ...

....

...

.

.

TAAAGGGCACAATGAATAATATATACCAACGGTAATCCTTCTACTTGTTTA .. .......... AAAGTGTACAATGAATAATATATACCAACGGTAATCCTTCTACTTGTTTA

...

.

.

...

CrnR-UCG

ATTAGTATAAAGGAACAACGTTACACGCGTGGATTGACCTATTTTTCACCAGAGTCCCGTGGCACCGGAAAGTAG8AGCTTACTTATCATGG AGTGGCC ........................ ATTAGTATAAAGGAACA

terY-GUA

c ACGTTACACGCGTGGATTGACCTATTTTTCACCAGAGTCCCGTGGCACCGGAAAGTAGAAGTTTACTTATCAT_

AGACTGTAAACTGCTGAAGGTTTTCTACGTAGGTTOGAATCCTGcCTCTCC AGTATACTTCGTATTTTTCAAATAGAGGCTGGGACCCAAGCCCCAAAAACCACAATATCT 15528 AGACTGTAAATCZ CSGAAGGmTC$ACGTAGGTTCGAASTC$G4C tC GTATACTTCGTATTTTTCAAATAGAGGCGAAGCACATATTTTTGTGTTTAACCCTTCAT 79255

(C)

trnYr UA

20052 CGCACCTACGAGCCTGAAGTGA TCCCTTTCGTCTACGGGTATAGGACATCGTCTTTTCATGTCGAAAACACGGGTTCAAATCCCGTAAGGGAT CTCTTCCTTACCTTC 18657 TTATATGGAGGTGCA TTTTGTTTTCTCGTTGCGA A CTTTTTTGGAA GACAGGGTT CAAATCGTAAGGGGCGAAGCACCT-ACCGTT

ACTAAGGTTGCTCTAAAAGCAAAGTGAAAAAAGCTTTCTGGCGCACGGGGGAGGTTTTGAAAAAAAAAAGAATAAACTTCAATGCTTTTAATTATCCTGGTTTTCGTTTGGTAC 20279 TATTATGCAAAAAGACAACTTG 18882 ACTACGGTTGCTCTAAAAGCAGAGCGAAAAG-GCTTTCTGGTGCACGGATTTTTACAAAAAAAAAATACTT (D) 145510 TCTTTTTTTGTTGGCTGT GCGGGATAGAGTAATTGGTAACTCGT CATAA GAATGTTGTG TCGATccACTccc GGTGATAAA 145407 34122 CGAAGCGTTAGACTTTGATGATGTTCGCGGGTAATTGGTAACTCGTCAGGCTCATAATCTGAATGTTGTAGGTTGGAAAGTTGCACGATGTATGAAGGGCGAGGGC 34227

Figure 3. Location and comparisons of repeated sequences of tRNA genes. (A) tmflM gene with the flanking regions, (B) tmY and trnR genes with their flanking regions, (C) partial repeated fragment of tmnE and (D) tinnM genes. Numbers indicate the positions of the repeated sequences in the complete nucleotide sequence (accession number M68929 in the GenBank Data Library).

3776 Nucleic Acids Research, Vol. 20, No. 14 Table 2. Total codon usage of functionally known protein genes and tRNA genes in the liverwort mitochondrial genome

UUU Phe 441 UUc Phe 129 trnF-GAA UUA Leu 350 tnL-UAA UUG Leu 170 tnL-CAA cUU Leu 164 CUC Leu 36 CUA Leu 97 trnL-UAG CUG AUU AUC AUA AUG

Leu Ile Ile Ile Met fMet GUU Val GUC Val

47 379 93 214 trnl-CAU 206 trnM-CAU

188 68 GUA Val 160 GUG Val 98

UCU Ser UCC Ser UCA Scr UCG Ser CCU Pro CCC Pro CCA Pro CCG Pro ACU Thr ACC Thr ACA Thr ACG Thr

148 80 114 43 122

trnS-UGA

60 89

trP-UGG

34 153 80 101 45

trnT-GGU

UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG

Tyr 238 Tyr 53 trnY-GUA 16 8 ter His 123 His 32 trnH-GUG Gln 209 trnQ-UUG Gln 48 Asn 220 Asn 68 trnN-GUU Lys 335 trnK-UUU Lys 101 ter

Cys Cys

GGU GGC GGA GGG

Gly 201 Gly 77 tmG-GCC Gly 192 tmG-UCC Gly 67

trjMf-CAU trnV-UAC

GCU GCC GCA GCG

Ala 235 Ala 86 Ala 142 tmA-UGC Ala 48

GAU GAC GAA GAG

Asp 163 Asp 51 tmD-GUC Glu Glu

176 tmnE-UUC 68

82 43 6

UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG

ter

trnC-GCA

Trp 125 tmW-CCA Arg 95 tn,R-ACG Arg 41 Arg 75 trnR-UCG Arg 12 Scr 123 Scr 65

Arg 104 Arg 31

trnS-GCU trnR-UCU

Termination codons (UAA, UAG, and UGA) are indicated by ter. The codon AUA for isoleucine is presumably decoded by an isoleucine tRNA with a LAU anticodon (9). han 60 kb apart on the opposite DNA strand. Interestingly, these repeated sequences also contain trnR genes 143 bp upstream apart from tmrYgenes, but the tmnR genes are not identical (Figure 3B). There are three nucleotide substitutions between the two trnR genes. One of the substitutions is located at the third position of the anticodon resulting in two different anticodons, UCU and UCG. The other substitutions are in the extra loop and acceptor stem and do not disrupt the clover leaf structure of the tRNAs. Since the two trnR genes are present in the regions of repeated sequences, one must be derived from the other by gene duplication. It is of interest to know which tRNA is an original copy. There are 6 codons corresponding to arginine in plant mitochondrial genomes. The liverwort tmnR-UCU gene is the sole tRNA gene corresponding to the AGR codons. On the other hand, there are the two tmR-UCG and trnR-ACG genes corresponding to the CGN codon box. In chloroplast genomes of higher plants, trnR-ACG is the only tRNA gene for the CGN codon box, and the tRNA has been shown to be able to recognize the four codons, since the tRNAArg (ACG) has in fact an ICG anticodon (15). In potato mitochondria, there is also one tRNA,rg (ICG) which is able to read the four codons in the CGN box (1). If this is a case in the liverwort mitochondria, the liverwort mitochondrial trnRUCG gene was possibly derived from trnR-UCU gene by gene duplication and mutation during evolution. We found another two duplications of portions of tRNA genes. One has the 3' portion of the trnE gene with its 3' flanking region (Figure 3C) and the other has only the central portion of the tmJM gene (Figure 3D). Neither of them can form a familiar cloverleaf structure, suggesting that they are not active tRNA genes.

Anticodon table Mitochondrial genomes of higher plants have been shown not to contain tRNA genes for leucine, threonine, alanine, and arginine, whereas the liverwort mitochondrial genome has 'native' tRNA genes corresponding to all amino acids including the four amino acids mentioned above. In spite of the smaller size of the liverwort mitochondrial genome, the number of tRNA genes is higher than those in higher plants. Table 2 is an anticodon table of all of the identified tRNA genes in the liverwort

mitochondrial genome with codon usage obtained from the 30 functional genes known (8). Mammalian and yeast mitochondrial genomes have a minimum set of tRNA genes for codon recognition. The four codons for a single amino acid in a four codon box are read by one species of tRNA using U:N wobbling between the first base of anticodon and the third base of the codon. The wheat mitochondrial genome has been reported to contain two species of tRNA genes, trnS-UGA and trnS-GGA for the UCN codons of serine. However, the latter is a 'chloroplast-like' tRNA that is thought to have been acquired from the chloroplast genome recently during evolution. This does not exclude the possibility that plant mitochondrial genomes originally had two or more species of tRNAs for a four codon box. In fact two 'native' tRNAs for the GGN codon box for glycine are found in the liverwort mitochondrial genome. This indicates either that the progenitor of mitochondria had more than the minimum set of tRNAs for codon recognition and that extra tRNA genes have been lost in mammalian and yeast mitochondria to economize their genomes, or that there has been more than one endosymbiotic event during evolution (16). There are two species of tRNAs for the CGN codon box for arginine in the liverwort genome. However, one of these tRNAs was duplicated from the trnR-UCU gene as mentioned above. Two synonymous codons NNY and NNR in two codon boxes are decoded by tRNA(GNN) and tRNA(UNN), respectively, in mammals and yeast. In the liverwort mitochondrial genome, the UUG codon in the UUR codon box for leucine can be recognized by tRNAIeU (CAA) in addition to tRNALeu (UAA). This indicates that the liverwort mitochondrial genome still has extra tRNA species. We could not find species of tRNAs differing in their sequences but with the same anticodon in the liverwort mitochondrial genome. All 61 sense codons are used in the genes of known proteins in the liverwort mitochondrial genome (Table 2). The 27 anticodons are capable of reading almost all the codons except for ACR (threonine) and AUY (isoleucine) even if G:U and U:N wobbling is allowed. We found only one kind of tRNA gene, the tmT-GGU gene for ACN codon box and the trnI-CAU gene for AUU, AUC, and AUA. However, the first letter of the

Nucleic Acids Research, Vol. 20, No. 14 3777 anticodon of tRNAIle (CAU) is post-transcriptionally modified from cytidine to a lysidine-like residue in potato mitochondria and the tRNAIle (L*AU) is supposed to recognize only the AUA codon (9). Therefore at least two species of tRNAs with anticodons complementary to ACR codons for threonine and AUY codon for isoleucine are thought to be required because the respective codons (ACR and AUY) are used in the liverwort mitochondrial genome (Table 2). There are several possibilities for recognition of ACR and AUY codons in the liverwort mitochondria. The first possibility is that the tRNAs transcribed from the identified tmT-GGU and tmI-CAU genes recognize the ACR and AUY codons, respectively, by a 'two out of three' mechanism (17). Genes of tRNAs for threonine and isoleucine have not been found in mitochondrial genomes of higher plants except for the trnI-L*AU gene. Therefore, those tRNA genes may have been lost from the mitochondrial genome in the land plants before the divergence from bryophytes. The second possibility is that there still are unidentified tRNA genes which do not form a clover-leaf structure as reported in a mammalian tRNAser (18) and worm mitochondrial tRNA genes (19). The third possibility is that we have not found the corresponding tRNA genes because of the presence of large introns in the coding regions. The fourth possibility is that liverwort mitochondria have plasmid-like DNA containing active tRNA genes, although we could not find plasmid-like molecules upon electrophoresis or electron microscopy (8). The last possibility is that the missing tRNAs for threonine and isoleucine may be encoded by the nuclear genome and imported into the mitochondria. There are several reports to support the conclusion that some of the tRNAs encoded by the nuclear genome are imported into plant mitochondria from the cytoplasm (1-4).

ACKNOWLEDGEMENTS This research was supported in part by a Grant-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Science, and Culture, and by The Yamada Science Foundation. The authors thank Professor Emeritus H.Ozeki, Kyoto University, and Professor R.B.Hallick, University of Arizona, for their critical reading of the manuscript.

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Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia ...
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