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Rapid nucleotide sequence analysis of the small subunit ribosomal RNA of Toxoplasma gondii: evolutionary implications for the Apicomplexa
Molecular and Biochemical Parasitology, 25 (1987) 239-246
239
Elsevier MBP 00854
Rapid nucleotide sequence analysis of the small subunit ribosomal R N A of Toxoplasma gondii: evolutionary implications for the Apicomplexa Alan M. Johnson, Peter J. Murray, Susana Illana and Peter J. Baverstock* Unit of Clinical Microbiology, School of Medicine, Flinders University of South Australia, Bedford Park, South Australia (Received 18 March 1987; accepted 18 May 1987)
A method for obtaining a large proportion of the nucleotide sequence of the small subunit ribosomal RNA (srRNA) was applied to the obligate intracellular protozoon Toxoplasma gondii. The method uses reverse transcription of as little as 8 ~g of total cellular RNA. This fast, efficient method has numerous advantages over traditional gene cloning methods when nucleotide sequences are required for evolutionary studies. A phylogenetic analysis of the srRNA sequence data showed that T. gondii is not especially closely related to any other organism for which srRNA sequences are available, including another member of the Apicomplexa. Key words: Ribosomal RNA; Toxoplasma gondii; Phylogeny; Protozoa; Apicomplexa
Introduction
While a primary division of cellular organisms into the Eukaryota and Prokaryota superkingdoms appears to be gaining acceptance [1,2], there is still much controversy at lower taxonomic levels. Depending upon which classification is used, the protozoa (used here as a term of convenience, not a respectable taxon [2]) are either placed into a kingdom of their own, into a subkingdom, or grouped with the green, red and brown algae into the kingdom Protista [2-5]. It is generally believed that of the about 30 000 living protozoan species, about 10000 are parasitic [3]. Many of the important protozoan parasites of humans and other mammals have been placed together in the phylum Apicomplexa because of structural similarities [3,6]. In particular, members of the genus Plasmodium cause significant mortality or morbidity in millions of huCorrespondence address: Dr. A.M. Johnson, Department of Clinical Microbiology, Flinders Medical Centre, Bedford Park, South Australia 5042. * On leave from the South Australian Museum, Adelaide. South Australia 5000.
Abbreviation: srRNA, small subunit ribosomal RNA.
mans, while members of the genera Babesia, Besnoitia, Eimeria, Isospora, Sarcocystis, and Theileria cause very significant financial loss in the livestock industry [6]. Members of other genera of protozoa such as Cryptosporidium and Toxoplasma are known to widely and frequently infect both humans and domestic animals [7,8], and members of the genera lsospora and Sarcocystis can infect humans as opportunists often during the acquired immunodeficiency syndrome (AIDS) [9,10]. However, their phylogenetic relationships are not well known because traditional classifications based on similarities in morphology, development or nutritional characteristics have been difficult to apply to such a diverse group of organisms. The more precise molecular methods usually used to infer phylogeny include isoenzyme electrophoresis, immunological methods, DNA/DNA hybridization, protein and D N A sequencing. However, isoenzyme electrophoresis can be used only for closely related (less than 5 million years) organisms [11]. Immunological and D N A / D N A hybridization techniques have the disadvantage that the building of an n x n matrix (i.e. 10 taxa require 100 reactions) and all reference taxa must be included also. Protein sequencing and DNA
0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
240
sequencing yield character state data that can be stored and used for future comparisons, but these techniques are technically difficult and very time consuming. These problems have led to the use of ribosomal R N A (rRNA) sequence comparisons to infer phylogeny. rRNA is universally functional and can therefore be used as the yardstick for all protozoa. In most eukaryotes, the genes for 18S, 5.8S, and 28S rRNAs are co-linear [12]. The small size of 5.8S rRNA makes the phylogenies determined from them statistically uncertain [13] and this has led to the use of the large subunit rRNA or small subunit rRNA (srRNA) sequences for phylogenetic analysis [14]. The srRNA sequences of about 20 eukaryotes, including one apicomplexan, Plasmodium berghei [15], are known [16]. In this study we have de-
termined the nucleotide sequence of the semiconserved regions of the srRNA of another apicomplexan, Toxoplasma gondii. This sequence was compared with published srRNA sequences in order to infer the phylogenetic position of Toxoplasma, especially with respect to Plasmo-
dium. Materials and Methods
RNA extraction. Bulk, cellular R N A was extracted and purified from tachyzoites of the RH strain of T. gondii as described elsewhere [17] and used at a final concentration of 2.8 mg ml-1
Oligonucleotide primers. The three rRNA primers were purchased from Boehringer Mannheim GmbH-Biochemical (Mannheim, F.R.G.) and
3~6 r. g. T. ~. P. b.
AUUACCCAAU AUUGGCCAAU AUUACCCAAU
CCUGAACCAG CCUAAUUCA[~ UCUAAAUAAG
GGAGG-AGUG GGAGCCAGUG AGAGGUAGUG
AC--GAAAUA ACAAGAAAUA ACAAGAAAUA
T . g. [ . L. P. b .
U-LICU-AGUG U -UCUACGGC GG-UUUUAUA
AUUGGAAUC~A AUUGAAAUGA AUUGGAALJE~A
UAGGA-AUCC G-AACAI3UGU U-GGGAAUUU
ACCCCCCUUU AAAUCUCUUA AAAAC]CULJCC
ACAACACUGG GCAAGCUGGG ACAAUAUAAG
AAA--UUUCU AAACUUACGU GCCAAAUUUU
U A .........A U G U A ....... A U G
4~8
597 I
I.g. F.t. F'.b.
AUAGAUAGAU AUA-AUGGAA AUA-ACAAAA
U C G C C ............... C U A - U U U UAGGACUA AGUCCAUUUU UUGAACAAGU CAAAUUUUUU
GU-GGUUUCU AUUGGUUCUU GUUCUUUUUU
AGGACUGAAG GGAUUU--GG CUUAUUUUGG
CUUAG[JUACG
-r. g. T.t. P. b.
AUUAAUAGGAUUAAUAGGG AUUAAUAGGA
A C G G U G ....... ACAGUU-GGG GUAGCUUGGG
GGG(]UUCGUA GGCAUUAGUA GGCAUUUGUA
UUUAACUGUC UUUAAUAGUC UUCAGAUGUC
AGAGGUGAAA AGAGGUGAAA AGAGGUGAAA
UUCUUAGAUU UUCUUGGAUU UUGUUAGAUU
T . g. T. t . P. b .
UGUUAAAGAC UAUUAA[~GAC UUCUGGAGAC
G-ACUACUGC UAACUAAUGC AAAC, A A C U G C
GAAAGCAUUU GAAAGACUUU GAAAGCAUULI
GGCAAAGAUG GCCAAAGAUG GGCUAAAAUA
UUUUCAUUAA UUUUCAUUAA CUUCCAUUAA
UCAAGAACGA UCAAGAACGA UCAAGAACGA
I.g . .t . P .b .
AAGUUAGGGG AAGUUAGGGG AAGUUAAGGG
CUCGAAGACG AUCAAAGACG AGUGAAGACG
AUC-GAUACC AUCAGAUACC AUCAGAUACC
GUCGUAGUCU GUCGUAGUCLI GUCGUAAUCU
UAACCAUAAA UAACUAUAAA UAACCAUAAA
CUAUGCCGAC CUAUACCGAC CUAUGCCGAC
T .g . b .
UAGAGAUAGG UCGGGAUGGG UAGGUUUUGG
A--AAACGUC CUG-GAAUAA AUG-AAAAULI
A .................................................. UGCU AU ............................................ GUCCA UUUAAAUAAG AUUUCCCUUC GGGGAUUUCU UAGAUUGCUU
T. g. r. t . F'. b.
UGACU-UCUC GUCGGCACCG CCUUCAGUAC
CC-CCUUAUG UAU .... GAG C U U ......A U G
AGAAAUC-AA AGAAGCUAAA AGAAAUCAAA
T .t . P.
887 I
GUGUUUGGG GUCUUUGGG GUCULIUGGG
241
used at a final concentration of 0.1 mg ml 1. The sequence compositions of each of the rRNA primers are: primer A (5'-GWATTACCGC GGCKGCTG-Y), primer B (5'-CCGTCAATI" CMTTTRAGTTT-3'), and primer C (5'ACGGGCGGTGTGTRC-3'). Their complementary binding sites in Escherichia coli using the Brosius et al. [18] srRNA numbering system are: primer A (positions 519-536), primer B (positions 907-926) and primer C (positions 1392-1406).
Dideoxynucleotide sequencing. The sequencing reactions are essentially the same as previously described by Lane et al. [19]. [~-35S]dATP was purchased from Amersham International plc
(Amersham, U.K.) and 35 I~Ci was used in each reaction. Avian Myeloblastosis Virus reverse transcriptase was purchased from Promega Biotec Pty. Ltd. (Wisconsin, U.S.A.) and used at a final value of 6.2 units per reaction. Reactions were heated to 90°C for 2 min prior to loading onto a 6% polyacrylamide, 8 M urea, 0.4 mm thick, non-gradient gel. After electrophoresis at 1200 V gels were fixed in 10% acetic acid, then 20% methanol, and baked onto the glass plate at 110°C for 1 h. The baked gels were then autoradiographed using Kodak X-OMAT X-ray film. At least four sequencing reactions and determinations were carried out for each primer to ensure the accuracy of the nucleotide sequence data obtained. Two reactions were electrophoresed for
1109 I
C~AUGCUAAA -AACC-UGCU -AACC-UGCU
UAGGAUCAGG A A C U A G ..... A A U U A G .....
ACUUCGCU .............................. UCUGCUUGU .................................. CGGCGAGUAC UCUAUAUCCU UUAUUGGGAG
GGGCAUAUU(3
AULIAAUCAAU
[.g. l.t. P.b,
AGACCU---A ACGAGACCUU ACGAGAUCUU
I.g.
- ................................
T.t. P. b .
- ............................... AUUGGUUUUI3 ACGUUUAI_IGI.J
i. g. T. t . P. b.
- .....................................................................
UUCAUUAUGA
UUCUUGCGUU
UACGACAUGC
CUUUUUUCUA
GUAAGGAUGU
UU[gU ........... AAA-UAACA AUUCGCUULJA
T. g. T. t. P. b.
UGUAUCACUU GGUUGUACLIU UUUAAUGCUU
CUUAGAGGG-CUUAGAGGGA CUUAGAGGAA
CU-.UUGCGUG CUAUUGLJGCA CGAUGUGUGU
UCUAACGCAA AUAAGCCAAU CUAACACAA-
GGAAGUUUGA GGAAGUUUAA GGAAGULJUAA
GGCAAUAACA GGCAAUAACA GGCAACAACA
T. g. [. t. P. b.
CGUCUGCUGA GGUCUG-UGA GGUCUG-UGA
UGCLX3UUAGA UGCCCCUAGA LIGUCCUUAGA
UGUUCUGGGC CGUGCUCGGC UAUACUAGGC
UAGCACGCGC C-GCACGCGC U-GCACGCGU
GCUACACUGA GUUACAAUGA GCUACACUGA
UGCAUCCAAC CUGGCGCAAA UAUGUAAAAC
T. g. T. t. P. b.
GAGUUUAUAA AAGUAUU--U GAGUGCUUAA
CCUUGGCCGA CCUGUCCUGG AUUUA--UAUC
UA---GGUCU --,GAAGGUAC --UGUGCUUA
-i[~ ....................................... -GGG ........................................... GGUGUUAAA[.~ CCUAUGLJUUC; AGUAUAUAEIU
] , g. l.t. P. b.
- .................................. A A - - U C U U - ..................................... U A A U C U U UUL]CCUCCAC UGAAAAGUGU AGGLJAALJ(]UU
GUGAGUAU-AUUAAUACCA UAUCAAU-AC
GU--[]GUGAU GU--CGLJGUU AUAUCGUGAU
UGGGGALIU---AGGGAUAGU -GGGGAUAGA
T. g • 7 . t. P. b .
--AUUGGCCA UCUUUG-GAA UUAUUG-CAA
UUAUUAAUCU UUGUGGAUCU UUAUUAAhICU
AUGCCUAGUA AUUUCUAGUA AUGCCUAGUA
GGCGCAAGGU AGUGCAAGUC AGCAUGAUUC
CGCUG---GC AUCAGCUUGC AUCAGAUUGU
T.g. T. t. P ,b .
GC--AUU-CG GUUGAUUAUG GCUGACUACG
UCC U(2C UCC
- ...................................................................
UUAAC-CCGA UGAACGAGGA UGAACGAGGA
U(iItilUUU A[I]CiU
AG
ULIUSCUC;~JULI
1352 I
Fig.
1.
Sequence blocks of T. gondii s r R N A ( T . g . ) aligned against those of Tetrahymena thermophila srRNA (T.t.) and P. berghei srRNA (P.b.). The numbering system is that of the E. coli srRNA sequence [ 1 8 ] .
242
2 ~ h and two reactions were electrophoresed for 6 h to extend the sequence obtained.
Organisms used for sequence comparison. Protozoan species from a broad range of groups whose srRNA sequences are known were used for analysis. According to the classification of Levine et al. [3], the groups are from the three major phyla: Sarcomastigophora; represented by Euglena gracilis [20], Trypanosoma brucei [20] and Dictyostelium discoideum [21]; Apicomplexa represented by T. gondii (this report) and P. berghei [15]; and the Ciliophora represented by Tetrahymena thermophila [22], Euplotes aediculatus [13] and Oxytricha nova [23]. The srRNA sequence of Homo sapiens, Xenopus laevis, and Saccharomyces cerevisiae were taken from Huysmans and De Wachter [16], and of Neurospora crassa from Sogin et al. [24]. Sequence analysis. The sequence blocks obtained from T. gondii were aligned with those of the other 11 species by eye. Only the semi-conserved regions in the primary structure were chosen for sequence analysis and only regions with less than six continuous nucleotide insertions or deletions per block per species were selected [23]. 526 positions were considered in the analysis.
Phylogenetic analysis. The sequence data for the 526 sites were analysed by two general phylogenetic methods - by calculating pairwise differences and by site-by-site character state analysis. Differences were assessed as Knuc values (the rate of nucleotide substitution per site) following the
formula of Hori [25]. Phylogenetic hypotheses were erected from these values by two phylogenetic methods - the Fitch-Margoliash method [26] using the PHYLIP package of Felsenstein, and the Distance Wagner procedure using Farris's [27] algorithm. The Fitch-Margoliash method aims to provide a tree whose topology and branch lengths are such that the distances between taxa on the tree match as closely as possible the input distances, according to the squared differences between input and output values [26]. The Distance Wagner procedure aims for a tree of minimum length [27]. Phylogenetic hypotheses were erected from the character state data according to two criteria - parsimony and compatibility. Parsimony aims for a tree whose topology minimizes the number of postulated changes per character over all characters [28]. Compatibility aims for a tree whose topology minimizes the number of characters that are incompatible with the tree [28]. For parsimony we used the D N A P A R S programme and for compatibility the D N A C O M P programme, both in Felsenstein's P H Y L I P package. Results
Fig. 1 shows the derived srRNA T. gondii sequence aligned against P. berghei srRNA [15] and T. thermophila srRNA [22]. Blocks chosen for analysis were those that were semi-conserved, i.e. showed limited variation relative to the other taxa used. The results of the comparative analysis of primary sequences are shown in Table 1.
TABLE I Knuc values based upon 526 semi-conserved nucleotide sites for T. gondii and 11 othcr taxa
Saccharomyces cerevisiae Dictyostelium discoideum Oxytricha nova Tetrahymena therrnophila Trypanosoma brucei Euglena gracilis Euplotes aediculatus Toxoplasma gondii Plasmodium berghei Neurospora crassa Homo sapiens Xenopus laevis
Sc
Dd
On
Tt
Tb
Eg
Ea
Tg
Pb
Nc
Hs
0.278 0.173 0.184 0.572 (I.523 (I.239 0.216 (I.273 0.127 0.275 0.266
0.289 0.320 0.521 0.527 0.363 0.38(t (I.383 (/.317 0,366 (/.353
0.154 (/.557 0.520 0.159 0.211 0.286 (I.18(I 0.314 0.308
O.588 0,539 (I.226 (I.271 0.333 0.216 0.338 0.326
0.523 0.619 0.614 0.612 0.529 (/.564 0.564
(/.563 0.566 0.593 0.526 0.571 0.571
0.270 0.342 0.236 (I.359 0.344
0.315 0.248 0.362 0.362
0.315 0.402 0,405
0.299 0.288
I).037
Xl
243 Fig. 2A shows the best fit Fitch-Margoliash tree obtained f r o m over 1000 trees tested. It has a % standard deviation of 3.65%. The tree was rooted assuming a trichotomy between T. brucei, E. gracilis, and all remaining taxa [20]. This tree shows Toxoplasma monophyletic with the Ciliophoran taxa Tetrahymena, Euplotes and Oxytricha, with the Fungi (Neurospora and Saccharomyces) the sister-taxon to this clade. The Distance Wagner tree is shown in Fig. 2B. H e r e , Toxoplasma is monophyletic with Plasmodium, and this clade is monophyletic with the Ciliophora. The tree derived using the m a x i m u m parsimony criterion for site-by-site comparison is shown in Fig. 2C. It agrees with the Distance Wagner analysis in showing Toxoplasma and Plasmodium as a clade,
but this time the sister-group to this clade is the Ciliophora along with the Fungi. Fig. 2D shows the tree derived using the m a x i m u m compatibility criterion. This tree agrees with the FitchMargoliash tree in showing monophyly of Toxoplasma with the Ciliophora, but differs in showing Plasmodium as the sister taxon to this clade. Thus with regard to Toxoplasma, the consensus of all four trees is monophyly of Fungi, Ciliophora, Plasmodium and Toxoplasma to the exclusion of Sarcodina as represented by Dictyostelium and Vertebrata as represented by Homo and Xenopus. However, the relationships among Fungi, Ciliophora, Plasmodium and Toxoplasma remain unresolved by the data.
B
NEUROSPORA
A
,~SICCHAROMYCES ----'~--~'~EUPLOTES
~
/,'~/TETRAHYMENA
/
\
N-" . ~
~
0.100
C
"sM°°'uM
/
NEUROSPORA
D
-
/
\ \
~','oxoP,,s.,
\ \
\
~~
-, ,....sMoo.u.
/
/
x .o,,us
'~
DICTYOSTELIUM
NEUROSPORA
SACCHAROMYCES ,~ ~ TETRAHYMENA ,/~ -,~--~-, EuP,o'res ~r~ ~ ~ OXYTRICHA
\\\-,oxoPL,SM, \\-,,sMoo,uM
",~ HOMO
\.
EUPLOTES
~ ~TRYPANOSOMA EUGLENA
SACCHAROMYCES TETRAHYMENA
,~
TETRAHYMENA
~.~
\
7 TRYPANOsOMA EUGLENA
/
NEUROSPORA SACCHAROMYCES
~
~
~ ~ "
~" HOMO XENOPUS
7 2 : 2 °.. Fig. 2. Trees resulting from Fitch-Margoliash analysis (A) and Distance Wagner analysis (B) of the K,uc distances, and maximum parsimony analysis (C) and maximum compatibility analysis (D) of 526 semi-conservedsites. In A and B, the branch lengths shown are proportional to the proposed amount of evolutionary change.
244
Discussion
Traditional taxonomic methods have struggled with the diversity of the protozoa. The classification of the group has been refined and altered many times [1]. The basic result of the taxonomic attempts has been an expansion of the higher taxa in order to incorporate the revelations of great diversity within the kingdom [3,5]. Unfortunately, no system based upon systematic or numerical taxonomy is totally satisfactory for protozoa due to their vast heterogeneity. Nucleotide sequence comparisons between molecules that all organisms possess, and are functionally universal, provide the most quantitative and efficient method of assessing relationships among protozoa. Although r R N A has been highly conserved throughout evolution, the rate of nucleotide substitutions, deletions or insertions at various positions in the srRNA, in particular, is not constant. Some regions are non-variable, unchanged over millions of years of evolution, others are highly variable [12]. The more closely related organisms are likely to be, so the more the variable regions will probably be required for comparison. The srRNA of about 30 organisms have been totally sequenced usually by cloning and then sequencing of the gene or its fragments [16]. However, the requirement to clone the srRNA gene or its fragments prior to sequencing lengthens the already long time period required to obtain the nucleotide sequence by standard D N A sequencing techniques. In addition, it is not necessary to sequence the whole gene to allow accurate phylogenetic comparisons to be made [14]. It is more important to compare certain specific regions such as the semi-conserved areas, rather than have the whole gene sequence [14]. To date this has been accomplished by comparing the semi-conserved regions of entire sequences of the srRNA from about 20 species of eukaryotes [16]. These sequences have been determined by sequencing cloned srRNA genes which can be a tedious and time consuming process. Even though we have cloned fragments of the srRNA gene of T. gondii [29], sequencing these would be a lengthy process. The srRNA can, however, provide large sequence blocks in a short space of time when re-
verse transcribed using universally conserved sites for priming [19]. In fact, primers A and C used here for T. gondii were also used as primers for sequencing the P. berghei srRNA coding region [15]. The simple R N A extraction method used by us is even shorter and more technically simple than that recommended by Lane et al. [19]. This means that we can go from parasite pellet to loading of a D N A sequencing gel in less than 5 days. We foresee a large expansion in the quantitative taxonomy of the protozoa. By simply partially sequencing the srRNA of members of each of the major groups proposed by Levine et al. [3] or Corliss [5], true relationships can be plotted. The result will probably be an increase in the number of higher taxa and a necessary rearrangement of current groupings. In addition, rapid srRNA sequencing of medically important organisms is likely to have great benefit in diagnosis. Specific, simple srRNA 'probes' can be readily identified from srRNA sequences, and species (or even strain) specific srRNA oligonucleotide 'probes' could be applied to human disease diagnosis [30]. We have compared T. gondii sequence blocks with 11 other taxa including three ciliates, a cellular slime mould, two flagellates, two fungi and two vertebrates, as well as another apicomplexan, and then assessed the sequence homologies and structural distances between them by four phylogenetic methods. The trees are in general agreement with previously published trees involving Trypanosorna. Euglena, Dictyosteliurn, Saccharornyces, ciliates and vertebrates [15,2023]. With regard to Toxoplasrna and Plasrnodium, all four trees agree in showing monophyly of Fungi, Ciliophora, Plasrnodiurn and Toxoplasrna to the exclusion of Trypanosoma, Euglena, Dictyosteliurn and vertebrates. However, there was no consensus among the four trees in the phylogenetic relationships among Fungi, Ciliophora, Plasmodium and Toxoplasrna. These data suggest, therefore, that the branches leading to these four taxa all diverged at about the same time. In particular, there is no evidence for an especially close relationship between 7". gondii and P.
berghei. The members of the Apicomplexa have been
245 g r o u p e d t o g e t h e r as a p h y l u m , b a s e d largely o n the possession, visible by e l e c t r o n m i c r o s c o p y , of a n apical c o m p l e x g e n e r a l l y consisting of p o l a r ring(s), r h o p t r i e s , m i c r o n e m e s , c o n o i d a n d subpellicular microtubules present at some stage [31]. T h e results of o u r studies p r e s e n t e d here are consistent with the h y p o t h e s i s that the i n c l u s i o n of o r g a n i s m s i n t o a p h y l u m b a s e d o n possession of s o m e c e r t a i n o r g a n e l l e s m a y n o t be valid. A l t e r natively, e i t h e r P. berghei or T. gondii m a y not be close to the rest of the A p i c o m p l e x a . M o r e studies such as the r a p i d s r R N A s e q u e n c i n g techn i q u e used here will be necessary to a n s w e r these questions.
Acknowledgements This work was partly f u n d e d by a grant to A . M . J . from the N a t i o n a l H e a l t h a n d Medical R e s e a r c h C o u n c i l of the C o m m o n w e a l t h of A u s tralia. P . J . M . is a recipient of a F l i n d e r s U n i v e r sity of South A u s t r a l i a S u m m e r Scholarship. W e t h a n k J. F e l s e n s t e i n , D e p a r t m e n t of G e n e t i c s , U n i v e r s i t y of W a s h i n g t o n , Seattle, for his P H Y L I P package a n d A. G u n j k o a n d M. A d a m s for c o m p u t i n g assistance. D e b b i Sullivan a n d Liz D a v i s o n skillfully typed the m a n u s c r i p t .
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23
24
25 26
hyrnena therrnophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin. J. Biol. Chem. 260, 6334-6340. Elwood, H.J., Olsen, G.J. and Sogin, M.L. (t985) The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata. Mol. Biol. Evol. 2, 399-410. Sogin, M.L., Miotto, K. and Miller, L. (1986) Primary structure of the Neurospora crassa small subunit ribosomal RNA coding region. Nucleic Acids Res. 14, 9540. Hori, H. (1975) Evolution of 5SRNA. J. Mol. Evol. 7, 75-86. Fitch, W.M. and Margoliash, E. (1967) Construction of phylogenetic trees. Science 155,279-284.
27 Farris, J.S. (1972) Estimating phylogenetic trees from distance matrices. Am. Nat. 106, 645-668. 28 Felsenstein, J. (1982) Numerical methods for inferring evolutionary trees. Q. Rev. Biol. 57, 379-404. 29 Johnson, A.M., Illana, S., Dubey, J.P. and Dame, J.B. (In Press) Toxoplasma gondii and Harnmondia hammondi: DNA comparison using cloned rRNA gene probes. Exp. Parasitol. 30 Stahl, D.A. (1986) Evolution, ecology and diagnosis: unity in variety. Biotechnology 4,623-628. 31 Levine, N.D. (1970) Taxonomy of the sporozoa. J~ Parasitol. 56, 208-209.
239
Elsevier MBP 00854
Rapid nucleotide sequence analysis of the small subunit ribosomal R N A of Toxoplasma gondii: evolutionary implications for the Apicomplexa Alan M. Johnson, Peter J. Murray, Susana Illana and Peter J. Baverstock* Unit of Clinical Microbiology, School of Medicine, Flinders University of South Australia, Bedford Park, South Australia (Received 18 March 1987; accepted 18 May 1987)
A method for obtaining a large proportion of the nucleotide sequence of the small subunit ribosomal RNA (srRNA) was applied to the obligate intracellular protozoon Toxoplasma gondii. The method uses reverse transcription of as little as 8 ~g of total cellular RNA. This fast, efficient method has numerous advantages over traditional gene cloning methods when nucleotide sequences are required for evolutionary studies. A phylogenetic analysis of the srRNA sequence data showed that T. gondii is not especially closely related to any other organism for which srRNA sequences are available, including another member of the Apicomplexa. Key words: Ribosomal RNA; Toxoplasma gondii; Phylogeny; Protozoa; Apicomplexa
Introduction
While a primary division of cellular organisms into the Eukaryota and Prokaryota superkingdoms appears to be gaining acceptance [1,2], there is still much controversy at lower taxonomic levels. Depending upon which classification is used, the protozoa (used here as a term of convenience, not a respectable taxon [2]) are either placed into a kingdom of their own, into a subkingdom, or grouped with the green, red and brown algae into the kingdom Protista [2-5]. It is generally believed that of the about 30 000 living protozoan species, about 10000 are parasitic [3]. Many of the important protozoan parasites of humans and other mammals have been placed together in the phylum Apicomplexa because of structural similarities [3,6]. In particular, members of the genus Plasmodium cause significant mortality or morbidity in millions of huCorrespondence address: Dr. A.M. Johnson, Department of Clinical Microbiology, Flinders Medical Centre, Bedford Park, South Australia 5042. * On leave from the South Australian Museum, Adelaide. South Australia 5000.
Abbreviation: srRNA, small subunit ribosomal RNA.
mans, while members of the genera Babesia, Besnoitia, Eimeria, Isospora, Sarcocystis, and Theileria cause very significant financial loss in the livestock industry [6]. Members of other genera of protozoa such as Cryptosporidium and Toxoplasma are known to widely and frequently infect both humans and domestic animals [7,8], and members of the genera lsospora and Sarcocystis can infect humans as opportunists often during the acquired immunodeficiency syndrome (AIDS) [9,10]. However, their phylogenetic relationships are not well known because traditional classifications based on similarities in morphology, development or nutritional characteristics have been difficult to apply to such a diverse group of organisms. The more precise molecular methods usually used to infer phylogeny include isoenzyme electrophoresis, immunological methods, DNA/DNA hybridization, protein and D N A sequencing. However, isoenzyme electrophoresis can be used only for closely related (less than 5 million years) organisms [11]. Immunological and D N A / D N A hybridization techniques have the disadvantage that the building of an n x n matrix (i.e. 10 taxa require 100 reactions) and all reference taxa must be included also. Protein sequencing and DNA
0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
240
sequencing yield character state data that can be stored and used for future comparisons, but these techniques are technically difficult and very time consuming. These problems have led to the use of ribosomal R N A (rRNA) sequence comparisons to infer phylogeny. rRNA is universally functional and can therefore be used as the yardstick for all protozoa. In most eukaryotes, the genes for 18S, 5.8S, and 28S rRNAs are co-linear [12]. The small size of 5.8S rRNA makes the phylogenies determined from them statistically uncertain [13] and this has led to the use of the large subunit rRNA or small subunit rRNA (srRNA) sequences for phylogenetic analysis [14]. The srRNA sequences of about 20 eukaryotes, including one apicomplexan, Plasmodium berghei [15], are known [16]. In this study we have de-
termined the nucleotide sequence of the semiconserved regions of the srRNA of another apicomplexan, Toxoplasma gondii. This sequence was compared with published srRNA sequences in order to infer the phylogenetic position of Toxoplasma, especially with respect to Plasmo-
dium. Materials and Methods
RNA extraction. Bulk, cellular R N A was extracted and purified from tachyzoites of the RH strain of T. gondii as described elsewhere [17] and used at a final concentration of 2.8 mg ml-1
Oligonucleotide primers. The three rRNA primers were purchased from Boehringer Mannheim GmbH-Biochemical (Mannheim, F.R.G.) and
3~6 r. g. T. ~. P. b.
AUUACCCAAU AUUGGCCAAU AUUACCCAAU
CCUGAACCAG CCUAAUUCA[~ UCUAAAUAAG
GGAGG-AGUG GGAGCCAGUG AGAGGUAGUG
AC--GAAAUA ACAAGAAAUA ACAAGAAAUA
T . g. [ . L. P. b .
U-LICU-AGUG U -UCUACGGC GG-UUUUAUA
AUUGGAAUC~A AUUGAAAUGA AUUGGAALJE~A
UAGGA-AUCC G-AACAI3UGU U-GGGAAUUU
ACCCCCCUUU AAAUCUCUUA AAAAC]CULJCC
ACAACACUGG GCAAGCUGGG ACAAUAUAAG
AAA--UUUCU AAACUUACGU GCCAAAUUUU
U A .........A U G U A ....... A U G
4~8
597 I
I.g. F.t. F'.b.
AUAGAUAGAU AUA-AUGGAA AUA-ACAAAA
U C G C C ............... C U A - U U U UAGGACUA AGUCCAUUUU UUGAACAAGU CAAAUUUUUU
GU-GGUUUCU AUUGGUUCUU GUUCUUUUUU
AGGACUGAAG GGAUUU--GG CUUAUUUUGG
CUUAG[JUACG
-r. g. T.t. P. b.
AUUAAUAGGAUUAAUAGGG AUUAAUAGGA
A C G G U G ....... ACAGUU-GGG GUAGCUUGGG
GGG(]UUCGUA GGCAUUAGUA GGCAUUUGUA
UUUAACUGUC UUUAAUAGUC UUCAGAUGUC
AGAGGUGAAA AGAGGUGAAA AGAGGUGAAA
UUCUUAGAUU UUCUUGGAUU UUGUUAGAUU
T . g. T. t . P. b .
UGUUAAAGAC UAUUAA[~GAC UUCUGGAGAC
G-ACUACUGC UAACUAAUGC AAAC, A A C U G C
GAAAGCAUUU GAAAGACUUU GAAAGCAUULI
GGCAAAGAUG GCCAAAGAUG GGCUAAAAUA
UUUUCAUUAA UUUUCAUUAA CUUCCAUUAA
UCAAGAACGA UCAAGAACGA UCAAGAACGA
I.g . .t . P .b .
AAGUUAGGGG AAGUUAGGGG AAGUUAAGGG
CUCGAAGACG AUCAAAGACG AGUGAAGACG
AUC-GAUACC AUCAGAUACC AUCAGAUACC
GUCGUAGUCU GUCGUAGUCLI GUCGUAAUCU
UAACCAUAAA UAACUAUAAA UAACCAUAAA
CUAUGCCGAC CUAUACCGAC CUAUGCCGAC
T .g . b .
UAGAGAUAGG UCGGGAUGGG UAGGUUUUGG
A--AAACGUC CUG-GAAUAA AUG-AAAAULI
A .................................................. UGCU AU ............................................ GUCCA UUUAAAUAAG AUUUCCCUUC GGGGAUUUCU UAGAUUGCUU
T. g. r. t . F'. b.
UGACU-UCUC GUCGGCACCG CCUUCAGUAC
CC-CCUUAUG UAU .... GAG C U U ......A U G
AGAAAUC-AA AGAAGCUAAA AGAAAUCAAA
T .t . P.
887 I
GUGUUUGGG GUCUUUGGG GUCULIUGGG
241
used at a final concentration of 0.1 mg ml 1. The sequence compositions of each of the rRNA primers are: primer A (5'-GWATTACCGC GGCKGCTG-Y), primer B (5'-CCGTCAATI" CMTTTRAGTTT-3'), and primer C (5'ACGGGCGGTGTGTRC-3'). Their complementary binding sites in Escherichia coli using the Brosius et al. [18] srRNA numbering system are: primer A (positions 519-536), primer B (positions 907-926) and primer C (positions 1392-1406).
Dideoxynucleotide sequencing. The sequencing reactions are essentially the same as previously described by Lane et al. [19]. [~-35S]dATP was purchased from Amersham International plc
(Amersham, U.K.) and 35 I~Ci was used in each reaction. Avian Myeloblastosis Virus reverse transcriptase was purchased from Promega Biotec Pty. Ltd. (Wisconsin, U.S.A.) and used at a final value of 6.2 units per reaction. Reactions were heated to 90°C for 2 min prior to loading onto a 6% polyacrylamide, 8 M urea, 0.4 mm thick, non-gradient gel. After electrophoresis at 1200 V gels were fixed in 10% acetic acid, then 20% methanol, and baked onto the glass plate at 110°C for 1 h. The baked gels were then autoradiographed using Kodak X-OMAT X-ray film. At least four sequencing reactions and determinations were carried out for each primer to ensure the accuracy of the nucleotide sequence data obtained. Two reactions were electrophoresed for
1109 I
C~AUGCUAAA -AACC-UGCU -AACC-UGCU
UAGGAUCAGG A A C U A G ..... A A U U A G .....
ACUUCGCU .............................. UCUGCUUGU .................................. CGGCGAGUAC UCUAUAUCCU UUAUUGGGAG
GGGCAUAUU(3
AULIAAUCAAU
[.g. l.t. P.b,
AGACCU---A ACGAGACCUU ACGAGAUCUU
I.g.
- ................................
T.t. P. b .
- ............................... AUUGGUUUUI3 ACGUUUAI_IGI.J
i. g. T. t . P. b.
- .....................................................................
UUCAUUAUGA
UUCUUGCGUU
UACGACAUGC
CUUUUUUCUA
GUAAGGAUGU
UU[gU ........... AAA-UAACA AUUCGCUULJA
T. g. T. t. P. b.
UGUAUCACUU GGUUGUACLIU UUUAAUGCUU
CUUAGAGGG-CUUAGAGGGA CUUAGAGGAA
CU-.UUGCGUG CUAUUGLJGCA CGAUGUGUGU
UCUAACGCAA AUAAGCCAAU CUAACACAA-
GGAAGUUUGA GGAAGUUUAA GGAAGULJUAA
GGCAAUAACA GGCAAUAACA GGCAACAACA
T. g. [. t. P. b.
CGUCUGCUGA GGUCUG-UGA GGUCUG-UGA
UGCLX3UUAGA UGCCCCUAGA LIGUCCUUAGA
UGUUCUGGGC CGUGCUCGGC UAUACUAGGC
UAGCACGCGC C-GCACGCGC U-GCACGCGU
GCUACACUGA GUUACAAUGA GCUACACUGA
UGCAUCCAAC CUGGCGCAAA UAUGUAAAAC
T. g. T. t. P. b.
GAGUUUAUAA AAGUAUU--U GAGUGCUUAA
CCUUGGCCGA CCUGUCCUGG AUUUA--UAUC
UA---GGUCU --,GAAGGUAC --UGUGCUUA
-i[~ ....................................... -GGG ........................................... GGUGUUAAA[.~ CCUAUGLJUUC; AGUAUAUAEIU
] , g. l.t. P. b.
- .................................. A A - - U C U U - ..................................... U A A U C U U UUL]CCUCCAC UGAAAAGUGU AGGLJAALJ(]UU
GUGAGUAU-AUUAAUACCA UAUCAAU-AC
GU--[]GUGAU GU--CGLJGUU AUAUCGUGAU
UGGGGALIU---AGGGAUAGU -GGGGAUAGA
T. g • 7 . t. P. b .
--AUUGGCCA UCUUUG-GAA UUAUUG-CAA
UUAUUAAUCU UUGUGGAUCU UUAUUAAhICU
AUGCCUAGUA AUUUCUAGUA AUGCCUAGUA
GGCGCAAGGU AGUGCAAGUC AGCAUGAUUC
CGCUG---GC AUCAGCUUGC AUCAGAUUGU
T.g. T. t. P ,b .
GC--AUU-CG GUUGAUUAUG GCUGACUACG
UCC U(2C UCC
- ...................................................................
UUAAC-CCGA UGAACGAGGA UGAACGAGGA
U(iItilUUU A[I]CiU
AG
ULIUSCUC;~JULI
1352 I
Fig.
1.
Sequence blocks of T. gondii s r R N A ( T . g . ) aligned against those of Tetrahymena thermophila srRNA (T.t.) and P. berghei srRNA (P.b.). The numbering system is that of the E. coli srRNA sequence [ 1 8 ] .
242
2 ~ h and two reactions were electrophoresed for 6 h to extend the sequence obtained.
Organisms used for sequence comparison. Protozoan species from a broad range of groups whose srRNA sequences are known were used for analysis. According to the classification of Levine et al. [3], the groups are from the three major phyla: Sarcomastigophora; represented by Euglena gracilis [20], Trypanosoma brucei [20] and Dictyostelium discoideum [21]; Apicomplexa represented by T. gondii (this report) and P. berghei [15]; and the Ciliophora represented by Tetrahymena thermophila [22], Euplotes aediculatus [13] and Oxytricha nova [23]. The srRNA sequence of Homo sapiens, Xenopus laevis, and Saccharomyces cerevisiae were taken from Huysmans and De Wachter [16], and of Neurospora crassa from Sogin et al. [24]. Sequence analysis. The sequence blocks obtained from T. gondii were aligned with those of the other 11 species by eye. Only the semi-conserved regions in the primary structure were chosen for sequence analysis and only regions with less than six continuous nucleotide insertions or deletions per block per species were selected [23]. 526 positions were considered in the analysis.
Phylogenetic analysis. The sequence data for the 526 sites were analysed by two general phylogenetic methods - by calculating pairwise differences and by site-by-site character state analysis. Differences were assessed as Knuc values (the rate of nucleotide substitution per site) following the
formula of Hori [25]. Phylogenetic hypotheses were erected from these values by two phylogenetic methods - the Fitch-Margoliash method [26] using the PHYLIP package of Felsenstein, and the Distance Wagner procedure using Farris's [27] algorithm. The Fitch-Margoliash method aims to provide a tree whose topology and branch lengths are such that the distances between taxa on the tree match as closely as possible the input distances, according to the squared differences between input and output values [26]. The Distance Wagner procedure aims for a tree of minimum length [27]. Phylogenetic hypotheses were erected from the character state data according to two criteria - parsimony and compatibility. Parsimony aims for a tree whose topology minimizes the number of postulated changes per character over all characters [28]. Compatibility aims for a tree whose topology minimizes the number of characters that are incompatible with the tree [28]. For parsimony we used the D N A P A R S programme and for compatibility the D N A C O M P programme, both in Felsenstein's P H Y L I P package. Results
Fig. 1 shows the derived srRNA T. gondii sequence aligned against P. berghei srRNA [15] and T. thermophila srRNA [22]. Blocks chosen for analysis were those that were semi-conserved, i.e. showed limited variation relative to the other taxa used. The results of the comparative analysis of primary sequences are shown in Table 1.
TABLE I Knuc values based upon 526 semi-conserved nucleotide sites for T. gondii and 11 othcr taxa
Saccharomyces cerevisiae Dictyostelium discoideum Oxytricha nova Tetrahymena therrnophila Trypanosoma brucei Euglena gracilis Euplotes aediculatus Toxoplasma gondii Plasmodium berghei Neurospora crassa Homo sapiens Xenopus laevis
Sc
Dd
On
Tt
Tb
Eg
Ea
Tg
Pb
Nc
Hs
0.278 0.173 0.184 0.572 (I.523 (I.239 0.216 (I.273 0.127 0.275 0.266
0.289 0.320 0.521 0.527 0.363 0.38(t (I.383 (/.317 0,366 (/.353
0.154 (/.557 0.520 0.159 0.211 0.286 (I.18(I 0.314 0.308
O.588 0,539 (I.226 (I.271 0.333 0.216 0.338 0.326
0.523 0.619 0.614 0.612 0.529 (/.564 0.564
(/.563 0.566 0.593 0.526 0.571 0.571
0.270 0.342 0.236 (I.359 0.344
0.315 0.248 0.362 0.362
0.315 0.402 0,405
0.299 0.288
I).037
Xl
243 Fig. 2A shows the best fit Fitch-Margoliash tree obtained f r o m over 1000 trees tested. It has a % standard deviation of 3.65%. The tree was rooted assuming a trichotomy between T. brucei, E. gracilis, and all remaining taxa [20]. This tree shows Toxoplasma monophyletic with the Ciliophoran taxa Tetrahymena, Euplotes and Oxytricha, with the Fungi (Neurospora and Saccharomyces) the sister-taxon to this clade. The Distance Wagner tree is shown in Fig. 2B. H e r e , Toxoplasma is monophyletic with Plasmodium, and this clade is monophyletic with the Ciliophora. The tree derived using the m a x i m u m parsimony criterion for site-by-site comparison is shown in Fig. 2C. It agrees with the Distance Wagner analysis in showing Toxoplasma and Plasmodium as a clade,
but this time the sister-group to this clade is the Ciliophora along with the Fungi. Fig. 2D shows the tree derived using the m a x i m u m compatibility criterion. This tree agrees with the FitchMargoliash tree in showing monophyly of Toxoplasma with the Ciliophora, but differs in showing Plasmodium as the sister taxon to this clade. Thus with regard to Toxoplasma, the consensus of all four trees is monophyly of Fungi, Ciliophora, Plasmodium and Toxoplasma to the exclusion of Sarcodina as represented by Dictyostelium and Vertebrata as represented by Homo and Xenopus. However, the relationships among Fungi, Ciliophora, Plasmodium and Toxoplasma remain unresolved by the data.
B
NEUROSPORA
A
,~SICCHAROMYCES ----'~--~'~EUPLOTES
~
/,'~/TETRAHYMENA
/
\
N-" . ~
~
0.100
C
"sM°°'uM
/
NEUROSPORA
D
-
/
\ \
~','oxoP,,s.,
\ \
\
~~
-, ,....sMoo.u.
/
/
x .o,,us
'~
DICTYOSTELIUM
NEUROSPORA
SACCHAROMYCES ,~ ~ TETRAHYMENA ,/~ -,~--~-, EuP,o'res ~r~ ~ ~ OXYTRICHA
\\\-,oxoPL,SM, \\-,,sMoo,uM
",~ HOMO
\.
EUPLOTES
~ ~TRYPANOSOMA EUGLENA
SACCHAROMYCES TETRAHYMENA
,~
TETRAHYMENA
~.~
\
7 TRYPANOsOMA EUGLENA
/
NEUROSPORA SACCHAROMYCES
~
~
~ ~ "
~" HOMO XENOPUS
7 2 : 2 °.. Fig. 2. Trees resulting from Fitch-Margoliash analysis (A) and Distance Wagner analysis (B) of the K,uc distances, and maximum parsimony analysis (C) and maximum compatibility analysis (D) of 526 semi-conservedsites. In A and B, the branch lengths shown are proportional to the proposed amount of evolutionary change.
244
Discussion
Traditional taxonomic methods have struggled with the diversity of the protozoa. The classification of the group has been refined and altered many times [1]. The basic result of the taxonomic attempts has been an expansion of the higher taxa in order to incorporate the revelations of great diversity within the kingdom [3,5]. Unfortunately, no system based upon systematic or numerical taxonomy is totally satisfactory for protozoa due to their vast heterogeneity. Nucleotide sequence comparisons between molecules that all organisms possess, and are functionally universal, provide the most quantitative and efficient method of assessing relationships among protozoa. Although r R N A has been highly conserved throughout evolution, the rate of nucleotide substitutions, deletions or insertions at various positions in the srRNA, in particular, is not constant. Some regions are non-variable, unchanged over millions of years of evolution, others are highly variable [12]. The more closely related organisms are likely to be, so the more the variable regions will probably be required for comparison. The srRNA of about 30 organisms have been totally sequenced usually by cloning and then sequencing of the gene or its fragments [16]. However, the requirement to clone the srRNA gene or its fragments prior to sequencing lengthens the already long time period required to obtain the nucleotide sequence by standard D N A sequencing techniques. In addition, it is not necessary to sequence the whole gene to allow accurate phylogenetic comparisons to be made [14]. It is more important to compare certain specific regions such as the semi-conserved areas, rather than have the whole gene sequence [14]. To date this has been accomplished by comparing the semi-conserved regions of entire sequences of the srRNA from about 20 species of eukaryotes [16]. These sequences have been determined by sequencing cloned srRNA genes which can be a tedious and time consuming process. Even though we have cloned fragments of the srRNA gene of T. gondii [29], sequencing these would be a lengthy process. The srRNA can, however, provide large sequence blocks in a short space of time when re-
verse transcribed using universally conserved sites for priming [19]. In fact, primers A and C used here for T. gondii were also used as primers for sequencing the P. berghei srRNA coding region [15]. The simple R N A extraction method used by us is even shorter and more technically simple than that recommended by Lane et al. [19]. This means that we can go from parasite pellet to loading of a D N A sequencing gel in less than 5 days. We foresee a large expansion in the quantitative taxonomy of the protozoa. By simply partially sequencing the srRNA of members of each of the major groups proposed by Levine et al. [3] or Corliss [5], true relationships can be plotted. The result will probably be an increase in the number of higher taxa and a necessary rearrangement of current groupings. In addition, rapid srRNA sequencing of medically important organisms is likely to have great benefit in diagnosis. Specific, simple srRNA 'probes' can be readily identified from srRNA sequences, and species (or even strain) specific srRNA oligonucleotide 'probes' could be applied to human disease diagnosis [30]. We have compared T. gondii sequence blocks with 11 other taxa including three ciliates, a cellular slime mould, two flagellates, two fungi and two vertebrates, as well as another apicomplexan, and then assessed the sequence homologies and structural distances between them by four phylogenetic methods. The trees are in general agreement with previously published trees involving Trypanosorna. Euglena, Dictyosteliurn, Saccharornyces, ciliates and vertebrates [15,2023]. With regard to Toxoplasrna and Plasrnodium, all four trees agree in showing monophyly of Fungi, Ciliophora, Plasrnodiurn and Toxoplasrna to the exclusion of Trypanosoma, Euglena, Dictyosteliurn and vertebrates. However, there was no consensus among the four trees in the phylogenetic relationships among Fungi, Ciliophora, Plasmodium and Toxoplasrna. These data suggest, therefore, that the branches leading to these four taxa all diverged at about the same time. In particular, there is no evidence for an especially close relationship between 7". gondii and P.
berghei. The members of the Apicomplexa have been
245 g r o u p e d t o g e t h e r as a p h y l u m , b a s e d largely o n the possession, visible by e l e c t r o n m i c r o s c o p y , of a n apical c o m p l e x g e n e r a l l y consisting of p o l a r ring(s), r h o p t r i e s , m i c r o n e m e s , c o n o i d a n d subpellicular microtubules present at some stage [31]. T h e results of o u r studies p r e s e n t e d here are consistent with the h y p o t h e s i s that the i n c l u s i o n of o r g a n i s m s i n t o a p h y l u m b a s e d o n possession of s o m e c e r t a i n o r g a n e l l e s m a y n o t be valid. A l t e r natively, e i t h e r P. berghei or T. gondii m a y not be close to the rest of the A p i c o m p l e x a . M o r e studies such as the r a p i d s r R N A s e q u e n c i n g techn i q u e used here will be necessary to a n s w e r these questions.
Acknowledgements This work was partly f u n d e d by a grant to A . M . J . from the N a t i o n a l H e a l t h a n d Medical R e s e a r c h C o u n c i l of the C o m m o n w e a l t h of A u s tralia. P . J . M . is a recipient of a F l i n d e r s U n i v e r sity of South A u s t r a l i a S u m m e r Scholarship. W e t h a n k J. F e l s e n s t e i n , D e p a r t m e n t of G e n e t i c s , U n i v e r s i t y of W a s h i n g t o n , Seattle, for his P H Y L I P package a n d A. G u n j k o a n d M. A d a m s for c o m p u t i n g assistance. D e b b i Sullivan a n d Liz D a v i s o n skillfully typed the m a n u s c r i p t .
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