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Small-Subunit Ribosomal RNA Sequence from Naegleria
gruberi Supports the Polyphyletic Origin of Amoebas’
C. Graham Clark and George A. M. Cross
Laboratory
of Molecular
Parasitology,
The Rockefeller
University
We have sequenced the small-subunit xibosomal RNA gene of the amoebo-flagellate
protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences
of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.
Introduction
Amoebas are a group of morphologically and biochemically very diverse protozoa
and are most likely a polyphyletic aggregation of species (Bovee and Jahn 1973). The
small number of classification criteria available-manner
of locomotion and pseudopod appearance, presence or absence of flagellated stages, nuclear structure, and
division patterns (Page 1976)-has
led to the placing of all pseudopodal amoeboid
organisms into the class Lobosea for convenience ( Lee et al. 1985 ) . Uncertainty about
the value of these criteria as phylogenetic markers leaves the evolutionary relationships
among groups of amoebas unclear.
The use of small-subunit ribosomal RNA (rRNA) sequence comparison has
greatly increased our understanding of evolutionary relationships among prokaryotes
and, more recently, among eukaryotes. However, only one amoeba, Acanthamoeba,
has so far been examined by this method (Gunderson and Sogin 1986).
Within the class Lobosea, the order Schizopyrenida is distinguished by, among
other characteristics, the presence of a number of amoebo-flagellate genera (Page 1976),
but it is unclear whether it is closely related to any other order of amoebas within its
class. We recently described the unique rRNA gene structure of the schizopyrenid
amoeba Naegleria gruberi (Clark and Cross 1987). In this organism and in related
genera (Clark and Cross 1988)) the rRNA genes ( rDNA) are carried only on multicopy
circular plasmids-no
chromosomal copy has been detected. Given the uniqueness
of this gene structure and uncertainty about the organism’s phylogenetic relationship
to other groups of amoebas, we decided to sequence its small-subunit rDNA. Our
results show that the lobose amoebas are indeed polyphyletic in origin and that the
Schizopyrenida are a relatively ancient eukaryotic group, with their closest relatives
being flagellates.
Material and Met hods
The recombinant plasmid pNgex27 contains an entire Naegleria gruberi rDNA
plasmid (Clark and Cross 1987). The small-subunit rRNA region was subcloned in
1. Key words: Naegleria gruberi, amoeba, 18s rRNA, eukaryote phylogeny.
Address for correspondence and reprints: C. Graham Clark, Laboratory of Molecular Parasitology, The
Rockefeller University, 1230 York Avenue, New York, New York 1002 l-6399; telephone (CGC): (212)
570-7574.
Mol. Biol. Evol. 5(5):512-5 18. 1988.
0 1988 by The University of Chicago. All rights reserved.
0737-4038/88/0505-0004$02.00
512
Naegleria Small Subunit rRNA Sequence
5 13
Table 1
Structural Similarity and Distance Data from Eukaryotic Small-Subunit
rRNA Sequence Comparisons
Cr
Cr.. . . .
Zm . . . .
AC.....
Xl.....
SC . . . .
Tt.. . . .
Pb.....
Dd . . . .
Tb .,..
Eg.. . . .
Ng . . .
EC. . . . .
0.076
0.094
0.155
0.118
0.185
0.185
0.211
0.327
0.319
0.282
0.720
Zm
AC
Xl
SC
Tt
Pd
Dd
Tb
Eg
Ng
Ec
0.928
0.912
0.928
0.860
0.864
0.853
0.891
0.904
0.899
0.853
0.836
0.867
0.860
0.812
0.856
0.836
0.839
0.837
0.783
0.832
0.807
0.816
0.820
0.844
0.797
0.820
0.796
0.793
0.735
0.737
0.734
0.713
0.726
0.702
0.700
0.728
0.740
0.740
0.731
0.7 13
0.734
0.710
0.710
0.705
0.728
0.765
0.754
0.763
0.752
0.762
0.732
0.755
0.737
0.691
0.697
0.537
0.525
0.535
0.5 17
0.528
0.5 11
0.521
0.522
0.505
0.5 19
0.520
0.076
0.150
0.103
0.146
0.181
0.181
0.324
0.319
0.298
0.752
0.164
0.108
0.155
0.184
0.175
0.329
0.333
0.285
0.726
0.164
0.216
0.256
0.237
0.362
0,362
0.301
0.775
0.160
0.190
0.206
0.341
0.328
0.286
0.744
0.223
0.238
0.380
0.367
0.325
0.791
0.242
0.383
0.367
0.297
0.763
0.338
0.375
0.324
0.761
0.338
0.398
0.809
0.388
0.769
0.766
NOTE.-The values above the diagonal are the structural similarities calculated according to the method of Elwood et
al. (1985) for the regions of the sequence considered to be unambiguously alignable. Below the diagonal are the structural
distances (substitutions/site) calculated according to the method of Jukes and Cantor (1969). For the Nuegleria gruberi (Ng)
sequence, the base numbers included in the analysis were l-62,98- 130,373-547,597-7 17,1023- 1208,1245- 15 17, I60017 15, 1744- 1920, and 1937-20 19 for a total of 1,227. These bases were aligned with the corresponding regions from the
small-subunit rRNAs of Chlurnydomonas reinhurdtii (Cr; Gunderson et al. 1987), Zeu muys (Zm; Messing et al. 1984),
Acunthumoebu custellunii (AC; Gunderson and Sogin 1986), Xenopus luevis (X1; Salim and Maden 1981), Succhuromyces
cerevisiue (SC; Mankin et al. 1986), Tetruhymenu thermophila (Tt; Spangler and Blackbum 1985), Plasmodium berghei
(Pb; Gunderson et al. 1986), Dictyostelium discoideum (Dd; McCarroll et al. 1983), Trypanosomu brucei and Euglenu
gracilis (Tb and Eg; Sogin et al. 1986), and Escherichia coli (EC; Brosius et al. 1978).
both orientations into pGEM-4 (Promega Biotec). Following digestion with the restriction enzymes SphI and SalI, both of which cut only in the polylinker region of
each clone, nested deletions were produced by the Exo III/S1 technique in the two
small-subunit plasmids (pNgSS6 and pNgSS55 ) by using an Erase-a-Base@ kit (Promega Biotec) and the provided protocol. Deletions of - 200 bases in size were generated
by sampling at 30-s time intervals. A small number of colonies from each time point
were screened, and a complete nested set of deletion clones was obtained from both
starting plasmids. DNA for sequencing was prepared from small-scale ( 5-40 ml) plasmid preparations by using the alkaline lysis method (Maniatis et al. 1982) but with
an additional phenol-CHC13 extraction and ethanol precipitation following RNase
treatment. After alkaline denaturation and hybridization of the SP6 promoter primer
(Promega Biotec), deletion clones were sequenced by the dideoxynucleotide chain
termination method with modified T7 DNA polymerase by using the Sequenase- kit
and protocol from US Biochemical Corporation and [ 35S]a-dATP. dITP was included
to eliminate compressions due to secondary structure (Barnes 1987 ) . Reactions were
run on 6% acrylamide-urea sequencing gels, fixed in 10% acetic acid, dried, and exposed
to Kodak XAR-5 film at room temperature. Complete overlapping sequence was
obtained for both strands of the DNA.
The Naegleria 18s rRNA sequence was deduced from the DNA sequence. The
5 ’and 3’ends of the 18s rRNA were inferred by sequence similarity, and the Naegleria
18s rRNA was aligned by eye to a number of other eukaryotic small-subunit rRNAs
by using secondary structure as a guide. Similarity values were calculated for each
pair of organisms by using the method described by Elwood et al. ( 1985 ) (see table
5 14 Clark and Cross
TACCTGGTTG
ATCCTGCCAG
TACTATATGC
TTGTCTCAAA
GCCTAAGCCA
AGATCAATCA
TATGCGGTTT
CGGCCGTGTA
TUTGATAGT
CTGTGGAAGG
CTCATTATAA
121
CAGTTATACT
CCTAGCCACT
GGAAAGTTTA
CAAGGATACC
ACCGTTAACT
GCAGCGATAT
1
61
TGCAA.ATGTA
181
ACTTGTTCCC
TTCGGGGTGG
TAATAGTATT
TGTGCTGAAG
CCTAGCTATT
GTAACCTAGT
241
TTTTCGGGTG
TGGCAACATA
TTCGGGGGAT
TAGGAATCGA
CCGCTAGCAG
GTGCCTTCGG
301
GCGCGGGAAA
GTGAATTAAC
AAGGTTTTCA
TAAGGCCTTT
CAGGTTTGCT
TTTTCTAGTG
361
GCCAGGCAGA
GGAGTTTCTT
ACCTATCAGC
TCGTTGTTTG
TTTAAAGGAC
AAACCAGGCT
421
TTGACGGGTA
CGGGGAATCA
GTGTTCGATT
CCGGAGAGGG
AGCCTGAGAA
ATCGCTACCA
481
CATCTAAGGA
CGGCAGCAGG
CGCGCAAATT
ACCCAATCTC
AATACGAGGA
GGTAGTGACA
5il
AGCTATAGTG
ACTCCACACC
ATTCGGTGGG
GAGGTATTGT
CTTCTGACGA
TTTTCCATGA
601
TTTGGGTGTA
GATAACCCTT
AGAGTAGCCA
TTGGAGGAAA
AGTCTGGTGC
CAGCACCCGC
661
GGTAATTCCA
GCTCCAAGAG
CGTATATTAA
TACTGCTGTA
GTTAUACGC
CCGTAGTATA
721
CCTAAGAGTG
GGTGTGTAGT
AATTAGTTTT
ACCAGAGGAC
GGTTGGCGAG
AGTTTATCAC
781
TCTTGTTTGC
CTACTTTTGG
TAGACTTTAG
TCGGGCTGGA
TCTTTGGTCC
TCGTCTGACA
841
GTTGCTACGT
ACTTACTTAC
CACGGTTCAT
CCGTGAGGCC
CTTGGCTTGC
AACTGTAAAT
901
UTCGTTGT
GCTTAAAGCG
GGCTATGATA
CTCTGCCAGA
GCGATTTAGC
ATGGGACTGC
981
AGAGTAGCTG
TATTTGAGCG
AAGGTTGCAC
CTCGGTGTGG
CTGGAGCTTG
GTACAGCGCT
1021
TGTAATGGAG
CTCAGGGTGA
GGCCCCGGGT
ACCATGAGGC
TAGAGGTGAA
ATTCTGAGAC
1081
CCTCATGTGA
CCAACTAAGG
CGAAAGCTGT
CGTGGGCCAC
CACAAGCTCG
TCTATCAGGG
1141
ACAAAAGTTG
GGGGATCGAA
GACGATTAGA
TACCGTCGTA
GTCCCAACTA
TAAACGATAC
1201
CAACCGAGTA
TTTGGGAAGA
CACTATCCCA
GCCATCTTCT
CAGAACTCAA
GGGMACCTT
1261
AAGTCTTTGG
GTTCTGGGGG
GAGTATAGTC
GCAAGACCGA
AACTTAAAGG
AATTGACGGA
1321
AAGGCACCAC
CAGGAGTGGA
GTCTGCGGCT
TAATTCGACT
CAACACGGGG
AAACTCACCA
1381
GGTCAGGACA
CAAGTTTGAT
TGACAGGTTA
ATAGCCCTTT
CTTGATTGTG
TGGTGGGTAG
1441
TGCATGGCCG
TTTCCAGTTC
GTGGAGTGAT
CTGTCTTGTT
AATTCAGATA
ACGAACGAGA
1501
CCTAAGCCTT
TAACTAGCCG
TAGGCCTTTT
CCTTCGGGGA
AGGGTTAGTT
TGTCGGAACA
1561
GGTTTCGGCC
TGTTCCAAM
CCTACGAGAC
TTTTGTCAGC
TTCTTAAAGG
GACTTCATTC
AGATGTCCTG
1021
GTAAACTAGG
ATGAGGAAGA
TTTAGGCCAT
AACAGGTCTG
TGATGCTCTT
1681
GGCTGCACGC
GTACTACAAT
AACGGTACCA
GCGAGCGCTA
TGGTTTTATA
ACCCCTTATC
1741
CTAATAGGAT
TGGGAAAACT
TTTCAAACAC
CGTTATGACA
GGGATCGAGG
ATTGGAACAT
1801
CCTCGTGAAC
GAGGAATTCC
TAGTAAGCGT
GGTTCATCAT
ACCACATTGA
TTACGTCCCT
1861
GCCTTTTGTA
CACACCGCCC
GTCGCTCCTA
CCGATGGGAC
GAAGAGATGA
ACCTGGCGGA
1921
CCGAlLCCGCA
AGGTAAGGGA
IuiCCAGTTAA
ATCTCTTCGT
CTGTAGGAAG
GAAAAGTCGT
1981
AACAAGGTCT
TCGTAGGTGA
ACCTGCGTAG
GGATCATTT
of the small-subunit rRNA region of Nuegleriu gruberi rDNA. Unambiguous sequence
FIG. 1.-Sequence
was obtained at each of the 2,019 positions. (GenBank accession no.: M18732.)
1). A total of 1,227 positions were considered to be unambiguously alignable. The
tree was constructed following the guidelines of Fitch and Margoliash (1967).
Results and Discussion
Sequencing of the Naegleria gruberi small-subunit rRNA gene gave an unambiguous sequence of 2,0 19 bases (fig. 1). The putative 5’ end is 173 bases upstream
of the previously mapped PstI site, and the putative 3’ end is 63 bases upstream of
the CZaI site in the 18S-28s spacer region (Clark and Cross 1987). All secondarystructure elements conserved among other eukaryotic small-subunit rRNAs were also
found in the NaegZeria sequence. The size of this amoeba’s 18s rRNA is greater by
-200 bases than those of many other eukaryotes, although it is smaller by 230-290
bases than those of Acanthamoeba, Euglena, and Trypanosoma. As might be predicted,
the size differences reside in regions previously identified as variable in length
(see table 2; discussed by Gunderson and Sogin [ 19861, Schnare et al. [ 19861, and
Clark [ 19871).
The tree constructed from the structural distance values (table 1) is shown in
figure 2. This tree is very similar to others derived by this method, for example that
of Gunderson et al. ( 1987). The node of the Naegleria branch is between those which
gave rise to the euglenid and trypanosomatid flagellate groups on one side and Dic-
Naegleria Small Subunit
rRNA Sequence
5 15
Table 2
Position and Comparative Length of the Variable Regions of Nuegferiu 18s rRNA
REGION LENGTH
REGION, Ng LOCATION
I, 143-361 .........
II, 606-630
........
III, 713-1022 .......
IV, 1207-1247 ......
V, 1522-1610
......
VI,1707-1769
......
VII, 1886-1972 .....
Total length of 18s
Ng
Eg
AC
Tt
EC
219
25
310
41
89
63
87
2,019
268
29
521
46
121
68
112
2,305
245
29
375
131
154
110
129
2,303
163
29
215
35
47
51
90
1,753
78
176
67
29
43
42
83
1,542
NOTE.-This table shows the location in N. gruberi of the seven major variable regions of 18s rRNA identified by
Gunderson and Sogin ( 1986) and the comparative length of these regions in a selection of other species: Eg and AC are the
two longest 18s rRNAs known; Tt is approximately average length for a eukaryote; and EC is prokaryotic (the secondary
structure of Noller et al. [ 19851 was used). Abbreviations are as in table 1.
tyostelium on the other. The NaegZeria sequence deviates at a number of previously
universally conserved positions. The early branch point may explain this result, since
a similar observation was made when the Euglena and Trypanosoma sequences were
determined (Sogin et al. 1986). It must also be remembered that only a single, cloned
copy of the approximately 4,000 genes per cell was sequenced and that variation
between genes may exist, although we have no evidence for this.
The unique structure of the Naegleria rDNA can be reassessed in light of the
branch-point data. Both EugZena and the trypanosomes have tandemly repeated 18S5X3-28s rDNA units, in contrast to the extrachromosomal circular rDNA of Naegleria
and the extrachromosomal linear rDNA of Dictyostelium (Cockburn et al. 1978).
However, the 5s rRNA genes of Euglena are also located within the rDNA tandem
array (Curtis and Rawson 198 1), while those of Trypanosoma are not (Cordingley
1985 ) . This may be an indication that rDNA tandem array formation, a process likely
to have occurred numerous times in the eukaryotic lineage (Clark 1987)) occurred
after divergence of the ancestors of these two flagellate organisms. Extrachromosomal
rRNA genes may therefore be the ancestral eukaryotic condition rather than a lineagespecific peculiarity.
The positioning of the NaegZeria branch node so close to that of Euglena and
Trypanosoma suggests a flagellate ancestry for the Schizopyrenida. A close relationship
of amoeba and flagellate is not without precedent: the amoeba Dientamoeba fragiZis
is generally considered to be an aberrant member of the otherwise flagellate order
Trichomonadida (Lee et al. 1985).
The relative positioning of Naegleria and Acanthamoeba on the rRNA tree is
supported by their respective forms of mitosis. Naegleria shares with EugZena and
Trypanosoma a promitotic form of nuclear division (Schuster 1979)-persistence
of
the nucleolus and nuclear membrane through mitosis and the absence of centrioles.
The metamitotic nuclear division of Acanthamoeba most closely resembles that of
plants and animals-nuclear
membrane and nucleolar disintegration and the presence
of centrioles during mitosis.
It is evident from the phylogenetic tree that the amoebas Acanthamoeba and
Naegleria have separate origins and probably nonamoeboid ancestors. This confirms
5 16 Clark and Cross
Chlamydomonas teinhardtii
/
brucei
FIG. 2.-Eukaryotic phylogeny inferred from mutational distances between small-subunit rRNA sequences. With the data in table 1, the tree was constructed following the method of Fitch and Margoliash
(1967). The % SD (Fitch and Margoliash 1967) for this tree is 4.24%, the lowest value obtained of the
approximately 30 trees examined. The best-fit values for branch lengths and evolutionary distance between
nodes are given alongside the relevant tree segment.
the suggested polyphyletic origins of the class Lobosea (Bovee and Jahn 1973), while
indicating that rRNA sequence comparison has potential as a method for producing
a phylogenetically based classification of this diverse group.
Acknowledgments
C.G.C. is supported by the David C. Scott Foundation.
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Received December 4, 1987; revision received March 2 1, 1988