Isolation and Molecular Phylogenetic Analysis of Actincoding Regions from Emiliania huxleyi, a Prymnesiophyte
Alga, by Reverse Transcriptase and PCR Methods’
Debashish Bhattacharya, 2 Shawn K. Stickel, and Mitchell L. Sogin
Center for Molecular
Evolution,
Marine
Biological Laboratory
Reverse transcriptase and polymerase chain reaction methods were used to amplify
and clone actin cDNAs from the chlorophylls
a+c-containing
unicellular alga,
Emiliania huxleyi (Prymnesiophyta).
Actins in E. huxleyi are defined by a gene
family containing at least six distinct coding regions that were derived from relatively
recent gene duplications.
Five of the coding regions (types 1, 2, and 4-6) varied
only among synonymous
codons. A nonsynonomous
change in a sixth coding
region (type 3 actin) produced a serine-to-phenylalanine
replacement.
The G+C
composition
of third positions in E. huxleyi actin genes is 98%, which contrasts
with the mean value of 50% G+C content for first and second positions. Distancematrix and parsimony analyses of actin genes identified the prymnesiophytes
as a
photosynthetic
lineage that is not already related to other eukaryotic algal groups.
Introduction
Phylogenetic
frameworks inferred from comparisons
of small-subunit
rRNA sequences suggest novel relationships
among photosynthetic
protist groups. The placement of the chlorophylls
a+c-containing
unicellular
alga, Emiliania huxleyi (Prymnesiophyta),
in a lineage separate from all other photosynthetic
protist lineages is
particularly
significant ( Bhattacharya
et al. 1992 ) . Prymnesiophytes
are considered
by some as chrysophyte
plankton
because they have tubular mitochondrial
cristae
(Taylor 1976), plastids with chlorophyll-c
and carotenoids
(Bjornland
and LiaaenJensen 1989; Jeffrey 1989 ) , and use p- 1,3 ( or - 1,6 )-linked glucans as a storage product
( Hellebust 1988 ) . Because of differences in swimming behavior, presence of a haptonemal complex not found in other groups, and lack of other ultrastructure
features
(Hibberd
1976)) some phycologists place prymnesiophytes
in a separate group described as “coccolithophorids.”
These scaly unicellular
organisms form calcium carbonate crystal structures and are responsible for a major part of the carbon transfer
between the atmosphere
and the ocean sediments. Coccolithophorids
are the origin
of extensive beds of Cretaceous chalk and limestone and, because of distinctive morphologies, these organisms have served as important
stratigraphic
makers.
Because relationships
described on the basis of shared ultrastructural
features are
divergence
in general agreement with the rRNA molecular frameworks, an independent
for E. huxleyi relative to that of chrysophytes is surprising. The placement of E. huxleyi
Emiliania huxleyi.
1. Key words: actin, evolution,
phylogeny,
2. Present address: Universittit
zu Kiiln Bo’tanisches
Institut,
Lehrstuhl
I Gyrhofstrasse
15, W-5000
Kiiln 4 1, Germany.
Address for correspondence
and reprints: Mitchell L. Sogin, Center for Molecular
Biological Laboratory,
Woods Hole, Massachusetts
02543.
Evolution,
Marine
Mol. Biol. Evol. 10(3):689-703. 1993.
0 1993 by The University of Chicago. All rights reserved.
0737~4038/93/1003-0014$02.00
689
690
Bhattacharya
et al.
in rRNA phylogenies may be aberrant, or convergent evolution may have produced
a lineage with ultrastructural
features similar to those of genetically unrelated chlorophylls a+c-containing
unicellular
alga. Comparative
molecular analyses of actins
might explain contradictory
positions of E. huxleyi in rRNA versus ultrastructure
phylogenies.
The ubiquity of actin and microfilament
proteins such as tropomyosin
and alpha-actinin
supports the hypothesis that the cytoskeleton
was well developed
in the ancestor of all eukaryotes (Searcy and Hixon 199 1). Comparative
studies of
such cytoskeletal protein-coding
regions may be useful in understanding
evolutionary
relationships
among even the most divergent eukaryotic lineages.
Actin-coding
regions and their organization
have been extensively studied in
representatives
of the three “higher kingdoms”
but more rarely in the protista. Actins
are most commonly
encoded by multiple-copy
genes. Mammals express at least six
different actins, including a-skeletal, a-cardiac, and p- and y-nonmuscle
actins ( Alonso
1987). Most plants contain a minimum
of 10 actin genes (Meagher and McLean
1990)) with as many as 200 copies in Petunia hybrida (McLean et al. 1990b). Divergent
plant actins are divisible into subfamilies that are sometimes differentially
expressed
and functionally
discrete (McLean et al. 1990a). In contrast, single-copy actin genes
have been reported for the higher fungi Aspergillus nidulans (Fidel et al. 1988) and
Saccharomyces cerevisiae (Ng and Abelson 1980)) as well as for several protist groups,
including the oomycetes Achlya bisexualis (Bhattacharya et al. 199 1) and Phytophthora
megasperma ( Dudler 1990)) the chlorophylls a+c-containing
alga Costaria costata
(Bhattacharya
et al. 199 1 ), the ciliated protozoan
Tetrahymena sp. (Cupples and
Pearlman
1986; Hirono et al. 1987), and the chlorophyte
Volvox carteri (Cresnar et
al. 1990).
Because multicellular
representatives
of the plant, animal, and fungal kingdoms
had their evolutionary
roots in protistan lineages, it may be possible to study the
origins of complex actin gene families by characterizing
their counterparts
in protists.
For example, the flowering green plants have complex, multiple-copy
actin gene families and are monophyletic
with chlorophytic
green algae (Gunderson
et al. 1987).
Limited studies of actins in green algae V. carteri (Cresnar et al. 1990) and Scherfilia
dubei (D. Bhattacharya, unpublished data) demonstrate single copies of actins. Flowering-plant
actin gene duplications
most likely occurred after the divergence of the
green algae. By studying other protistan groups that bridge the evolutionary
distance
between the divergence of the green algae and flowering plants (e.g., charophytes),
it
may be possible to identify the precise position of actin gene duplication and to correlate
this event with the diversification
of multicellularity
in flowering plants.
Phylogenetic
analyses of actin-coding
regions have demonstrated
variable rates
of sequence divergence in different lineages. Green-plant
actins evolve approximately
five times faster than plant rDNAs. In contrast, substitution
rates in metazoan actin
genes are approximately
five times less than those in rDNA coding regions in the same
genomes ( Bhattacharya
et al. 199 1). In spite of such variability in rates of divergence,
copy number, and function in different eukaryotes, actins are highly conserved proteins
and can be used as markers of gene and species evolution
(Vandekerckhove
and
Weber 1984; Hightower and Meagher 1986; Drouin and Dover 1990). Phylogenetic
analyses of actin-coding
regions corroborate results of small-subunit
ribosomal RNA
( 16S-like rRNA) sequence comparisons.
For example, the animals, fungi, green plants,
ciliates, and chromophytes
/ oomycetes are represented
by discrete evolutionary
assemblages in both rDNA and actin trees (Gunderson
et al. 1987; Bhattacharya
et al.
199 1; Gajadhar et al. 199 1) .
*
Phylogenetic
Analysis of Actin from E. huxleyi
691
Unlike rDNA, actin-coding
regions have not been sequenced from diverse protist
lineages. In this paper we report the characterization
and phylogenetic analysis of actin
cDNA sequences from the chlorophylls
a+c-containing
unicellular
alga, E. huxleyi
(Prymnesiophyta)
.
Material and Methods
DNA Extraction and Amplification
of Actin DNA
Emiliania Huxleyi (strain CCMP379) was obtained from the algal collection at
Bigelow Laboratory for Ocean Sciences. Nucleic acids were extracted from exponentially growing axenic cultures that were monitored
for contamination
by using light
microscopy
and sequence analysis of polymerase
chain reaction ( PCR )-amplified
rRNA genes. Cells were pelleted and equilibrated
with STE ( 10 mM Tris pH 8.5, 100
mM NaCl, 1 mM EDTA), washed three times and suspended in IO-ml STE buffer,
and extracted according to the method of Bhattacharya
et al. ( 199 1) . Approximately
0.5% of the nucleic acids was DNA; the remainder was intact RNA suitable for cDNA
synthesis. The synthesis of cDNAs was initiated using oligo (dT) 15 and 200 units of
Md.) . Reaction conditions
M-MLV reverse transcriptase
(RT) (BRL, Gaithersburg,
were as described by Hu et al. ( 199 1) .
The cDNAs were amplified via PCR techniques (Mullis and Faloona 1987; Saiki
et al. 1988) using amplification
primers that are complementary
to highly conserved
coding sequences near the 5’ and 3’ termini of actin-coding
regions. The 5’ actin
amplification
primer (GAATTCTGCA
GAC AAC GGY TCS GGY ATGTGC)
contains a polylinker for EcoRI and PstI and is complementary
to the DNA sequence
encoding amino acids 12- 18. The 3’ actin amplification
primer (CTCGAGGATCC
GAA GCA YTT GCG RTG SAC RAT) contains a polylinker for XhoI and BamHI
and is complementary
to the DNA sequence encoding amino acids 358-364 (relative
to the actin sequence of Achlya bisexualis; Bhattacharya
et al. 199 1). Third positions
of the majority of the codons in the amplification
primers are degenerate, and the
polylinkers are compatible
with Bluescript and Ml3 cloning sites. These actin PCR
primers were designed to amplify isoforms of actin in diverse eukaryotic lineages. The
utility of the primers was tested in PCR reactions that included first-strand cDNA or
genomic DNA from the lower fungal (Oomycete)
species A. bisexualis, Lagenidium
giganteum, and Pythium irregular-e; the chromophyte
algae Costaria costata and
Vaucheria sessilis; the dinoflagellate Prorocentrum micans; and the sponge Microciona
spp. Actin-coding
regions were successfully amplified from these species by using PCR
reaction and amplification
conditions described by Medlin et al. ( 1988).
Characterization
and Phylogenetic
Analyses
of E. huxleyi Actin
Amplified E. huxleyi actin was used to probe Southern blots of E. huxleyi genomic
DNA. The actin probe was radiolabeled
with 32P-dCTP by using an oligo-labeling
kit
(Pharmacia)
and incubated with a genomic Southern blot of E. huxleyi DNA digested
with BamHI and KpnI. Annealings
were incubated overnight at 65°C in 5 X SSC ( 1
X SSC = 0.15 M NaCl, 0.0 15 M Na3 citrate) and 1 X Denhardt’s
[ 0.02% (w/v) bovine
serum albumin, Ficoll (molecular
weight 400,000), polyvinylpyrrolidone]
, 100 mM
sodium phosphate (pH 7.0), 0.5% SDS. Filters were washed at 60°C in 1 X SSC, 0.1%
SDS and air-dried, and radioactive fragments were located by autoradiography.
The polylinker sites in the actin primers were used to clone amplified E. huxleyi
actin-coding regions into single-stranded
M 13 bacteriophages. Oligonucleotide
primers,
complementary
to conserved sequence islands in actin-coding
regions (Bhattacharya
0yL marracnarya et al.
et al. 199 1), were used to initiate DNA synthesis in the dideoxy-mediated
chaintermination
sequencing
protocol (Sanger et al. 1977). As many as 11 independent
recombinant
M 13 clones were pooled and used to prepare a mixed sequencing template.
This strategy effectively samples the population
of PCR products. Heterogeneities
in
actin genes, artifacts of PCR amplification,
and possible contamination
by actin
mRNAs from other evolutionary
lineages are revealed as coincident migrating bands.
If heterogeneities
are found, sequence can be determined
for individual clones.
The actin nucleotide
sequence of E. huxleyi was aligned with those of 22 eukaryotes, including members of the major eukaryotic assemblages (i.e., animals, green
plants, ciliates, and fungi), and several independent
protist lineages. Phylogenetic
trees were inferred using distance-matrix
and maximum-parsimony
methods. Only
first and second positions of codons were used in the sequence comparisons,
because
ofmutational
saturation at third positions ( Hightower and Meagher 1986; Bhattacharya
et al. 199 1). Bootstrap methods (Felsenstein
1985) (using 100 samplings of the nucleotide sequences)
were used to assess the fraction of positions that support a
given clade.
For the distance technique (Fitch and Margoliash 1967 ), pairwise comparisons
of sequences were used to calculate similarity values. These similarity values were
converted to phylogenetic
trees as described by Olsen ( 1988). Maximum-parsimony
analysis of actin sequences was implemented
with the PAUP computer package (PAUP,
3.OL; Swofford 1990). Heuristic procedures using a branch-swapping
algorithm (tree
bisection-reconnection)
and the MULPARS
option were utilized.
Results and Discussion
Emiliania huxleyi actin cDNAs were amplified using the RT and PCR protocols.
The major PCR product contained
1,095 bp (fig. 1, lane 1). PCR products of similar
size are generated when actin primers are used to amplify E. huxleyi genomic DNA
(result not shown). Southern blots probed with the amplified cDNA clones demonstrated that E. huxleyi actin-coding
regions are members of a gene family (fig. 2). In
BamHI restriction digests two major bands, 2.1 kb and 12.2 kb, annealed with the
actin probe (fig. 2, lane 1). A complex array of lower-intensity
actin-annealing
fragments are observed in both BamHI and KpnI genomic digests. These may correspond
to variable numbers of actin-coding
regions at dispersed sites or may be artifacts of
incomplete
digestion of E. huxleyi DNA with restriction endonucleases.
Because the
dispersed banding pattern persisted after stringent washes, it is unlikely that the E.
huxleyi actin probe is binding to dispersed sequences of low similarity. Furthermore,
the isolation of nearly identical actin sequences (see below) and a unique rRNA sequence (identical to that of an independent
E. huxleyi isolate; L. Medlin, personal
communication)
in PCR cloning experiments
(Bhattacharya
et al. 1992) essentially rules out weak banding artifacts from contaminated
cultures. McLean et al.
( 1990b) found a similar result in the analysis of the actin gene family in Petunia
hybrida. Six divergent actin gene subfamilies were identified in P. hybrida, and they
are dispersed at five chromosomal
locations. Copy numbers within subfamilies ranged
from 1 to 12 members.
Sequence analysis of a mixture of 11 M 13 / mp 18 clones and 1 M 13 /mp 19 clone
demonstrated
sequence heterogeneity in E. huxleyi actin. Six structurally distinct actin
cDNAs (types l-6 ) were identified by characterizing
subsets of the original 12 clones.
The complete sequence of type 1 actin was determined for both coding and noncoding
strands (fig. 3). Alignment
of this DNA sequence with those of diverse eukaryotes
12216
4012
3054
2036
1636
1018
517
demonstrated
that the sequenced portion of the type 1 coding region was typical of
actin, with an open reading frame of 365 amino acids. Partial (629 nucleotides),
single-strand
sequences were determined
from the 5’ termini (starting with the 5’
amplification
primer) of E. huxleyiactin types 2-6, and these were aligned with the
type 1 sequence (fig. 4). Variability among these actin cDNA sequences ranged from
1 change (types 3-5 and types 4-5) to 25-29 changes between type 6 and the other
cDNAs (table 1) The nucleotide substitutions
were distributed throughout the coding
4072 -
3054 -
2036 1636 -
region, but most changes were confined to third codon positions and, except for type
3 actin, produced identical amino acid products. A second-position
C-T
substitution
in type 3 actin produced a serine-to-phenylalanine
replacement.
That nearly all sequence differences correspond to silent third-position
changes indicates that variations
in actin sequences are not artifacts introduced by RT or PCR methods. Variation in
actin cDNAs from E. huxkyi was not exhaustively
explored, and it is possible that,
because of variation at the amplification
primer sites, highly divergent actin cDNAs
were not amplified.
gacaacggttccggcatgtgcAAGGCGGGCTTCGCGGGCGACGACGCNCCGCGTGCGGTCTTCCCCTCCATCATCGGCCGCCCGCGCCAGCCCGGCGTG
AspAsnGlySerGlyMetCysLysAlaGlyPheAlaGlyAspAspAlaProArgAlaValPheProSerIleIleGlyArgProArgGlnProGlyVal
ATGGTCGGCATGGGCCAGAAGGACTCGTACGTCGGCGACGAGGCGCAGTCC~GCGTGGCATCCTCACGCTC~GTACCCGATCGAGCGCGGCATCGTC
MetValGlyMetGlyGlnLysAspSerTyrValGlyAspGluAlaGlnSerLysArgGlyIleLeuThrLeuLysTyrProIleGluArgGlyIleVal
ACGAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTAC~CGAGCTGCGCGTGGCACCCGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCG
ThrAsnTrpAspAspMetGluLysIleTrpHisHisThrPheTyrAsnGluLeuArgValAlaProGluGluHisProValLeuLeuThrGluAlaPro
CTCAACCCCAAGGCCAACCGCGAGAAGATGACGCAGATCATGTTTGAGACCTTC~CGTGCCCGCCATGTACGTCGCCATCCAGGCCGTCCTCTCGCTG
LeuAsnProLysAlaAsnArgGluLysMetThrGlnIleMetPheGluThrPheAsnValProAlaMetTyrValAlaIleGlnAlaValLeuSerLeu
TACGCGTCGGGCCGCACGACCGGCATCGTGATGGACTCGGGCGACGGCGTCTC~ACACCGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATC
TyrAlaSerGlyArgThrThrGlyIleValMetAspSerGlyAspGlyValSer.. ~~?ThrValProIleTyrG1uGlyTyrAlaValProHisAlaIle
CTCCGCCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTC~GCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATC
LeuArgLeuAspLeuAlaGlyArgAspLeuThrAspTrpMetValLysLeuLeuThrGluArgGlyTyrSerPheThrThrThrAlaGluArgGluIle
GTGCGCGACA~CAAGGAGAAGCTGGCGTACGTGGCGCTCGACTTTGACCAGGAGATGCAGACCGCCGCCTCCTCCTCCTCGCTCGAG~GTCGTACGAG
ValArgAspIleLysGluLysLeuAlaTyrValAlaLeuAspPheAspGlnGluMetGlnThrAlaAlaSerSerSerSerLeuGluLysSerTyrGlu
CTGCCCGACGGCCAGGTCATCACCATCGGCAACGAGCGCTTCCGCTGCCCCGAGGCGCTCTTCCAGCCCTCCTTCCTGGGGATGGAGTCGGCGGGCGTG
LeuProAspGlyGlnValIleThrIleGlyAsnGluArgPheArgCysProGluAlaLeuPheGlnProSerPheLeuGlyMetGluSerAlaGlyVal
CACGAGACGACGTACAACTCGATCATGAAGTGCGACGTCGACATCCGC~GGACCTCTACGCC~CGTCGTCCTCTCGGGCGGCACCACCATGTACGCC
HisGluThrThrTyrAsnSerIleMetLysCysAspValAspIleArgLysAspLeuTyrAlaAsnValValLeuSerGlyGlyThrThrMetTyrAla
tgcttc
CysPhe
FIG.3.-Nucleotide
andaminoacid
sequences
oftbecloned
type1 actin-coding
region
fromEmiliunia huxleyi. Thesequenced
coding
region
is1,095
acids).
The 5’ 34 nucleotides were not determined. PCR-amplification primer sequences are shown in lowercase letters, and ambiguous positions are indicated
nucleotide sequence and by “?’ in the amino acid sequence.
Tvpe
T&z
Type
Type
Type
Type
1
2
3
4
5
6
qacaacqqttccqqtatqtqcAAGGCGGGCTTCGCGGGCGACGACGCNCCGCGTGCGGTCTTCCCCTCCATCATCGGCCGCCCGCGCCAGCCCGGCGTGA
gacaacggttccggtatgtgcAAGGCGGGCTTCGCGGGCGACGACGNNCCGCGCGCGGTCTTCCCCTCCATCATCGGCCGGCCGCGCCAGCCCGGCGTGA
gacaacggttccggtatgtgcAAGGCGGGCTTCGCGGGCGACGACGNNCCGCGCGCCGTCTTCCCGTCCATCATCGGCCGGCCGCGCCAGCCTGGCGTGA
gacaacggctcgggtatgtgcAAGGCGGGCTTCGCGGGCGACGACGNNCCGCGCGCCGTCTTCCCGTCCATCATCGGCCGGCCGCGCCAGCCTGGCGTGA
gacaacggctccggtatgtgcAAGGCGGCGGGCTTCGCGGGCGACGACGNNCCGCGCGCCGTCTTCCCGTCCATCATCGGCCGGCCGCGCCAGCCTGGCGTGA
gacaacggctcgggtatgtgcAAGGCNGGNTTCGCCGGCGACGACGNNCCGCGCGCGGTCTTCCCCTCCATCATCGGCCGGCCGCGTCAGCCGGGCGTGA
*
* *
*
*
*
*
Type
Type
Type
Type
Type
Type
__
1
2
3
4
5
6
TGGTCGGCATGGGCCAGAAGGACTCGTACGTCGGCGACGAGGCGCAGTCC~GCGTGGCATCCTCACGCTCAAGTACCCGATCGAGCGCGGCATCGTCAC
TGGTCGGCATGGGCCAGAAGGACTCGTACGTCGGCGACGAGGCGCAGTCC~GCGCGGCATCCTCACGCTCAAGTACCCGATCGAGCGCGGCATCGTCAC
TGGTCGGCATGGGGCAGAAGGACTCGTATGTCGGCGACGAGGCGCAGTCCAAGCGTGGCATCCTCACGCTCAAGTACCCGATCGAGCGCGGCATCGTCAC
TGGTCGGCATGGGGCAGAAAGACTCGTATGTCGGCGACGAGGCGCAGTCCAAGCGTGGCATCCTCACGCTCAAGTACCCGATCGAGCGCGGCATCGTCAC
TGGTCGGCATGGGGCAGAAGGACTCGTATGTCGGCGACGAGGCGCAGTCC~GCGTGGCATCCTCACGCTCAAGTACCCGATCGAGCGCGGCATCGTCAC
TGGTCGGGATGGGTCAGAAGGACTCGTACGTGGGAGATGAGGCGCAGTCC~GCGTGGCATCCTGACGCTCAAGTACCCGATCGAGCGCGGCATTGTCAC
Type
Type
T-me
T;pe
Type
1
2
3
4
5
GAACTGGGACGACATGGAGAGATCTGGCACCACACCTTCTAC~CGAGCTGCGCGTGGCACCCGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCGCTC
CAACTGGGACGACATGGAGAGATCTGGCACCACACCTTCTAC~CGAGCTGCGCGTGGCGCCGGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCGCTC
CAACTGGGATGACATGGAGAGATCTGGCACCACACCTTCTAC~CGAGCTGCGCGTGGCGCCGGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCGCTC
CAACTGGGATGACATGGAGAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTGGCGCCGGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCGCTC
CAACTGGGATGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTGGCGCCGGAGGAGCACCCGGTCCTGCTGACCGAGGCGCCGCTC
CAATTGGGACGACATGGAGAAAATCTGGCACCACACCTTCTAC~TGAGCTGCGCGTGGCGCCTGAGGAGCACCCCGTCCTACTGACCGAGGCGCCGCTC
*
* *
*
*
*
*
*
*
Type 6
Type
Type
T-me
T$pe
Type
Type
1
2
3
4
5
6
Type
Type
1
2
3
T;pe 4
Type 5
T&e
6
TVDe
*
*
*
* * * *
*
*
AACCCCAAGGCCAACCGCGAGAAGATGATGACGCAGATCATGTTTGAGACCTTC~CGTGCCCGCCATGTACGTCGCCATCCAGGCCGTCCTCTCGCTGTACG
AACCCCAAGGCCAACCGCGAGAAGATGATGACGCAGATCATGTTTGAGACCTTCAACGTGCCCGCCATGTACGTCGCCATCCAGGCCGTCCTCTCGCTGTACG
AACCCCAAGGCCAACCGCGAGAAGATGATGACCCAGATCATGTTTGAGACCTTCAACGTGCCCGCCATGTACGTCGCCATCCAGGCGGTCCTCTCGCTGTACG
AACCCCAAGGCCAACCGCGAGAAGATGATGACCCAGATCATGTTTGAGACCTTCAACGTGCCCGCCATGTACGTCGCCATCCAGGCGGTCCTCTCGCTGTACG
AACCCCAAGGCCAACCGCGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACGTGCCCGCCATGTACGTCGCCATCCAGGCGGTCCTCTCGCTGTACG
AACCCCAAGGCCAACCGCGAGAAGATGACGCAGATCATGTTTGAGACCTTCAACGTGCCCGCCATGTACGTCGCCATCCAGGCGGTGCTCTCGCTCTACG
*
* *
CGTCGGGCCGCACGACCGGCATCGTGATGGACTCGGGCGACGGCGTCTCNNACACCGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTCCG
CGTCGGGCCGCACGACCGGCATCGTGATGGACTCGGGCGACGGCGTCTCNNACACGGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTCCG
CGTCGGGCCGCACCACCGGCATCGTGATGGACTCGGGCGACGGCGTCTCNNACACCGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTGCG
CGTCGGGCCGCACCACCGGCATCGTGATGGACTCGGGCGACGGCGTCTCNNACACCGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTGCG
CGTCGGGCCGCACCACCGGCATCGTGATGGACTCGGGCGACGGCGTCTCNNACACCGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTGCG
CGTCGGGCCGCACCACCGGCATCGTGATGGATTCGGGCGACGGCGTCTCNNACACGGTGCCCATCTACGAGGGCTACGCCGTGCCGCACGCCATCCTGCG
*
*
*
*
*
l
Type 1 CCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
TVDe
2 CCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
T;'pe
Type
Type
Type
3
4
5
6
CCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTTCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
CCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
CCTCGACCTCGCCGGCCGCGACCTGACCGACTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
GCTCGACCTGGCCGGCCGCGACCTGACCGATTGGATGGTCAAGCTGCTGACCGAGCGCGGCTACTCCTTCACCACCACCGCCGAGCGCGAGATCGTGCGC
*
*
*
*
Tvoe 1 GACATCAAGGAGAAGCTGGCGTACGTGGC
T&e
2 GACATCAAGGAGAAGCTGGCGTACGTGGC
Type 3 GACATCAAGGAGAAGCTGGCGTACGTGGC
Type 4 GACATCAAGGAGAAGCTGGCGTACGTGGC
Type 5 GACATCAAGGAGAAGCTGGCGTACGTGGC
Type 6 GACATCAAGGAGAAGCTGGCGTACGTGGC
FIG. 4.-Aligned partial (629-nucleotide) sequences of actin cDNAs from EmiliaCa huxleyi. Sequences were determined from the 5’ terminus only on the noncoding
strand. The sequ<xe of the 5’ amplification primer is shown in lowercase letters, and ambiguous positions are indicated by “N.” The variable sites in this alignment are denoted
by an asterisk (* ), and all but one are located in silent, third positions of codons. Nucleotide 566 in type 3 actin specifies a second-position substitution resulting in a serineto-phenylalanine replacement.
Phylogenetic
Analysis of Actin from E. huxleyi
697
Table 1
Comparisons of Emiliania huxleyi and Achlya bisexualis (Ab) Actin Genes
1
Type 1
Type 2
Type
Type
Type
Type
Ab
3
4
5
6
...
2
98.8
7
16
16
15
29
144
13
13
12
25
144
4
3
97.3
97.8
2
1
28
144
5
6
Ab
97.3
97.8
97.4
98.0
95.1
95.7
75.5
75.5
99.7
99.8
99.8
95.2
95.3
95.6
75.5
75.5
75.6
74.4
1
28
144
27
143
150
NOTE.-Pairwise
sequence comparisons are shown for all codon positions in six actin genie sequences from E. hux/eyi
(types l-6) and in the Ab single-copy actin gene. Percent similarities are above the diagonal, and numbers of changes are
below the diagonal.
The high amino acid sequence conservation observed in E. huxleyi actin contrasts
with that in green-plant
and animal coding regions. In Glycirte max, amino acid replacements range from 6% to 9% between members of its actin subfamilies. Vertebrate
actins display - 1% amino acid replacements
between muscle actins (Hightower and
Meagher 1986). The six actin cDNA types identified in this analysis are nearly identical
members of a gene family. Homogenization
of actin-coding
regions within this family
that are due to unequal crossing-over ( Dover 1982 ) would maintain overall subfamily
identity though some variation within subfamilies can be detected (i.e., there are seven
nucleotide differences between the type 1 and type 2 actin genes). Maximum-parsimony
analysis of E. huxleyi actin types 1-6, using all positions of codons, identifies three
clades that have arisen from duplications
and drift from an ancestral gene (fig. 5).
Type 6 is the most divergent actin cDNA and represents the product of a gene duplication that also resulted in the type l-5 cluster of actin cDNAs. A second, more recent
gene duplication
resulted in the clades represented
by types 1+2 (seven nucleotide
substitutions)
and by types 3+4+5 (one or two nucleotide substitutions).
5
3
Type 1
7
I
2
Type 2
1 Type 3
7
1
Type 4
Type 5
23
Type 6
analysis (PAUP, 3.OL; Swofford 1990) of actin types l-6 from Emiliania
method with type 6 actin as outgroup. Partial (629-nucleotide)
sequences were used in this analysis, and the results demonstrate
three clades within the actin gene family
of E. huxleyi. Values above branches indicate the number of nucleotide changes.
FIG. 5.-Maximum-parsimony
huxleyi, by using the exhaustive-search
B
CostariaA!&lYabisexualis
Phvtoohthorameeasoerma
-uYQxsxkti
72
100
Glvcinem
_
Acanthamoebom
Dirtvosteliumdiscoideum
r
-Emilianahuwlevi
44
44
__
ILQmPsaniens
IiQm!i!saoleas
55
-BombnrmQti
50
Phylogenetic Analysis of Actm from &. huxleyz
bYY
Table 2
G+C Composition in Actin-coding Regions
G+C FREQUENCY
Emiliania huxleyi . . . . .
Achlya bisexualis . . _. . . . . . .
Costaria costata
...... ....
Arabidopsis thaliana . . .
..
Volvox carteri
.... ... ....
Acanthamoeba castellanii . . .
Bombyx mori . . . . , . . . . . . . .
Dictyostelium discoideum
.
Entamoeba histolytica . . . . .
Saccharomyces cerevisiae
Tetrahymena thermophila . . . .
First and Second Positions
Third Position
0.50
0.98
0.59
0.94
0.48
0.72
0.90
0.83
0.36
0.22
0.44
0.49
0.47
0.48
0.48
0.49
0.49
0.48
0.46
0.45
0.44
0.44
Analysis of the G+C content of E. huxZeyi actin cDNAs reveals a significant bias
in third-position
nucleotide
composition.
The third positions of E. huxZeyi type 1
actin have a G+C content of 98% (table 2). This number contrasts with the value of
50% G+C content when only first and second positions are considered in E. huxleyi
type 1 actin. Third-position
G+C contents of actin-coding
regions vary widely in
eukaryotes, ranging from 22% in Entamoeba histolytica (Edman et al. 1987) to 98%
in E. huxleyi (table 2). D’Onoftio
et al. ( 199 1) concluded that, in eukaryotic genes,
G+C content of first plus second positions is positively correlated with G+C content
of third positions. Actins of E. huxleyi differ from those of other eukaryotes in this
regard. The significance of extreme third codon position G+C bias is unknown,
but
it reduces by nearly 50% the number of codons that is used in E. huxleyi actin genes.
FIG. 6.-Phylogenetic analyses of actin-coding regions from E. huxleyi (type 1) and diverse eukaryotes.
A, Tree based on nucleotide differences between first and second codon positions of actin genes. A total of
662 positions were considered. Distances between taxa are represented by the horizontal component of their
separation. The distance that corresponds to 10 differences/ 100 nucleotide positions, or 66 total differences,
is indicated by the scale. Maximum-parsimony analysis of actin-coding regions by using a heuristic procedure
with a branch-swapping algorithm ( PAUP, 3.OL; Swofford 1990) identified a single parsimonious phylogram
(B). T. brucei was used as the outgroup to root both trees. Bootstrap values, based on 100 replications, are
indicated on horizontal line segments. The number of nucleotide changes is proportional to the length of
horizontal line segments, with the scale bar corresponding to 50 nucleotide changes. Clusters that were not
supported in >20% of the replications do not have bootstrap values indicated. Sequences came from the
following sources: A. bisexualis and C. costata (Bhattacharya et al. 1991 ), Acanthamoeba castellanii (actin-i;
Nellen and Gallwitz 1982)) Arabidopsis thaliana-AAc 1 (Nairn et al. 1988)) Bombyx mori actin 1 (Mounier
et al. 1987 ) , D. discoideum A8 ( Romans and Firtel 1985), Drosophila melanogaster locus 42A (Fyrberg et
al. 198 1)) E. huxleyi (present study), Entamoeba histolytica EhAct-g 1 ( Edman et al. 1987 ) , G. max (SAC 1;
Shah et al. 1983), Homo sapiens Al-smooth
muscle alpha-actin (Kamada and Kakunaga 1989); H.
sapiens A2-skeletal
muscle alpha-actin (Gunning et al. 1983); H. sapiens Bl-cytoplasmic
beta-actin
(Ponte et al. 1984), Kluyveromyces luctis (Deshler et al. 1989), 0. fallax (Kaine and Spear 1982), Oryza
sativa RAcl (McElroy et al. 1990), P. falciparum actin I (Wesseling et al. 1988), P. megasperma (Dudler
1990)) Physarum polycephalum Ppa5 (Gonzales-y-Merchand and Cox 1988)) S. cerevisiae (Ng and Abelson
1980)) Schizosaccharomyces pombe (Mertins and Gallwitz 1987), T. brucei actin- 1 (Amar et al. 1988), T.
thermophila (Cupples and Pearlman 1986)) V. carteri (Cresnar et al. 1990)) and Zea mays MAC 1 (Shah
et al. 1983).
/VU
anarkicnarya
tx
al.
Phylogenetic
analyses of the E. huxleyi type 1 actin-coding
regions identify the
prymnesiophytes
as a chlorophylls a+c-containing
photosynthetic
lineage that is not
specifically related to any of the major photosynthetic
or heterotrophic
protist groups
(fig. 6). The divergence of E. huxleyi occurs in the period that corresponds
to the
nearly simultaneous
radiation of many eukaryotic lineages. The bootstrap values in
the distance-matrix
(fig. 6A) and maximum-parsimony
(fig. 6B) analyses indicate the
uncertain position of the prymnesiophyte
lineage. There are no significant bootstrap
values that group E. huxleyi with any other eukaryotic clade. These results agree with
distance-matrix
and maximum-parsimony
analyses of the E. huxleyi small-subunit
rDNA coding region (Bhattacharya
et al. 1992).
The general branching patterns in the distance-matrix
and maximum-parsimony
analyses of actin-coding
regions are similar. The precise branching order for chromophytes/oomycetes
and green plants relative to other recently diverged groups and the
placement of Plasmodium falciparum is uncertain. The unresolved branching pattern
for chromophytes/
oomycetes, animals, green plants, and fungi is reflected by low
bootstrap values and different branching patterns in parsimony and distance analyses
of actin-coding
regions. Internal nodes separating these groups are very short, which
agrees with lack of resolution
in the “crown”
of the eukaryotic tree inferred from
ribosomal RNA sequence similarities.
Uncertain placement of P. falciparum in the actin molecular trees is more difficult
to explain. Phylogenetic
analyses based on 16S-like rRNA sequences place the Apicomplexans
(including P. falciparum) within a complex evolutionary
assemblage that
contains ciliates and dinoflagellates
(Gunderson
et al. 1987; Gajadhar et al. 199 1;
Sogin 199 1). The distance analysis of actin-coding
regions correctly positions the P.
falciparum sequence with Tetrahymena thermophila and Oxytricha fallax. The bootstrap analysis does not, however, significantly support monophyly of this lineage ( 56%).
The maximum-parsimony
analysis positions P. falciparum outside the ciliate cluster,
as an independent
lineage. These results may reflect the differing rates of sequence
divergence of actin-coding
regions in these organisms and may be resolved by the
inclusion
of a dinoflagellate
sequence or of additional
apicomplexan
sequences in
future analyses.
A variety of statistical methods have been devised to assess the reliability of
phylogenetic
trees inferred from molecular sequence data. Another mechanism
for
interpreting
the reliability of molecular frameworks is to examine congruence
with
molecular phylogenies inferred from genetically distinct genes and/or to evaluate the
distribution
patterns of independent
biological features on the gene tree. Here we have
applied RT and PCR techniques to phylogenetic
studies of actin cDNAs. Analysis of
expressed actin-coding
regions avoids potential complications
introduced
by intervening sequences in coding regions or by the cloning of pseudogenes
or truncated
genomic clones. The phylogenetic placement of E. huxleyi in trees inferred from actincoding regions supports a similar placement
in trees inferred from comparisons
of
ribosomal
RNAs. Both frameworks
describe E. huxleyi as an evolutionary
lineage
that is not clearly related to other algal groups.
The commonly cited ultrastructure
features that link prymnesiophytes
with other
chromophytes
are associated with plastids and the mitochondria.
These organelles
were introduced into the eukaryotic cell by ancient eubacterial endosymbionts.
Agreement between the actin- and rRNA-based
phylogenies instills confidence about the
independent
divergence of the prymnesiophyte
evolutionary
lineage. Disagreement
between the molecular trees and inferences based on organellar characteristics
is con-
Phylogenetic Analysis of Actin from E. huxleyi
701
sistent with hypotheses of convergent
evolution for some ultrastructure
features or
with multiple, independent
endosymbiotic
events leading to formation of stable mitochondria
and chloroplasts.
Acknowledgments
This research was supported
M.L.S. and by Sloan Foundation
1-6 ME to D.B.
LITERATURE
by National
Fellowship
Institutes of Health grant GM32964 to
for Molecular Studies of Evolution 89-
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Received
February
Accepted
December
editor
10, 1992; revision
1, 1992
received
December
1, 1992