Protist. Vol. 150, 33-42, March 1999 © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/protist
Protist
ORIGINAL PAPER
Phylogenetic Affinities of Dip/onema within the
Euglenozoa as Inferred from the SSU rRNA Gene
and Partial COl Protein Sequences
Dmitri A. Maslova,1, Shinji Yasuhira b,2, and Larry Simpson b
aDepartment of Biology, University of California, Riverside, CA 92521, USA
bHoward Hughes Medical Institute, Department of Molecular, Cell and Developmental Biology and Department of
Medical Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 900951662, USA
Submitted November 4, 1998; Accepted February 6, 1999
Monitoring Editor: Mitchell L. Sogin
In order to shed light on the phylogenetic position of diplonemids within the phylum Euglenozoa, we
have sequenced small subunit rRNA (SSU rRNA) genes from Dip/onema (syn. /sonema) papillatum
and Dip/onema sp. We have also analyzed a partial sequence of the mitochondrial gene for cytochrome c oxidase subunit I from D. papillatum. With both markers, the maximum likelihood method
favored a closer grouping of diplonemids with kinetoplastids, while the parsimony and distance suggested a closer relationship of diplonemids with euglenoids. In each case, the differences between
the best tree and the alternative trees were small. The frequency of codon usage in the partial D.
papillatum COl was different from both related groups; however, as is the case in kinetoplastids but
not in Eug/ena, both the non-canonical UGA codon and the canonical UGG codon were used to encode tryptophan in Dip/onema.
Introduction
The phylum, Euglenozoa, includes four major
groups: euglenoids, kinetoplastids (consisting of
bodonids and trypanosomatids), diplonemids (including the genera Diplonema (syn. Isonema) and
Rhynchopus) and Postgaardi (incertae sedis) (Cavalier-Smith 1993; Corliss 1994; Simpson 1997). This
grouping is supported by a large amount of morphological and ultrastructural data; however, the relationship among these groups is not resolved. In par1 Corresponding author;
fax 1-909-787-4286
e-mail maslov@ucrac1.ucr.edu
2 Present address: Research Reactor Institute, Kyoto University, Kumatori-cho, Noda, Sennan-gun, Osaka 590-04, Japan
ticular, the phylogenetic affinity of Diplonema is uncertain, with these organisms sharing a variety of
features with both euglenoids and kinetoplastids,
but clearly differing from either group (Triemer 1992;
Triemer and Farmer 1991; Triemer and Ott 1990). Initially it was proposed that Diplonema represents an
evolutionary link between bodonids, which were
considered ancestral to the entire group, and euglenoids (Kivic and Walne 1984). Subsequent scenarios considered an early divergence of all three lineages from the common ancestor, and in one case
Diplonema was proposed to be a close relative of
euglenoids (Willey et al. 1988), while in another it
represented a separate branch (Triemer 1992; Triemer and Farmer 1991). Although morphological data
1434-4610/99/150/01-33 $ 12.00/0
34
D. A. Maslov, S. Yasuhira, and L. Simpson
proved to be insufficient to corroborate or refute any
of these hypotheses, no molecular data on diplonemids has been obtained until now. An usual base (J)
was recently found in the nuclear DNA of Dip/onema
and kinetoplastids, but no search was performed for
this base in euglenoids (Van Leeuwen et al. 1998). In
the present work, we have attempted to resolve the
relationships among the major euglenozoan groups
by a molecular phylogenetic analysis of the small
subunit (SSU) ribosomal RNA and cytochrome c oxidase subunit I (COl) polypeptide sequences.
Results
Phylogenetic Position of Diplonema Based
on the SSU Data
The SSU rRNA gene sequences from D. papillatum
and Dip/onema sp. were amplified from the total cell
DNA with the 5' and 3' conserved sequence oligonucleotides, S762 and S763, respectively, and
aligned with the sequences from kinetoplastids and
euglenoids. The alignment contained 17 taxa and
1349 alignable characters including gaps. The kine-
COl
56
100
D. papillatum
toplastids included representatives of the major trypanosomatid clades (Leishmania tarento/ae, Herpetomonas muscarum, Phytomonas serpens, B/astocrithidia culicis and Trypanosoma bruce/) (Hollar et
al. 1998) and four currently available bodonids (Dimastigella trypaniformis, Rhynchobodo sp., Trypanop/asma borreli and Bodo caudatus). Four euglenoid sequences (Eug/ena gracilis, Khawkinea
quartana, Lepocinc/is ovata and Peta/omonas cantuscygm) , were retrieved from the GenBank™ database. The alignment contained two slowly evolving
outgroup sequences - Physarum po/ycepha/um and
Saccharomyces cerevisiae.
Analysis of the data with the maximum likelihood
method using the PAUP* program produced the tree
shown in Figure 1. Three major groups of Euglenozoa are represented by the corresponding monophyletic clades, and the bootstrap support for their
monophyly is high. The clade of Dip/onema is shown
as a sister-group of kinetoplastids. However this
affinity is not supported by the bootstrap analysis.
Indeed, the maximum likelihood consensus majority
tree (not shown) shows the clade of diplonemids as
a sister-group of euglenoids with a 77% bootstrap
level. The branching order which united kinetoplas-
SSU
79
95
Leishmania tarentolae
Figure 1. Maximum likelihood
SSU
rRNA and COl protein phyPhytomonas serpens
86
logenetic trees of Euglenozoa.
100
L. tarentolae
Blastocrithidia culicis
Ln likelihood of the SSU tree is 79
9936.69451. Bootstrap values
Trypanosoma brucei
T. brucei
Dimastigella trypaniformis
were obtained for 100 pseu79
doreplicates. The sequences
E. gracilis
Rhynchobodo sp.
have the following Genbank™
100
T. aestivum
accession numbers: L. tarentoTrypanoplasma borreli
/ae - M84225, H. muscarum <50
Bodo caudatus
S. cerevisiae
L18872, P. serpens - AF016323,
Diplonema papillatum
B. culicis - U05679, T. brucei 100
M12676, D. trypaniformis Diplonema sp.
100
X76494, Rhynchobodo sp. Euglena gracilis
U67183, T. borreli - L14840, B.
caudatus - X53910, D. papillaKhawkinea quarlana
tum - AF119811 (this work),
Lepocinclis ovata
Dip/onema sp. - AF119812 (this
' - - - - - - Petalomonas cantuscygni
work), E. gracilis - M12677, K.
quartana
- U84732, L. ovata ' - - - - - - Physarum polycephalum
AF061338, P. cantuscygni Saccharomyces cerevisiae
U84731, P. po/ycepha/um X13160, S. cerevisiae - J01353.
Ln likelihood of the COl tree is -3572.64 +/- 97.57. Bootstrap values were obtained for 1000 pseudoreplicates. The
corresponding Genbank™ accession numbers are: L. tarento/ae - M1 0126, T. brucei - M94286, T. borreli - U11683,
D. papillatum - AF119813 (this work), E. gracilis - U49052, T. aestivum - Y00417, S. cerevisiae - M97514.
T. borreli
91
Herpetomonas muscarum
Phylogeny of Dip/onema
tids and diplonemids as shown in Fig. 1 occurred in
only 19% of the bootstrap replicates. This value may
be an underestimate of the actual support level: in
order to ease the computations, the bootstrap analysis, unlike the search for the best tree, was performed assuming that all sites evolve with an equal
rate, and this simplified condition favors the affinity
of diplonemids with euglenoids.
In order to additionally address possible effects of
unequal rates of sequence evolution in different lineages (Felsenstein 1978) on the support of the
diplonemid-kinetoplastid clade, we also performed
a bootstrap analysis after omitting the long branch
of P. cantuscygni and the short trypanosomatid
branches. Although the support level increased to
47% (not shown), it remained statistically irrelevant.
With three monophyletic euglenozoan clades diplonemids (D), kinetoplastids (I<) and euglenoids
(E) - and the clade of an outgroup, there can be only
three alternative trees. Each of these alternatives
was evaluated using the likelihood, parsimony and
distance algorithms by comparing trees without
topological constraints with trees constructed under
the corresponding user-defined constraints. The results are presented in Table 1. The best unconstrained maximum likelihood tree shows diplonemids and kinetoplastids as sister-groups - ((K,D),E)
(see also Fig. 1), while the shortest unconstrained
parsimony and distance trees contain diplonemids
as the closest relatives of euglenoids - (K,(D,E)).
However, the trees with enforced alternative topologies - (K,(D,E)) for likelihood and ((K,D),E) for parsimony and distance - are not significantly different
from the corresponding unconstrained trees. With
each of these methods, the topology which shows
35
diplonemids as an earliest branch of Euglenozoa (D,(K,E)) - was the least favorable.
It should also be noticed that using the SSU rRNA
sequences of a diplomonad (Giardia /amblia) and a
microsporidian (Vairimorpha necatrix) as outgroups
substantially increases a bootstrap support for the
diplonemid-kinetoplastid clade (up to 90% with
some alignments, data not shown). However, such a
high level of support may be an artefact associated
with a biased nucleotide composition and fast
substitution rates observed in the outgroup sequences.
Cloning and Analysis of the COl mRNA
Sequence
In order to obtain additional information for resolving
the phylogenetic position of Dip/onema, we analyzed the sequence of the mitochondrial COl protein. The usefulness of this marker is validated by
previous work which showed that mitochondria are
monophyletic and that nuclear and mitochondrial
phylogenies are congruent (Inagaki et al. 1997;
Sicheritz-PontEm et al. 1998; Tessier et al. 1997;
Viale and Arakaki 1994; Yasuhira and Simpson
1997).
Total cell RNA was isolated from D. papillatum.
cDNA to polyadenylated mRNA was synthesized by
reverse transcription using dT20 N primer, and a partial COl mRNA was amplified by PCR using the
oligonucleotides C112 and dT2o N. The conserved
sequence-specific oligonucleotide C112 anneals to
a site within the central region of the kinetoplastid
COl mRNA approximately 700 nt from the 5' end or
800-900 nt from the 3' end. The PCR yielded several
Table 1. Parameters of the phylogenetic trees of Euglenozoa with or without specific topological constraints.
Tree topology
Marker
Likelihood 1
Parsimony2
Distance3
((K, D), E)
SSU
COl
SSU
COl
SSU
COl
-9936.69451
-3572.64
-9936.72057
-3575.73
-9938.29060
-3574.26
1912
536
1900
534
1914
537
1.48893 (2 trees)
(K, (0, E))
((K, E), D)
1.48734 (2 trees)
1.52350 (2 trees)
Ln likelihood value found by a heuristic search using empirical nucleotide frequencies, assuming two substitution
types, and estimating a proportion of invariable sites and a transition/transversion ratio via maximum likelihood.
2 Performed by a branch-and-bound search. The score indicates a number of steps.
3 Minimal evolution score found by a heuristic search with Kimura 3-parameter distances and allowing for among
site variation; starting tree obtained through neighbor-joining followed by global rearrangements. The two trees in
each case differ only by arrangement of L. tarento/ae, H. muscarum and P. serpens branches. The similar differences in tree scores were also obtained for sums of unweighted least squares.
For each optimality criterion, the underlined tree corresponds to the constrained tree which coincides with the best
unconstrained tree.
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Figure 2. Partial mRNA and deduced protein sequence of the COl from D. papillatum. All tryptophan codons are boxed, UGA codons are additionally
shown in bold face and the UGA codon conserved among kinetoplastids is underlined.
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Phylogeny of Diplonema
121
2
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37
B
Hoechst
Figure 3. Fractionation of the total cell DNA from D. papillatum by equilibrium density centrifugation in CsCI gradients with ethidium bromide (A) and Hoechst 33258 (8). The gradient-purified DNA from the upper and lower band of
each gradient, either undigested (lane 1) or digested with Mspl (lane 2) was analyzed in a 1% agarose gel shown to
the left of the corresponding gradient.
Results of the restriction digests shown in Figure
4A and 4C indicate that each digest contains a discrete set of nonstoichiometric fragments with the
sizes not exceeding several kilobases. Some hexanucleotide recognizing enzymes (8glll, Hindlll,
EcoRI and others) apparently do not digest this
DNA, while most tetranucleotide cutters do. There is
no evidence for the presence of uniform length minicircle-like molecules as those seen in digests of
kinetoplast DNA from trypanosomatids. Therefore,
this mtDNA is probably composed of several types
of circles. The exact size, number of types and topological arrangement of these molecules remain to be
investigated.
Probing of the mtDNA digests with the cloned
COl cDNA revealed hybridization with one or a few
restriction fragments or hybridization with undigested molecules, depending on the enzyme used
(Fig. 48). Specific restriction fragments producing
hybridization signals could be identified in most
cases. Cloning and sequence analysis of the mitochondrial genomic COl sequence are in progress.
Figure 2 shows the cloned 3' end segment of the
COl mRNA along with the deduced protein. The
polypeptide sequence is 62% similar (41 % identical)
to the corresponding portion of the E. gracilis COl
sequence and 65% similar (37% identical) to the T.
borreli sequence. There is no similarity at the C-ter-
minus. The amplified COl sequence is likely to be
derived from a polyadenylated mRNA and there is
only a single nucleotide between the UAG termination codon and the poly(A) tail. This arrangement
was identical in all three cDNA clones sequenced.
However, there is also a possibility that the 3' UTR
was indeed longer and contained an A-rich sequence which acted as an annealing site for the
dT20 N primer. Future studies should clarify this question.
An important feature of the COl sequence is the
use of UGA codons as tryptophan instead of termination. The sequence contains seven deduced tryptophan residues, four of which are encoded by standard UGG codons, while three tryptophans, including one conserved residue, are encoded by noncanonical UGA codons.
The codon usage is summarized in Table 2. The
usage is biased towards codons with G or C in the
3rd position (65.3% of all codons). This reflects the
55.3% G+C content of this fragment and correlates
with a relatively high G+C-richness of this mtDNA as
mentioned above.
Phylogenetic Analysis of COl Sequences
The amplified 3' partial sequence of D. papillatum
COl and additional 53 residues from the 5' region of
38
D. A. Maslov, S. Yasuhira, and L. Simpson
the sequence were aligned with
the corresponding sequences
from E. gracilis, T. borreli, T.
brucei and L. tarento/ae. The
yeast and wheat cal sequences were used as outgroups. After elimination of the
nonalignable C-terminus and a
few short internal regions, the
alignment contained 293 characters. As for the SSU genes,
analysis of the data with maximum likelihood (PROTML) resulted in a tree which showed
Dip/onema as a sister group to
kinetoplastids (Fig. 1). However,
two alternative topologies were
not significantly different (Table
1). Protein parsimony analysis
performed with PHYLIP resulted in two equally short
trees, one of them containing
Dip/onema as a sister lineage to
kinetoplastids and another one
showing it as a sister lineage to
E. gracilis (data not shown).
Protein parsimony analysis performed with PAUP slightly favored the second of these two
options (Table 1). Protein distance analysis done with
PHYLIP using the Kimura distance formula or the Dayhoff
PAM matrix for distance computation combined with two different methods of tree construction (least squares and
neighbor-joining),
showed
Dip/onema as the closest relative of kinetoplastids (not
shown).
01
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Discussion
II I I I
C! C!
II)
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OeD
C'i";
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C!
...
I
"'!
o
We have analyzed the phylogenetic relationships of Dip/onema with other Euglenozoa
using SSU rRNA gene and mitochondrial cal protein sequences. Confirming results of
the previous morphological
studies (Triemer 1992; Triemer
and Farmer 1991), our analysis
showed that diplonemids rep-
Phylogeny of Dip/onema
39
Table 2. Codon frequency in the partial COl mRNA of D. papillatum.
Gly
Gly
Gly
Gly
Glu
Glu
Asp
Asp
Val
Val
Val
Val
Ala
Ala
Ala
Ala
GGG
GGA
GGU
GGC
GAG
GAA
GAU
GAC
GUG
GUA
GUU
GUC
GCG
GCA
GCU
GCC
6.00
7.00
8.00
3.00
5.00
0.00
4.00
4.00
16.00
9.00
0.00
0.00
3.00
8.00
3.00
10.00
Arg
Arg
Ser
Ser
Lys
Lys
Asn
Asn
Met
lie
lie
lie
Thr
Thr
Thr
Thr
AGG
AGA
AGU
AGC
AAG
AM
AAU
AAC
AUG
AUA
AUU
AUC
ACG
ACA
ACU
ACC
4.00
2.00
3.00
7.00
1.00
0.00
0.00
5.00
14.00
5.00
0.00
3.00
3.00
5.00
3.00
7.00
resent a third lineage within Euglenozoa in addition
to kinetoplastids and euglenoids, and this lineage
branches deeply within this group. However, molecular phylogeny, as well as the previous morphological studies, fail to provide an unequivocal answer to
the question which of these two clades represents a
closest relative of diplonemids. Maximum likelihood,
a proven powerful and accurate method of phylogenetic reconstruction, favors a closer relationship of
diplonemids with kinetoplastids rather than euglenoids with both markers, while parsimony and
distance supported the alternative topology. However, differences between the best tree and suboptimal trees are small with each method.
One interesting feature of the mitochondrial genetic code of D. papillatum , the use of UGA and
UGG as tryptophan codons in cal mRNA is also
shared by kinetoplastids (de la Cruz et al. 1984;
Simpson et al. 1987). In cal from E. gracilis and the
euglenoid, Eutreptiella gymnastica, only the UGG
codon is used for this purpose (Inagaki et al. 1997;
Tessier et al. 1997; Yasuhira and Simpson 1997). In
addition, cal mRNA in E. gracilis does not contain a
poly-A tail (Yasuhira and Simpson 1997), while the
D. papillatum cal mRNA apparently is polyadenylated, as are mitochondrial messengers in kinetoplastids (Bhat et al. 1992). Although these observations support a closer affinity of diplonemids with
kinetoplastids, they are based only on a single mitochondrial gene and need to be verified by analysis of
additional genes.
Our preliminary analysis of the mitochondrial DNA
from D. papillatum indicates that it contains covalently closed molecules. No evidence for a kinetoplast-like network could be seen, although existence of some catenation needs to be investigated.
Trp
Trp
Cys
Cys
End
End
Tyr
Tyr
Leu
Leu
Phe
Phe
Ser
Ser
Ser
Ser
UGG
UGA
UGU
UGC
UAG
UAA
UAU
UAC
UUG
UUA
UUU
UUC
UCG
UCA
UCU
UCC
4.00
3.00
3.00
3.00
1.00
0.00
3.00
8.00
5.00
2.00
1.00
10.00
1.00
2.00
2.00
10.00
Arg
Arg
Arg
Arg
Gin
Gin
His
His
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
CGG
CGA
CGU
CGC
CAG
CAA
CAU
CAC
CUG
CUA
CUU
CUC
CCG
CCA
CCU
CCC
1.00
1.00
4.00
0.00
4.00
0.00
5.00
11.00
15.00
10.00
2.00
13.00
1.00
2.00
3.00
0.00
It is unclear if topologically relaxed molecules also
present in our preparations are due to the damage
caused by handling or whether they represent replication intermediates. In any case, isolated mtDNA of
D. papillatum is quite dissimilar from the heterogeneous linear molecules isolated from mitochondria
of E. gracilis (yasuhira and Simpson 1997), and also
from trypanosomatid networks (Simpson 1986;
Simpson 1987). Although only limited information is
available on the molecular organization of mtDNA of
bodonids (Blom et al. 1998; Hajduk et al. 1986;
Lukes et al. 1998; Yasuhira and Simpson 1996), it
seems the non-catenated or weakly catenated
structure is more similar to that seen in Dip/onema.
Finally, although the data obtained in this work
favor a closer association of diplonemids with kinetoplastids than with euglenoids, it is evident that
more work is required in order to fully resolve phylogenetic relationships among these organisms.
Methods
Strains and cultivation conditions: Dip/onema
(syn. /sonema) papillatum (ATCC50162) and Dip/onema sp. 2, strain IIIGPC, (ATCC50224) were obtained from the American Type Culture Collection.
The strains were cultivated at 27°C in an enriched
/sonema medium (ATCC Culture Medium 1728) containing 10% heat-inactivated fetal bovine serum in
stationary flasks. For cultures of D. papillatum , stationary phase of growth was achieved at 107 cells/ml
on the fourth day after inoculation with a starting cell
density of 0.5-1.0 x 106 cells/ml. For Dip/onema sp.,
only a ten fold lower density of cells could be obtained.
40
D. A. Maslov, S. Yasuhira, and L. Simpson
Isolation of total cell DNA and RNA: Cells from
stationary cultures were washed with 10 mM TrisHCI (pH 8.0), 150 mM NaCI, 100 mM EDTA, and
lysed with 2% Sarcosyl, 0.5 mg/ml pronase at 65 DC
for 30 min. The lysate was extracted with buffered
(pH 8.0) phenol, phenol-chloroform (1 :1) and chloroform. Nucleic acids were precipitated with an equal
volume of isopropanol. The pellet was rinsed with
ethanol, dissolved and reprecipitated with ethanol.
Total cell RNA was isolated by guanidinium thiocyanate/phenol-chloroform extraction procedure
using the RNA Isolation Kit (Stratagene).
Isolation of mitochondrial DNA: Total cell DNA
isolated from 0.5 x 1010 cells of D. papillatum was
fractionated using CsCI-Hoechst 33258 or CsCIethidium bromide equilibrium density centrifugation
in a VTi50 rotor at 45,000 rpm for 20 h. Other conditions were as described previously (Maslov and
Simpson 1994; Simpson 1979).
peR amplification and sequencing of the SSU
genes: Small subunit rRNA genes were amplified
using the oligonucleotides S762 (GACTITTGCTTCCTCTAWTG) and S763 (CATATGCTTGTTTCAAGGAC), cloned in the vector pT7Blue (Novagen), and
sequenced using the Sequenase kit (version 2.0,
Amersham) as described previously (Maslov et al.
1996). The first strand of the SSU gene of D. papillatum was sequenced with the following oligonucleotide primers (listed in the order of occurrence):
S847: CATATGCTTGTTTCAAGGACTWAGCCATGCATGCC;
S1404: CTGAGAACGGCTACCACATC;
S713: CCGCGGTAATTCCAGCTCC;
S1381: ACGGTCGACCACCGATGTTA;
S757: TCAGGGGGGAGTACGTTCGC;
S1402: TTGTAGGGGGTGTCTITTGG;
S828: CAACAGCAGGTCTGTGATGC;
The second strand was sequenced with the primers:
S1401: AGCAACGACGGGCGGTGTGT;
S829: GCATCACAGACCTGCTGTTG;
S1378: CACACAATTCATCGAGAAAG;
S714: CGTCAATTTCTTTAAGTTTC;
S1382: GAAACTCAAAAGAGAACCGC;
S1403: CACCATTACCACCGTTCATA;
S1380: TCAGCAGTGTTGCTATTGGG;
For the first strand of the Dip/onema sp. SSU gene
we used:
S847 (see above); S1645: CCCGCAAGAGTATCTGCCCTATC;
S1736: GGAATTAGGGTTCGATTCCG; S713;
S1644: ~GTCA GTCA
S757 and S828.
For the second strand of the same gene we used:
S829; S714; S1643: GGTTTGGAGCCTTACCTTAAATTAT;
S1739: CGGGTITTGATCTTCAACAG;
S755: CTACGAACCCTTTAACAGCA;
S1646: GATGTGGTAGCCGTTTCTCAGGCT.
The SSU rRNA sequences are deposited in the
GenBank™ database under the following accession
numbers: AF119811 (D. papillatum) and AF119812
(Dip/onema sp.).
Other nucleic acid manipulations: The conditions for RT-PCR and TA-cloning were as described
previously (Simpson et al. 1996). Briefly, the cDNA
from D. papillatum was synthesized using total cell
RNA and an oligo-dT primer (dT20 N). The oligonucleotide C112 (TTYTGRTTYTTYGGNCAYCCNGA)
(Lukes et al. 1994), which corresponds to a highly
conserved region of eukaryotic cal sequences, was
used together with the oligonucleotide dT20 N to PCR
amplify a partial cal mRNA. The determined partial
cal mRNA sequence is deposited in GenBank™
database under the accession number AF119813.
Agarose gel electrophoresis, restriction digestion
and Southern blotting were performed by standard
protocols. Hybridization with a DNA probe labeled
by random priming (Stratagene) was performed at
68 DC in 6 x SSC, 5 x Denhardt's solution, 100 mg/
ml salmon sperm DNA and 0.5% SDS.
Phylogenetic analysis: Alignments were generated manually using the program Seq Edit, version 3.1
(Olsen 1990). The SSU and cal alignments are available on request or can be downloaded from the following URL: http://www.lifesci.ucla.edu/RNAItrypanosome/alignments.html. Maximum likelihood,
parsimony and distance analyses were performed as
described previously (Lukes et al. 1997) using PAUP*
4.0 beta version (Swofford 1998) (Sinauer Associates,
Inc.) and PHYLIP 3.5 (Felsenstein 1995). In addition,
maximum likelihood analysis of the cal polypeptide
sequences was performed with PROTML, version
2.2, (Adachi et al. 1992) with the JTT amino acid substitution matrix (Jones et al. 1992).
Acknowledgements
We would like to thank Dr. J. Lukes for the oligonucleotide C112. This work was supported in part by
the Burroughs Wellcome New Investigator Award in
Molecular Parasitology to D.M. and the grant
AI09102 from National Institutes of Health to L.S.
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