mitochondrial genetic code journal of molecular evolution

7/28/2019 Mitochondrial Genetic Code Journal of Molecular Evolution

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J Mol Evol (1992) 34:254-258 J o u r n a l o fM o l e c u l a r E v o l u t io n~) Springer-VerlagNew York Inc. 1992

G e n e t ic C o d e a n d P h y l o g e n e t ic O r i g in o f O o m y c e t o u s M i to c h o n d r i aPetr Karlovsky and Berthold Fartman nInstitute of Plant Pathology, University of Goettingen, Grisebachstrasse 6, W-3400 Goettingen, Germany

Summar y. We sequenced the 3'-terminal part ofth e C O X 3 gene encoding cytoch rome c oxidase sub-unit 3 from mitochondria of P h y t o p h t h o r a p a r a s i-t i c a (phylum Oomycota , k ingdom Protoc t i s ta ) .Comparison of the sequence with known C O X 3genes revealed that UGG is used as a tryptophancodon in contrast to UG A in the mitochondrial codesof most organisms other than green plants. A v eryhigh AT mutation pressure operates on the mito-chondrial genome of P h y t o p h t h o r a , as revealed bycodon usage and by A+ T content of noncodingregions, which seems paradoxical because AT pres-sure causes tryptophan codon reassignment fromUG G to UGA in mitochondria of most species. Thegenetic code and ot her data suggest that mito chon-dria of Oomycota share a direct commo n ancestorwith mitochondria of plants and that mitochondriaof the ancestor of Planta and Oomyco ta were ac-quired in a second endosymbiotic event, which oc-curred later than the acquisition o f mitoc hondr ia byother eukaryotes.Key words: Genetic code -- M itochondria --Oomycetes -- AT pressure -- Codon usage -- Cy-tochrome oxidase -- Phylogeny -- Endosymbiotichypothesis -- Polyphyletic mitoch ondri al originIntroductionThe phylogenetic origin of mitochondria as eubac-terial endos ymbi onts is generally accepted (Margulis1981). Mito chondr ial genom es o f different eukary-otes exhibit an extraordinary diversity, which sug-gests a poly phyl etic origin (Gray 1989). A clear de-marcat ion line can be drawn between mitochon drialgenomes of plants and that of other eukaryotes. PlantOffprint requ ests to: P. Karlovsky

mitocho ndrial genomes are ve ry large (up to the sizeof a eubacterial genome); even the smallest oneknown is larger than the largest mitochondria l DNA(mtDNA) of a nonplant organism. The open struc-ture of plant mitoc hondri al genomes with its genesfloating in a sea of noncoding sequences stronglycontrasts with the economized structure found inmitochondrial genomes of other organisms. Inter-genic sequences in plant mtDNA contain recom-binogenic repeats that pro mote fr equent genome re-arrangements or even the coexistence of variousDNA molecules in equilibrium with the masterchro moso me (Gray 1989; Palmer 1990). In contrastto the frequent recombination and the resultingstructural divergence, the rate of sequence changeby point mutations is extremely low in plant mi-tochondria, aroun d 100 times slower than for chlo-roplast genomes or animal mitochondria (Palmerand Herbon 1988; Palmer 1990). Additional char-acteristics that distinguish plant mitochondrial ge-nomes from those o f other organisms are the pres-ence of genes for the a subun it o f F1-ATPase, a 5SRNA, and a protein homo logous to the S 13 ribo-somal protein o f E s c h e r i c h i a c o li ; larger rRNAs; andfrequent acquisition o f genes from nuclei and chlo-roplasts, resulting in the forma tion of mosaic ge-nomes (for a review see Levings and Brown 1989).

The most generally applicable metho d for infer-ring long-range phylogenetic relationships within themitochondrial lineage is the comparison of rRNAsequences. The results of such studies indicate thatplant mitochondria, or at least their rRNA genes,originated at a later time in evolution than did themitochondria or their rRNA genes in other organ-isms (Gray 1989; Gray et al. 1989).

Another feature relevant to the evolution of themitocho ndrial geno me is the genetic code. Plant mi-tochondria retain the universal UG G code for tryp-


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Ph. parasiticaDros. yakubaD. melanogasterAsp. nidulansPod. anserinaNeur. crassaOen. berterianaGlycine maxTrit. aestivumZe a maysO r y z a s a t i v a

Ph. paraslticaDros. yakubaO. melanogasterAsp. nidulansP o d . a n s e r i n aN e u r . c r a s s a0 e n , b e r t e r i a n aG l y c i n e m a xT r i t . a e s t i v u mZea maysO r y z a s a t i v a

Ph. parasiticaDros. yakubaD. melanogasterAsp. nidulansPod. anserinaNeur. c r a s s aO e n . b e r t e r i a n aGlycine maxTrit. aestivumZea maysOryza sativa

A A G A T T A A T T A A T C A C C A T T T T A C A A A A C A A C A C C A T T T C G G T T T T G A A G C TA C G T C A T T T A A A T A A T C A T T T T T C A A A A A A T C A T C A T T T T G G A T T T G A A G C AA C G A C A T T T A A A T A A T C A C T T C T C A A A A A A T C A T C A T T T T G G T T T T G A A G O AA O G T T T A G C T G C T T A C C A C T T A A C T G A T C A T C A T C A T C T A G G A T A C G A A T C AA A G A A T A T T T G C T T A C C A T T T A A C T G A O A A C C A T C A T G T A G G T T T T G A A G G TA A G A A T A T T T G C T T A C C A T T T A A C T G A C A A O C A T C A T G T A G G T T T T G A G G G TT C G C C A A T A T C TT G G T O A T C T G A C AA A G G A G C A T C A C G T TG G C T T T G A A G C ATCGCCAATATCTTGGTCATCTGACOAAGGAGCATCACGTTGGCTTTGAAGCAT C G C C A A T A T C T T G G T C A G A T G A C C A A G A A G C A T O A C G T T G G C T T T G A A G O AT C G C C A A T A T C T G G G T O A T e T G A C O A A G A A G C A T C A C G T T G G O T T T G A A G C ATCGCCAATATCTTGGTCATCTGACCAAGAAGCATOACGTTGGCTTTGAAGCA

W W . . . . . . W . . . . .G C T G C A T G G T A O T G G C A T T T TG T T G A T G T T G T T T G G T TA T T T T T A T T TG T A GG C T G C A T . C - ~ T A C T G & C A T T T T G T T G A T G T A G T T T G A T T A T T T T T A T A T A T C AG C T G C A T ~ T A T T G A C A T T T T G T C G A T G T A G T T T G A T T A T T T T T A T A T A T C AG G A A T C T T A T A C I . C I ~ , O A T T T T G T T G A T G T T G T A T ~ u ~ T T A T T T T T A T A T A T A TG G T A T T T T A T A T T G A C A T T T T G T G G A T G T A G T T T ~ e T T T T T T T A T A T G T T TG G A A T T T T A T A O T T . . C a ~ C A T T T T G T A G A T G T T G T T ~ C T A T T T T T G T A C A T T TGCTGOATQGTACTGGCATTTTGTAGACGTGGTTCG6TTATTCCTATTTGTATG C T G C A T G Q T A C T G G C A T T T T G T A G A C G T G G T T O G G T T A T T C C T A T T T G T C TG C T G C A T G G T A C T ( ~ t O A T T T T G T A G A C G T G G T T C C ~ I T T A T T C O C A T T T G T C TG C T G C A T C - ~ T A C T G G C A T T T T G T A G A C G T G G T T C G G T T A T T C C C A T T T G T C TG C T G C A T G G T A C T G G C A T T T T G T A G A C G T G G T T C ~ { ~ T T A T T C C Q A T T T G T C T. . , W W , . .CTGTTTACTGGTGGGGOGGTAATTAATTTATATTTAATAAATTA-ACAATTTC A A T T T A C T G A T G A G G A G G G - - - T A A C C T T T T A T T A T T A A T T A O A T A T O T A TC A A T T T A C T G A T G A G G A G G A - - - T A A - - T T A T A T T A T T A A T T A A A T A T C T A TC A G T A T A T T A C T G A G G T T A T - - - T A A A T T - - - A A A A T T T A T C A O A T A G - T A TCAATTTATTAT.T_C~GGTTCT---TAGTAGATTATTCATOATTTOCAAATTA-C C G T A T A O T A T ~ G G T T C T - - - T ~ T A A A A A T A A T C G A G T A A G G T T G T G A TC T A T O T A T T G G T G C - K 3 G A G G T A T A ~ . . . . G G T T AG A A TC A G T GG A T TG A

C TATC TATTG G TG G G G AG G TATATG .8 . A . . . . . G G AAC G AAAC AG TG G ATTG AC T A T C T A T T G G T ~G G A G G T A T A T G a ~ A A G A A G G G A AC G A A T A A G T G G A T T G ACTATTTATTGGTGGGGAGGCACAT.T_.~AAGAAGGCAACCAACAAGAGCATCGAC T A T T T A T T G G T G G G G A G G T A T A ~ T A - A T . . . A AC . . . .. . . G G A A T - -

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F i g . 1 . M u l t i p le a l i g n m e n t o f 3 ' p a r t s o f m i t o c h o n -d r i a l g e n e s e n c o d i n g c y t o c h r o m e c o x i d a s e s u b u n i t 3a n d t h e i r d o w n s t r e a m r e g io n s . R e a d i n g f r a m e ( - ) a n df i ve c o n s e r v a t i v e t r y p t o p h a n r e s i d u e s ( W ) a r e i n d i c a t e da b o v e t h e s e q u e n c e s . T r y p t o p h a n c o d o n s a n d s t o p c o -d o n s o f t h e c y t o c h r o m e o x i d a s e g e n e s a re i n b o l d t y p e ,T G A c o d o n s a r e u n d e r l i n e d . N o t e t h a t t h e C G G c o -d o n s a r e e d it e d to U G G i n p l a nt m i t o c h o n d r i a l m R N A .A s t e r i s k s i n d i c a t e p l a c e s w i t h i d e n t i c a l b a s e s i n a l l s e -q u e n c e s . F u ll n a m e s o f o r g a n i s m s a n d a c c e s s i o n n u m -b e r s o f th e r e s p e c t iv e s e q u e n ce s i n G e n B a n k a n d E M B Ll i b ra r i e s a re A sperg i l l u s n idu lans ( J 0 1 3 9 0 ) , Drosoph i lam e l a n o g a s t e r ( M 3 7 2 7 5 ) , D r o s o p h il a y a k u b a ( c o m p l e t em i t o c h o n d r i a l g e n o m e s e q ue n c e c o m p i l e d u n d e rD R Y M T C G ) , G l y c i n e m a x ( X 1 5 1 3 1 ) , Neurosporacrassa ( J0 1 4 3 0 ) , Oeno thera ber t er iana ( X 0 4 7 6 4 ) , O r y -za sa t i va (X 1 7 0 4 0 ) , P odospora anser ina (X 1 4 7 3 4 ) , Tri-t i c u m a e s t iv u m ( X 1 5 9 4 4 ) , a n d Z e a m a y s ( X 1 2 7 2 8 ) .

t o p h a n ( T r p ), w h e r e a s m o s t n o n p l a n t m i t o c h o n d r i au s e U G A , a s w e l l a s o t h er n o n s ta n d a r d a m i n o a c idc o d o n s ( r e v i e w e d b y J u k e s a n d O s a w a 1 9 9 0 ) .

In th i s pape r , s imi lar i ti e s be twe e n the ge ne t i c c od ei n m i t o c h o n d r i a o f th e o o m y c e t o u s p l a n t p a th o g e nPhytophthora parasitica a n d t h a t o f g r e en p l a n t s i si n t e r p r e t e d w i t h r e s p e c t t o e n d o s y m b i o t i c t h e o r ya n d i m p l i c a t i o n s f o r th e p h y l o g e n y o f o o m y c e t e s .

M a t e r i a l s a n d M e t h o d sA c l o ne d f r ag m e n t fr o m m t D N A o f P . paras i t i ca D S M 1 8 2 9( d e n o t e d H i n d - A R S I ) , s e l e c t e d f o r it s a b i l i t y t o d r i v e a u t o n o m o u sp l a s m i d r e p l i c a t i o n i n y e a s t ( M f i h l b a u e r 1 9 8 8) , w a s p a r t i a l l y d i-g e s t e d w i t h D N a s e I i n t h e p r e s e n c e o f M n 2+ ( C a m p b e l l a n dJ a c k s o n 1 9 8 0) . F r a g m e n t s 1 5 0 - 2 4 0 b p l o n g w e r e i s o la t e d o n a na g a r o s e ge l, r e p a i r e d b y i n c u b a t i o n w i t h K l e n o w f r a g m e n t o f E .coli D N A p o l y m e r a s e I a n d a l l f o u r d e o x y r i b o n u c l e o t i d e t r i -p h o s p h a t e s , a n d c l o n e d i n t o t h e S i n a i s i te o f p A R S E Q . T h ec l o n i n g v e c t o r p A R S E Q i s a d e r i v a t i v e o f p U C 1 9 , w l hi ch c a r r i e sa y e a s t m a r k e r g e n e U R A 3 a n d a p o l y l i n k e r f r o m p S P 6 4 C S ( E c k -e f t 1 9 8 7) . T h i s p o l y l i n k e r c o n t a i n s o n e S i n a i s i t e fl a n k e d o n b o t hs i d e s b y T th 1 1 l I r e c o g n i t i o n se q u e n c e s w i t h d i f fe re n t c e n t ra lb a s e s . A f t e r t h e c l e a v a g e o f t h e c o n s t r u c t s w i t h T th 1 1 1 I , eache n d o f t h e c l o n e d f r a g m e n t w a s s e p a r a t e l y la b e l e d u s i n g K l e n o wf r a g m e n t a n d a 3 2 p - d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e c o m p l e -m e n t a r y t o t h e respec t i ve p r o t r u d i n g 5 ' n u c l e o t id e . L a b e l e d f r a g -m e n t s w e r e s e q u e n c e d u s i n g a m o d i f i e d c h e m i c a l d e g r a d a t i o np r o t o c o l . A f t e r c o m p a r i n g t h e sequence a s s e m b l e d f r o m o v e r -l a p p in g f r a g m e n t s w i t h G e n B a n k a n d E M B L l ib r a r ie s , a c o d i n gs e q u e nc e f o r th e c a r b o x y t e r m i n a l p a r t o f s u b u n i t 3 o f c y t o c h r o m ec o x i d a s e w a s i d e n ti f i e d . T h e s e c o n d s e q u e n c e u s e d i n t h i s s t u d y( p a r t o f t h e c o d i n g s e q u e n c e f o r t h e a s u b u n i t o f t h e F I - A T P a s e ,A c c . N o . X 1 4 3 5 1 ) w a s f o u n d b y s e a r c h i n g c o m p u t e r l i b r a r i e s f o rP h y t o p h t h o r a m i t o c h o n d r i a l s e q u en c e s .

R e s u l t s a n d D i s c u s s i o nGenetic Code in Mitochondria ofPhytophthora parasiticaI n th e c o u r s e o f a n a ly s i s o f A R S ( a u t o n o m o u s l yr e p l ic a t in g s e q u e n c e s ) f r o m m i t o c h o n d r i a o f P . par-asitica, we se qu e nc e d a 3 ' part o f the ge ne for c y -t o c h r o m e c o x i d a s e s u b u n i t 3 (COX3). F igur e 1s h o w s a n a l ig n m e n t o f th i s s e q u e n c e w i t h h o m o l -o g o u s s e q u e n c e s f r o m o t h e r o r g a n i s m s a n d t h e p o -s i t io n o f f iv e c o n s e r v a t i v e T I p r e s i d u e s . I n Phyto-phthora, a l l o f t h e s e T r p a r e e n c o d e d b y U G Gc o d o n s . B y c o m p a r i n g t h e a l i g n e d s e q u e n c e s w ef o u n d n o d i f f e r e n c e b e t w e e n t h e Phytophthora m i-t o c h o n d r i a l c o d o n a s s i g n m e n t a n d t h e u n i v e r s a lc o d e .

T h e o n l y k n o w n m i t o c h o n d r i a l p r o t e i n c o d i n gs e q u e n c e o f Phytophthora i n a d d i t i o n t o COX3 i s apar t o f the ge ne for the oL subu ni t o f F 1-A TP ase( F r r s te r et a l. 1 9 8 9 , A c e . N o . X 1 4 3 5 1 ) . T h e r e i s n oT r p c o d o n i n t h is s e q u e n c e . A c o m p a r i s o n w i t h h o -m o l o g o u s s e q u e n c e s r e v e a l ed n o d i f fe r e n c e f r o m t h eu n i v e r s a l c o d e ( d a ta n o t s h o w n ) . G r e e n p l a n t s w e r et h e o n l y o r g a n i s m s p r e v i o u s l y k n o w n t o u s e t h eu n i v e r s a l c o d e i n t h e i r m i t o c h o n d r i a ( J u k e s a n dO s a w a 1 9 9 0 ) .

O n e o f t h e f iv e c o n s e r v a t i v e T r p r e s i d u e s i n t h ec a r b o x y t e r m i n a l e n d o f t h e COX3 p r o d u c t i s c o d e df o r b y C G G i n p l a n t m i t o c h o n d r i a ( F i g . 1 ). T h i sc o d o n i s e d i te d to U G G i n m R N A s e q u e n c es (r e-v i e w e d b y C a t t a n e o 1 9 9 0 ) . A n o t h e r d i f f e re n c e b e -t w e e n Phytophthora a n d p l a n t COX3 ge ne s i s the


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256Table 1. Codons or the most frequentlyused amino acids in sequenced parts of COX3and of the gene encoding he a subunit ofthe F,-ATPase from mitoehondrialDNA ofPhytophthoraL euc ine Alan ine Glyc ine Va l ine I so leuc ineTTA 13 GCT 13 GG T 11 GTT 8 ATT 11T T G - - GCC - - GGC 1 GT C - - AT C - -CTT -- GCA 3 GGA 1 GTA 4 ATA --CT C - - GCG 1 GGG - - GT G - -C T AC T G

use of the stop codons UAA (Phytophthora) an dUGA (plants). We do not know whether the UGAtriplet would still functio n in Phytophthora mtDNAproperly as a translation termination signal. How-ever, we predict that because of the extremely highAT pressure (see the next section), most if not allUGA stops have been converted to UAA.

Some species are known to use both UGG andUGA Trp codons in their mitochondria (Andersonet al. 1982; Roe et al. 1985). Because these mito-chondria evolved under a high AT mutatio nal pres-sure, only a small fraction of Trp codons is repre-sented by UGG. Regarding this, we examined theC O X 3 genes used in the alignment in Fig. I. Wecounted 50 Trp codons in C O X 3 genes of two Dro-sophila and three fungal species, 49 were UG A and1 was UGG (in the gene of Podospora anserina).We predict that no UGA Trp codon will be foundin mtDNA of Phytophthora because at least someof the UGG codons found in C O X 3 should havealready been converted to UGA under a strong ATpressure if it were possible. More than 90% of co-dons allowed to contain A or T in the third pos itionare actually NNA or NNT (see the next section).Therefore, we predict that the CCA TIp anticodon(and not UCA) will be found in mitochondria ofPhytophthora. The definitive answer can be onlyobtain ed by analyzing the respective t RN A Tn' se-quences.

A T Pressure and Codon UsageAT pressure is the cause of the UG A capture by Trpin fungal and animal mitochondria and in somebacteria with a very high A/T content (Osawa andJukes 1989; Osawa et al. 1990). Th e presence o f ATor GC pressure can be ascertain ed by calculating theG/C content ofnon codi ng regions and by analyzingcodon usage, especially with respect to the frequencyof A/T versus G/C in the third codon position. Ex-cluding methi onin e and Trp, whi ch possess only onecodon each, codons used in both of the Phytophtho-ra mitochondrial protein coding sequences men-tioned above have A or T in their third position92.4% of the time. This is an indication of verystrong AT pressure. Codons for the five most fre-quently used amin o acids are shown in Table 1. Two

hierarchically ordered preferences governing the co-don usage are evident.1) Preference for A and T ove r G and C in all codon

positions. (Only one leucine codo n consisting ex-clusively of A and T nucleotides is used out ofsix possible codons. The A/T-richest codonsavailable are used in all other cases.)

2) Preference for T ove r A. (In all four amino acidswhere two codons share the same high A/T con-tent, the codon with T in the third position isstrongly preferred.)In the first noncoding region of Phytophthora

mtD NA, the beginning of which is shown in Fig. 1,83 out o f 103 bases following the stop c odon ofC O X 3 are A or T. Two othe r sequenced noncodingfragments with ARS activities, located at differentplaces in the mitochondrial genome, also have ahigh A/T content: ARSII, with a length of 414 bpand 87.7% A+ T, and ARSIII, with a length of 368bp and 78.8% A+ T (Karlovsky and Fartmann, un-published).Fragments with ARS ac tivity are special selectedsequences that may not be representative of the wholemitochondrial genome. Digestion with restrictionenzymes whose target sequences consist exclusivelyof A and T nucleotides can be used to test the dis-tribution of A/T-rich sequences in the whole ge-nome. F6rster et al. (1987) found that digestion ofPhytophthora mtDNA (40-45 kb) with DraI (rec-ognition sequence TTTAAA) produced numeroussmall fragments , all of them less than 1.3 kb. Es-sentially the same result was obtained with AsnI(target sequence ATTAAT; de Cock and Kar lovsky,unpublished), strongly suggesting tha t sequences witha very high A/T content are scattered throughoutthe whole mitochondrial genome, perhaps encom-passing all intergenic spacers.Phylogeny o f Oomycetous MitochondriaThe genetic code is the most conservative evolu-tionary feature for inferring evolutionary relation-ships that can be exploited by analyzing DNA se-quences. Many time-coordinated mutat ional changesare required to achieve a codo n reassignment with-out killing the cell throug h the a ccumul ation o f de-


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P h . p a r a s i t i c a R L I N H H F T K Q H H F G F E A A A W Y W H F V D V V W L F L F V A V Y W W G GN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .D ros . yakuba . H L. N . . S . N . . . . . . . . . . . . . . . . . . . . . . Y I TI . . . . . - 32D . me l anogas t e r . H L , N . . S . N . . . . . . . . . . . . . . . . . . . . . . Y I T I . . . . . - 32Oen, ber t e r i ana , QY LG. L , . E , . V . . . . . . . . . . . . . . . . . . . . . S I . . . . . I 32G l yc i ne max , QY LG. L . . E . . V . . . . . . . . . . . . . . . . . . . . . S I . . . . . I 32Zea mays . QY LG. L . . K . , V . . . . . . . . . . . . . . . . . . P . . S I . . . . . T 31Oryza sa t i va . qY LG. L , , K , . V . . . . . . . . . . . . . . . . . . P , . S I . . . . . I 31Tr i t , aes t i vum . QY LGQM. . K , . V . . . . . . . . . . . . . . . . . . P , . S I . . . . . I 30ASp, nidulans , . A A Y . L . D H . . L . Y . S GI L . . . . . . . . . . . . Y MS . . Y . . Y - 25P od. anser i na . MFA Y . L . D N , . V . . . GGI L . . . . . . . . . . . . Y . S I . Y , , S - 25N eur , c rassa . N FA Y . L . D N . , V . . . GGI L . . . . . . . . . . . . Y I S . . Y . . S - 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

fective proteins (Osawa and Jukes 1989), includingchanges in the codon in question at relevant posi-tions in all genes encoding essential proteins, mu-tations in tRNA genes, and in some cases changesin the specificity of aminoacyl-tRNA synthetasesand release factors.

Alterations in the genetic code are probably re-cent evol utio nary events (Osawa et al. 1990). Cap-ture of the stop codon UGA by Trp in mitochondrialcodes must have occurred after the separation ofmitochondria of nonplant organisms from an an-cestor shared with plant mitochondria (Jukes andOsawa 1990). The multistage process involved incodon reassign ment probably did not occur two suc-cessive times in oomy cetous mitoc hondria : first bythe capture of UG A by Trp and then by reassigningthe stop co don again. In this case, the second changewould have been contraselected by AT pressure.Evidently, oomycetous mitochondria developedfrom the same ancestor as did plant mitochondr iaafter its separation from the nonp lant lineage. Anal-ysis of rRNA sequences an d other specific featuresof plant mitochondria support the origin of thesemitochondria in a second endosymbiotic event. Thepresence of the gene for the a subunit of the F~-ATPase in mitochondria of Phy t oph t hor a (F6rsteret al. 1989) and our results concerning the geneticcode suggest that the second endosymbiotic eventgave rise to an endos ymbio tic lineage that divergedinto plant and o omycetous mitochondria and pos-sibly also into mitochondria of heterokont algae.There was not enough time for the AT pressure inthe evolution of oomyc etes to cause the codon reas-signment.

An alternative hypothesis is that an oomycetousancestor, living in close parasitic contact with a plant,captured a new mitochondrion from the host andsubsequently lost its own. The difficulty with thishypothesis is the assumption that all Phy t oph t hor aspecies with mito chon drial geno mes closely relatedto P. paras i t i ca developed from one plant parasitespecies. However, primiti ve mari ne species, whichare phylogenetically older than species that parasit-ize land plants, have restriction patterns of mtDN Asimilar to that of P. paras i t i ca (de Cock and Bahn-weg 1989). More molecular data on different P h y -t oph t hor a and other oo mycetes species are requiredbefore the hypothesis can be definitively disproved.

257

Fig. 2. Comparisonof carboxyterminalparts of cy-tochrome c oxidasesubunit 3 proteinsas deduced rommitochondrial DNA sequences (see Fig. 1). Aminoac-ids identicalwith hose at homologouspositions of hePhytophthoraparasiticaprotein are indicated by dots;nonidentical residues are abbreviated using the stan-dard one-letter code. The number of positions withidentical residues as compared withPhytophthora(to-tal of 42 positions) is indicated o the right of the se-quences.After the separation of the oomycetous and plant

mitochondrial lineages, AT mutation pressure leadto the non rando m nucleotide composition of non-coding sequences and to a biased codon usage inoomycetous mitochondrial genomes. Genomiceconomization formed mtDNA into a small mol-ecule resembling fungal and animal mitochondrialgenomes. The AT pressure and genomic economi-zation are connected with the replication and repairof DNA and with the competition for limited supplyof energy. The c ommo n d enomina tor of these fac-tors could be the absorbtive nutri tional mode, whichwas the main evolutionary force governing the di-vergence of oomycetes from plants. In any case,these factors have not operated up on the mitoch on-drial genome o f oomycetes long enough to reassignthe Trp codon.

Do other features of oomycetous mtDNA con-firm their close relationship to plant mitoch ondr ia?Unfortunately, molecular analysis of oomycetousmitochondria is in the early stages. Alignment ofthe protein sequence of the fragme nt translated fromC O X 3 of P. paras i t i ca with other c ytochrome oxi-dase sequences (Fig. 2) indicates that oomycetousmitochondria are not more related to mitoch ondriaof Fungi than to that of Metazoa and Planta. How-ever, extensive sequencing is required before aquantitative phylogenetic conclusion can be drawnfrom such sequence comparisons.

The phylogeny of oomycetes is a controversialissue even at the level of kingdoms. Traditionally,these species were grouped in the class Oomyceteswithin Phycomycetes or "lower fungi" within thekingdo m Fungi (Ban" 1983). I n the mod ern systems,they were given the status of phylum (Oomycota)and were first placed in Fungi (Whi ttaker 1969) onlyto be later mov ed into Protoctista (Margulis 1981;Margulis and Schwartz 1988). Protoctista is a poly-phyletic kingdom of mostly unicellular eukaryotesdefined by exclusion (neither plants nor animals no rfungi). It comprises such different groups as proto-zoa, algae, and slime molds. To avoid this artificialgrouping, the suggestion was made to raise majorprotoctist phyla to k ingdom s (Leedale 1974). How-ever, even these efforts did not lead to a satisfyingplacemen t o f oomycetes, e.g., according to the 13-kingdom system ofLeedale, oomycetes are groupedwith brown algae and diatoms (Margulis 1981).


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2 5 8T h e p h y l o g e n e t i c r e l a t i o n s h i p b e t w e e n o o m y -

c e t e s a n d h e t e r o k o n t a l g a e ( e s p e c ia l l y c h r y s o p h y t e sa n d x a n t h o p h y t e s ) i s, h o w e v e r , a l o n g e s t a b l i s h e dc o n c e p t s u p p o r t e d b y n u m e r o u s u l t r a s t r u c t u r a l a n db i o c h e m i c a l c h a r a c t e r s ( r e v i e w e d i n B a r r 1 9 8 3 ). F o re x a m p l e , o o m y c e t e s c o n t a i n c e l lu l o s e i n th e i r c e l lw a l l a n d s y n t h e s i z e l y s in e t h r o u g h d i a m i n o p i m e l i ca c i d - l i k e p l a n t s a n d n o t t h r o u g h a m i n o a d i p i ca c i d - l i k e f u n g i ( L e J o h n 1 9 7 1) . A n o t h e r r e l e v a n tp h y l o g e n e t i c f e a t u r e w a s e s t a b l i s h e d a f t e r a l o n g a n dc o n t r o v e r s i a l d e b a t e ( r e v i e w e d b y S h a w 1 9 83 ) ; t h eo o m y c e t o u s l if e c y c le h a s k a r y o g a m y i m m e d i a t e l yf o l l o w i n g m e i o s i s , a n d t h e i r v e g e t a t i v e p h a s e i s d i p -l o i d . T h e f ir st m o l e c u l a r d a t a c o n c e r n i n g o o m y c e -t o u s p h y l o g e n y e m e r g e d r e c e n t l y ; a s t u d y o f sm a l l -s u b u n i t r R N A s e q u e n c e s d e m o n s t r a t e d a v e r y c l o s er e l a t io n s h i p b e t w e e n Achlya a n d c h r y s o p h y t e s( G u n d e r s o n e t a l. 1 9 8 7 ). T h e a n a l y s i s o f t h e c o d i n gs e q u e n c e s f o r t w o e n z y m e s o f t h e T r p b i o s y n t h e t i cp a t h w a y r e s u l t e d i n p h y l o g e n e t i c t r e e s w i t hPhytophthora s e q u e n c e s l o c a t e d o u t s i d e t h e c l u s te r so f s e v e n f u n g a l g e n e ra , w h i c h i n c l u d e r e p r e s e n t a -t i v e s o f a s c o m y c e t e s , b a s i d i o m y c e t e s , a n d z y g o -m y c e t e s ( K a r l o v s k y a n d P r e l l 1 9 9 1) . A c o m p a r i s o no f a c t in s e q u e n c e s d e m o n s t r a t e d h o m o l o g y o f Phy-tophthora a c t i n s e q u e n c e s t o t h o s e o f Arabidopsis,Hydra, a n d Dictyostelium r a t h e r t h a n t o t h o s e o fAspergillus a n d Saccharomyces ( U n k l e s e t a l . 1 9 9 1 ) .

T h e u s e o f t h e u n i v e r s a l g e n e t i c c o d e i n t h e m i -t o c h o n d r i a of Phytophthora c o n f i r m s t h e c l o se p h y -l o g e n e t i c r e l a t i o n s h i p b e t w e e n t h e s e o r g a n e l l e s i no o m y c e t e s a n d p l a n t s . T h u s , t h e g e n o m e s o f o r g a n -e l le s p r o v i d e i m p o r t a n t c l u e s fo r t h e s t u d y o f e v o -l n t io n . T h i s s h o u l d n o t s u r p r i s e u s w h e n w e b e a r i nm i n d t h a t g e n o m i c m o s a i c i s m i s a b a s i c fe a t u r e o fe u k a r y o t i c o r g a n i s m s a n d t h a t i t p o s s e s s e s a n e v o -l u t i o n a r y r o l e , w h i c h i s v a l u e d e v e n h i g h e r t h a n t h a to f D a r w i n i a n s e l e c t i o n b y s o m e r e s e a rc h e r s ( M a n n1 9 9 1 ) .Acknowledgments. We are gr a t efu l t o H .H. PreU for consul t a-t i o n r e g a r d in g th e m a n u s c r i p t a n d t o M a r g a r e t N e m e c f o r i m -p r o v i n g t h e E n g l is h. P a r t o f th e w o r k w a s d o n e a t Z e n t r a l e sI s o t o p e n l a b o r a t o r i u m f a r b i o l o g i s c h e u n d m e d i z i n i s c h e F o r s c h -ung de r Univers it~ it G6 t t i ngen. Th e wo rk was par t i a l l y funde db y a g r a n t f r o m D e u t s c h e F o r s c h u n g s g e m e i n sc h a f t.Re f e r e n c e sA n d e r s o n S , d e B r u i j n M H L , C o u l s o n A R , E p e r o n I C , S a n g e r F ,Y o u n g I G ( 19 8 2 ) C o m p l e t e s e q u e n ce o f b o v i n e m i t o c h o n -d r i a l D N A c o n s e rv e d f e at u re o f t h e m a m m a l i a n m i t o c h o n -d r i a l g e n o m e . J M o l B i o l 1 5 6 : 6 8 3 -7 1 7B a r r D J S ( 19 8 3) T h e z o o s p o r i c g r o u p i n g o f p l a n t p a t h o g e n s.I n : B u c z a c k i S T ( e d) Z o o s p o r i c p l a n t p a t h o g e n s. A c a d e m i cP r e s s, L o n d o n , p p 4 3 - 8 3C a m p b e l l V W , J a c k s o n D A ( 19 8 0) T h e e ff ec t o f d i v a l e n t c a t i o n so n t h e m o d e o f a c t i o n o f D N a s e I . J B i o l C h e m 2 5 5 : 3 7 2 6 -3735

C a t t a n e o R ( 19 9 0) M e s s e n g e r R N A e d i t in g a n d t h e g e n et icc o d e . E x p e r i e n t ia 4 6 : 1 1 4 2 - 1 1 4 8d e C o c k A W A M , B a h nw e g G ( 19 8 9 ) M o l e c u l a r t a x o n o m y o fPhytophthora. In : Phytophthora-An I n t e r n a t i o n a l S y m p o -s ium, Tr in i ty Coll ege , Dub l in , p 22E c k e r t R ( 19 8 7 ) N e w v e c t o r s f o r r a p i d s e q ue n c i n g o f D N Af r a g m e n t s b y c h e m i c a l d e g r a d a t io n . G e n e 5 1 : 2 4 7 - 2 5 4F r r s t e r H , K i n s c h e r f T G , L e o n g S A , M a x w e l l D P ( 19 8 7) M o -l e c u l a r a n a l y s is o f t h e m i t o c h o n d r i a l g e n o m e of Phytophthora.C u r r G e n e t 1 2 : 2 1 5 -2 1 8F r r s t e r H , K i n s c h e r f T G , L e o n g S ( 19 8 9 ) F t - A T P a s e a s u b u n i ti n t h e m i t o c h o n d r i a l g e n o m e o f t h e P y t h i a c e a e . N u c l e i c A c i d sRes 17:7109G r a y M W ( 19 89 ) O r i gi n a n d e v o l u ti o n o f m i t o c h o n d r i a l D N A .A n n u R e v C e l l B i o l 5 : 2 5 - 4 0G r a y M W , C e d e r g re n R , A b e l Y , S a n k o f f D ( 19 8 9) O n t h ee v o l u t i o n a r y o r ig i n o f t h e p l a n t m i t o c h o n d r i o n a n d i t s g e -h o m e . P r o c N a t l A c a d S c i U S A 8 6 : 2 2 6 7 - 2 2 7 1G u n d e r s o n J H , E l w o o d H , I n g o l d A , K i n d l e K , S o gi n M L ( 19 8 7)P h y l o g e n e t i c r e l a ti o n s h i p b e t w e e n c h l o r o p h y t e s , c h r y s o -p h y t e s a n d o o m y c e t e s . P r o c N a t l A c a d S c i U S A 8 4 : 5 8 2 3 -5827J u k e s T H , O s a w a S ( 19 9 0 ) T h e g e n e t ic c o d e in m i t o c h o n d r i aand chloroplas t s . Exper i ent i a 46:1117-1126

K a r l o v s k y P , P re U H H ( 19 9 1 ) T h e T R P I g e n e of Phytophthoraparasitica e n c o d i n g i n d o l e - 3 - g l y c e r o l - p h o s p h a t e s y n t h a s eN - ( 5 ' - p h o s p h o r i b o s y l ) - a n t h r a n i l a te s o m e r a s e : s t ru c t u r e a n de v o l u t i o n a r y d i s ta n c e f r o m h o m o l o g o u s f u n g a l ge n es . G e n e ,in pr es sL e e d a l e G ( 19 7 4 ) H o w m a n y a r e th e k i n g d o m s o f o r g a n i sm s ?T a x o n 2 3 : 2 6 1 - 2 7 0LeJoh n HB (1971) Enzy me r egula t ion , l ys ine pathw ays and ce l lw a l l s t ru c t u r e s a s i n d i c a t o r s o f m a j o r l i n e s o f e v o l u t i o n o ffungi . Nature 231:164-168L e v i n g s C S I I I , B r o w n G G ( 19 8 9) M o l e c u l a r b i o l o g y o f p l a n tm i t o c h o n d r i a . C e l l 5 6 : 1 7 1 -1 7 9M ann C (1991) Lyn n Margul i s : Sci ence ' s unruly ear th mo ther .Sci ence 252:378-381Margu l i s L (1981) Sym bios i s i n ce ll evolu t ion . Freeman , SanF r a n c i s c oMargu l i s L , Schwar t z KV (1988) F ive k ingdoms . Freem an, SanF r a n c i s c oMi ih lbau er E (1988) Mo lekulargenet i s che Unter suchu ngen anPhytophthora s p e c . P h D d i s s e r ta t i o n , U n i v e r s i t y o f G r t t i n -g e n, G e r m a n y , p p 1 9 3 - 2 0 0O s a w a S , J u k e s T H ( 19 8 9) C o d o n r e a s s i g n m e n t ( c o d o n c a p t u r e )i n e v o l u t i o n . J M o l E v o l 2 8 : 2 7 1 - 2 7 8O s a w a S , M u t o A , J u k e s T H , O h a m a T ( 19 9 0) E v o l u t i o n a r yc h a n g e s i n t h e g e n e t i c c o d e . P r o c R S o c L o n d B 2 4 1 : 1 9 - 2 8P a l m e r JD ( 19 9 0) C o n t r a s ti n g m o d e s a n d t e m p o s o f g e n o m eevolut ion in l and p l ant organel l es . Trends Genet 6 :115-120P a l m e r J D , H e r b o n L ( 19 88 ) P l a n t m i t o c h o n d r i a l D N A e v o l v e sr apid ly in s t ruc ture , but s lowly in s equence . J M ol Evol 28:

8 7 - 9 7R o e B A , M a D - P , W i l s o n R K , W o n g J F - H ( 19 8 5 ) T h e c o m p l e t en u c l e o t i d e s e q u en c e o f t h e Xenopus laevism i t o c h o n d r i a l ge -n o m e . J B i o l C h e m 2 6 0 : 9 7 5 9 - 9 7 7 4Shaw DS (1983) The peren osp ora l e s - - a fungal genet i c i s t ' sn ightmare . I n : Buc zacki ST (ed) Zoospor i c p l ant pa thogens .A c a d e m i c P re s s , L o n d o n , p p 8 5 - 1 2 1U n k l e s SE , M o o n R P , H a w k i n s A R , D u n c a n J M , K i n g h o r n J R(1991) Ac t in i n t he oom ycetou s fungus Phytophthora infes-tans i s the prod uct of s evera l genes. Ge ne 100:105-112W h i t t a k e r R H ( 19 6 9 ) N e w c o n c e p t s o f k i n g d o m s o f o r g a n is m s .Science 163:150-160R e c e i v e d J u n e I 0 , 1 9 9 1 / R e v i s e d S e p t e m b e r 1 6 , 1 9 9 1

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