A deviant mitochondrial genetic code in prymnesiophytes (yellow-algae): UGA codon for tryptophan

Curr Genet (1997) 32: 296–299

© Springer-Verlag 1997

O R I G I N A L PA P E R

Yasuko Hayashi-Ishimaru · Megumi Ehara Yuji Inagaki · Takeshi Ohama

A deviant mitochondrial genetic code in prymnesiophytes (yellow-algae): UGA codon for tryptophan

Received: 23 December 1996 / 23 June 1997

Abstract The sequence of a representative mitochondrial gene COXI, encoding cytochrome c oxidase subunit I, was determined in five species that cover all the orders of the Prymnesiophyta with the exception of the Pavlovales. Through this analysis, we noticed that the ‘stop’ codon UGA appears frequently and, specifically, at conserved tryptophan (Trp) sites of the gene. We showed these sites were not edited in the corresponding mRNA in one of these species, Isochrysis galbana. Therefore, it is most likely that the UGA codon is used for Trp, and not as a stop codon, in prymnesiophytes. All the analyzed prymnesiophytes made a tight cluster on the COXI phylogenetic tree which includes representative species of green-algae, land plants, yellow-green algae, eustigmatophytes and a redalga. This suggests a monophyletic origin for the prymnesiophytes. The same deviant genetic code, i.e. UGA for Trp, has also been found in the red-alga, Chondrus crispus. In spite of the fact that this red-alga and the prymnesiophytes, share the same deviant genetic code for Trp, close affinity between the two groups was not statistically supported by the phylogenetic analysis of COXI sequences. Key words Prymnesiophyta · Deviant genetic code · Cytochrome c oxidase subunit I · The codon-capture theory

Y. Hayashi-Ishimaru1 · Y. Inagaki · T. Ohama (½) Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-11, Japan M. Ehara Department of Biology, Faculty of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560, Japan Present address: 1 Department of Cell Biology, National Institute for Basic Biology, Okazaki, Aichi 444, Japan Communicated by M. Yamamoto

Introduction

The mitochondria (mt) of land plants and a brown alga Pylaiella littoralis (Fontaine et al. 1995) use the universal genetic code where UGA functions as a stop codon, triggering the release of nascent peptides from ribosomes. In contrast, the mt of fungi, protozoans and animals utilize the UGA codon for Trp (for a review, see Osawa et al. 1992) or, as sometimes happens, this codon is not used throughout their mitochondrial genome (Feagin et al. 1992; Kario et al. 1994; Paquin and Lang 1996). Considering the close relationship between green-algae and land plants, it has been generally assumed that the genetic code of greenalgae mitochondria would resemble that of land plants. However, this viewpoint changed when Hayashi-Ishimaru et al. (1996) revealed the existence of deviant genetic codes in several colony forming green-algae. In these the UAG codon is used for leucine or alanine depending on the species. We also reported another type of mitochondrial genetic-code change in xanthophytes, where the AUA codon is used for methionine (Ehara et al. 1997). The first report of a mitochondrial deviant code in algae was that of UGA being used for Trp (UGA/Trp) in a red-alga, Chondrus crispus (Boyen et al. 1994). We have now found the same type of mitochondrial deviant code in algae belonging to the Prymnesiophyta, a phylum of yellow-algae.

Materials and methods Organisms. The algal strains used in this study were purchased from the National Institute for Environmental Studies (NIES, Japan) or the Center for Culture of Marine Phytoplankton (CCMP, USA). They were cultured following the conditions recommended by the stock centers that furnished them. Collected cells were stored at –120°C until use. The traditional classification of the organisms analyzed in this study is as follows: Syracosphaera sp. (CCMP 875) is a member of the order Coccosphaerales; Cricosphaera roscoffensis (NIES 8), Isochrysis galbana (CCMP 1323) and Gephyrocapsa oceanica (NIES 353) belong to the order Isochrysidales, and Phaeocystis pouchetii (NIES 388) is a member of the order Prymnesiales.

297 1 min at 50°C and 2 min at 70°C using a thermal cycler model 480 (Perkin-Elmer, USA). The expected size of PCR product was cloned into the TA-vector (Invitrogen, USA). DNA sequences were determined by the dye-terminator cycle sequencing method, using a DNA sequencer model 377A (Perkin-Elmer, USA) following the manufacturer’s instruction. For Syracosphaera sp. and P. pouchetii, a 101-bp region was first amplified using another set of primers (p2A and p2B, Fig. 1 B). Then, in order to extend the region towards the 5′-upper (or 3′-lower) end, PCR was carried out with p1A (or p1B) and a specific primer (syA, syB, phA or phB1) located inside the previously sequenced region (Fig. 1 B). The merged sequence gave a total of 366 bp. For P. pouchetii, we further extended the sequenced region to 1059 bp using a non-degenerate primer (phB2) and a degenerate primer p1C (Fig. 1 B). Fig. 1A–C Schema of the analyzed region of the COXI gene and location of the primers used for PCR. An arrowhead indicates the location and direction of the primers. A for Isochrysidales. B for Syracosphaera sp. and P. pouchetii. C for the coxI cDNA of I. galbana. The sequences of the primers are as follows: p1A: 5′-TTYTTYGGNCAYCCNGARGTNTA-3′, p1B: 5′-GCNACNACRTARTA NGTRTCRTG-3′, p1C: 5′-TGGTTNTTYTCNACNAAYCAYAARGAYAT-3′, p2A: 5’-GGWTTTTTAGGWTTTATWGTWTGA-3′, p2B: 5′-CCACCAAACATWGTWGCAATT-3′, igA: 5′-GCAATAT CTAGTCCTGAATTTGA-3′, igB: 5′-ACCAGCATTTGGAATAG TTTCAC-3′, phA: 5′-CTACAGGAATCAAAATTTTTAGTTG-3′, phB1: 5′-ACCTACAGTATACATATGGTGAGC-3′, phB2: 5′-ACCGAAAACAGCCTTTGAGCTAAA-3′, syA: 5′-CTACAGGCGTTAAAATTTTCAGTTG-3′ and syB: 5′-TCCAACAGTATACATATGATGCGC-3′. R: A or G; Y: C or T; W: A or T; and N is either T, C, A, or G

Preparation of DNA. Frozen cells were pulverized in a Teflon capsule with a tungsten ball by mechanical shaking using a micro-dismembrator (B. Brown, Germany). Total DNA was extracted as described previously (Hayashi-Ishimaru et al. 1996) and used as a template DNA for the polymerase chain reaction (PCR) after purification by silica gel. PCR and sequencing analysis. To obtain a DNA fragment of the mitochondrial COXI gene (approximately 400 bp) from each of the three algae classified as Isochrysidales, we synthesized the degenerate primers, p1A and p1B (Fig. 1 A). For the amplification of a 1059-bp region, p1C was used with p1B. PCR was performed in a 100 µl reaction mixture containing 2.5 mM of each deoxyribonucleoside, the set of primers described above (1.0 µΜ each), 2 units of Ex Taq DNA polymerase (Takara Shuzo, Japan) and 0.25 µg of total DNA as a template. Amplification was achieved by 35 cycles of 1 min at 94°C,

Table 1 TGA at conserved Trp sites in prymnesiophytes

cDNA analysis. Frozen cells of I. galbana were pulverized as mentioned above. Total RNA was extracted by the QuickPrep Total RNA Extraction Kit (Pharmacia Biotech, Sweden), and used as a template for reverse transcriptase after RNase-free DNase (Promega, USA) treatment. First-strand synthesis was carried out with the primer, igA. After the addition of the second primer, igB, the single-strand DNA was converted to double-stranded cDNA (Fig. 1 C). It was amplified under the same conditions as described above to obtain a sufficient quantity of the 305-bp cDNA fragment containing two TGA sites in the corresponding genome DNA (site no. 274 and 309 in Table 1). As a control experiment, first-strand synthesis was carried out without the first primer, igA. The PCR product was cloned and sequenced as described above. Construction of a COXI phylogenetic tree. The sequences obtained from the prymnesiophytes were analyzed phylogenetically along with the published or registered COXI sequences of land plants, green-algae, a red-alga, yellow-green algae and eustigmatophytes. The phylogenetic tree based on the deduced amino-acid sequences was constructed by the neighbor-joining method (Saitou and Nei 1987) utilizing programs in the DNA sequence analysis package SINCA (Fujitsu System Engineering, Japan). Bootstrap re-sampling (500 times) (Felsenstein 1985) was carried out to quantify the relative support for branches of the inferred phylogenetic tree. For all analyses, all TGA sites in prymnesiophytes were treated as Trp.

Results and discussion

We determined a part of the COXI gene (366 bp) for five species belonging to the Prymnesiophyta. These species cover all the orders of the Prymnesiophyta (Green et al. 1989) except for the order Pavlovales. To increase the de-

Site no.a

Species

90

113

173

223

274

309

Land plant Green alga Red alga

Marchantia polymorpha Prototheca wickerhamii Chondrus crispus

W W Wb

W W W

W W W

W W W

W W W

W W W

Prymnesiophyta

Isochrysis galbana Cricosphaera roscoffensis Gephyrocapsa oceanica Syracosphaera sp. Phaeocystis pouchetii

TGA –c – – W

TGA – – – TGA

TGA – – – TGA

W – – – TGA

TGA TGA TGA TGA W

TGA TGA TGA TGA W

a

The deduced amino-acid sequence was numbered based on 359 amino-acid residues (1059 bp) of I. galbana Trp coded by TGA codon (Boyen et al. 1994), while “W” is coded by TGG c No sequence data b

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Fig. 2 The COXI tree based on the deduced 122 amino acids. Evolutionary distances were calculated by Kimura’s two-parameter method (Kimura 1980), and the tree was constructed by the neighbor-joining method (Saitou and Nei 1987). The lineage presumed to possess the deviant genetic code (UGA/Trp) is shown by a dotted line. Bootstrap values are shown at each node. Species included in the trees and their accession numbers are as follows. Green-Algae 1: Chlamydomonas reinhardtii (U03843) and Volvox sp. (D63661). Green-Algae 2: Coelastrum microporum (D63656), Hydrodictyon reticulatum (D63654), Kirchneriella lunaris (D63653), Pediastrum boryanum (D63659), Scenedesmus quadricauda (D63658) and Tetraedron bitridens (D63657). Land Plants: Marchantia polymorpha (M68929), Oenothera berteriana (X05465), Oryza sativa (M57903) and Solanum lycopersicum (X54738). Xanthophytes: Botrydiopsis alpina (AB000203), Botrydium granulatum (AB000204), Heterococcus caespitosus (AB000206), Mischococcus sphaerocephalus (AB000208), Tribonema aequale (AB000211) and Vaucheria sessilis (AB000212). Eustigmatophytes: Eustigmatos magnus (AB000205), Monodus sp. (AB000207), Nannochloropsis oculata (AB000209) and Ophiocytium majus (AB000210). Monodus sp. (CCMP 505) and Ophiocytium majus (CCAP 855/1), which traditionally have been classified as members of the Xanthophyta, are now presumed to belong to the Eustigmatophyta (Ehara et al. 1997). Prymnesiophytes: Syracosphaera sp. (AB000213), Cricosphaera roscoffensis (AB000117), Isochrysis galbana (AB000119), Gephyrocapsa oceanica (AB000118) and Phaeocystis pouchetii (AB000120). Red alga: Chondrus crispus (Z47547)

teced TGA sites in the gene, the sequencing region was extended to 1059 bp for I. galbana (Isochrysidales) and P. pouchetii (Prymnesiales). Phylogenetic analysis of COXI sequences showed that all of the prymnesiophytes make a monophyletic tight cluster with a high bootstrap value of 97% (Fig. 2). We tried to obtain COXI sequences from algae (Pavlova gyrans CCMP 608 and Diacronema vlkianum CCAP914/1) belonging to the order Pavlovales. However, preliminary analysis showed that all the analyzed PCR products were of bacterial origin, probably arising from bacteria resident inside the algae. The deduced COXI amino-acid sequences of prymnesiophytes were compared with those of a liverwort (Oda et al. 1992), a red-alga (Leblanc et al. 1995) and a greenalga (Wolff et al. 1994). Through this analysis, we noticed that the UGA ‘stop’ codon in the five prymnesiophytes appears frequently at conserved Trp sites of the COXI gene (Table 1). It is well known that RNA editing is carried out extensively in the mt of higher land plants (for a review, see Covello and Gray 1989). However, no difference was detected between the genomic DNA and the cDNA se-

quence of I. galbana, providing evidence that TGA sites are not edited in the mRNA of this species. These results suggest that the UGA codon is used for Trp in the Prymnesiophyta. As five species of this phylum share the same deviant code, it seems reasonable to suggest that this noncanonical code existed in a very early ancestor of the prymnesiophytes. Based mainly on their fine structure, C. roscoffensis, I. galbana and G. oceanica have been classified in the same order, Isochrysidales (Green et al. 1989). However, in our COXI tree, one of the Isochrysidales, C. roscoffensis, made a stable pair (bootstrap value 100%) with Syracosphaera sp. which has been classified as a Coccosphaerales (Fig. 2). The other two Isochrysidales, G. oceanica and I. galbana, made up another pair (bootstrap value 85%). Further investigation will be needed to solve the contradictions between the results of the molecular phylogenetic analysis and traditional classification, which is based mainly on morphological characterization. It has been reported that the UGA codon is utilized for Trp in a red-alga, C. crispus (Boyen et al. 1994), and a TrptRNA gene for UGA and UGG codons has been detected in its mitochondrial genome (Leblanc et al. 1995). To the best of our knowledge, in the genetic-code system of greenalgae mitochondria, this codon is used neither as a stop nor for Trp (Boer and Gray 1988; Wolff et al. 1994), whereas it is apparently used as a stop in the mitochondrial genes of land plants and in the NADH dehydrogenase subunit 7 gene of a brown alga (P. littoralis) (Fontaine et al. 1995). Moreover, UGA is used as a Trp codon in the genetic code of mitochondria from animals (for a review, see Wolstenholme 1992), protozoans, yeasts and eubacteria belonging to the genus Mycoplasma or Spiroplasma (Osawa et al. 1992). In eubacteria, the point where UGA/Trp-codon capture occurred has been clearly shown through the analyses of the recognition ability of release factors (Inagaki et al. 1993) and Trp-tRNA genes (Osawa et al. 1992). It is apparent that the frequent occurrence of UGA/Trp-codon capture suggests the existence of a mechanism which specifies the amino acid to be captured by the UGA codon. However, unfortunately, neither our COXI tree (Fig. 2) nor a tree based on small ribosomal RNA sequences (Van de Peer et al. 1996) was reliable enough to statistically deduce how many times such codon capture has occurred in the mitochondrial lineage. Therefore, we will refrain from discussing whether the UGA codon was necessarily captured by a Trp-tRNA, or just by chance, until the frequency of such codon capture becomes clearer. The difference in the genetic code system between mitochondria and the nuclear genome could have functioned as a genetic barrier, blocking gene translocation between the two genomes. Actually, in animals, which share the deviant code UGA/Trp, mitochondrially encoded protein genes are common (Wolstenholme 1992) and probably such genes have been retained in the mitochondrial genome because their length was sufficient to contain at least one non-canonical codon constantly. These considerations suggest that, among the mitochondrial protein genes of prymnesiophytes, sequences long enough to contain at least one

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deviant codon might have resulted in their selective retention in the mitochondrial genome. Acknowledgements M. E. was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. We are pleased to acknowledge the excellent assistance of Ms. Hideko Tanaka.

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