Lactate Dehydrogenase (LDH) Gene Duplication During Chordate
Evolution: The cDNA Sequence of the LDH of the Tunicate StyeZaplicata
David W. Stock, *iclJoseph M. Quattro, *2 Gregory S. Whitt,t_ and Dennis A. Powers*
*Department of Biological Sciences, Hopkins Marine Station, Stanford University;
Evolution, University of Illinois at Urbana-Champaign
and TDepartment
of Ecology,
Ethology
and
L-Lactate dehydrogenase
(L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with
the exception of lampreys, which have a single LDH locus. Biochemical characterizations
of LDH proteins have
suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional
locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic
studies of LDH protein
sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of
studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have
diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about
the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences
have been available for comparison. We have attempted to provide further data on the timing of gene duplications
leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styelu plicatu.
Phylogenetic
analyses of this and other LDH sequences provide strong support for the duplications giving rise to
multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH
duplications is consistent with data from a number of other gene families suggesting widespread gene duplication
near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent
with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some
of the main lineages of vertebrate LDHs were not resolved in our analyses.
Introduction
Comparisons
of nuclear genome sizes and numbers
of loci encoding isozymes and other proteins in a variety
of chordates led Ohno (1970) to propose that episodes
of large-scale gene duplication had occurred during the
evolution of the group. The more recent discovery that
many genes involved in the control of development
are
members of multigene families with more representatives in vertebrates than in invertebrates
has led to increased interest in documenting
the patterns of gene duplication in chordate evolution (Holland 1992; Holland
et al. 1994). The sizes of some of these families, coupled
with the difficulty in determining gene number in a large
sample of taxa, suggest the value of continued examination of smaller, more extensively
studied gene families in detecting episodes of gene duplication in chordate
evolution. One such gene family encodes L-lactate dehydrogenase
(L-LDH, E.C. 1.1.1.27), which catalyzes
the interconversion
of pyruvate (the end product of glycolysis) and L-lactate with the concomitant interconversion of NADH and NAD+ (Markert 1984). Extensive
surveys of LDH locus number, expression pattern, kiand immunochemical
similarities
in
netic properties,
representatives
of the major taxa of jawed vertebrates
(Marker& Shaklee, and Whitt 1975; Fisher et al 1980;
Whitt 1984) have suggested that they all possess orthologs of Ldh-A and Ldh-B. LDH-A is expressed pre’Present address: Department
State University.
of Anthropology,
* Present address: Department of Biology,
ence, University of South Carolina.
Key words: tunicate,
molecular
phylogeny,
The Pennsylvania
Program
in Marine Sci-
gene duplication.
Address for correspondence
and reprints: David W. Stock, Department of Anthropology,
The Pennsylvania
State University, University Park, Pennsylvania
16802. E-mail: [email protected].
Mol. Biol.
Evol. 14(12): 1273-1284.
1997
0 1997 by the Society for Molecular Biology
and Evolution.
ISSN: 0737-4038
dominantly
in white skeletal muscle and is kinetically
suited to the reduction of pyruvate to lactate under conditions of low oxygen availability, while LDH-B is predominantly expressed in more aerobic tissues and is tailored to the oxidation of lactate to pyruvate (Markert
1984). An additional locus encoding an isozyme with
more variable kinetic properties (LDH-C) is expressed
in the sperm of mammals and columbid birds and in a
variety of tissues or the liver or eye of ray-finned fishes
(Markert, Shaklee, and Whitt 1975). Jawless fishes possess either a single locus (lampreys) or, in the case of
hagfishes, two loci with tissue expression patterns similar to those of jawed vertebrate Ldh-A and Ldh-B (Markert, Shaklee, and Whitt 1975). Markert, Shaklee, and
Whitt (1975) proposed that an initial gene duplication
gave rise to Ldh-A and Ldh-B early in vertebrate evolution and that Ldh-C is the result of a more recent duplication.
Subsequent
phylogenetic
analyses
of vertebrate
LDH sequence data have supported some aspects of this
“traditional”
scenario for LDH evolution
and challenged others (Li et al. 1983; Hendriks et al. 1988;
Crawford, Constantino,
and Powers 1989; Stock and
Whitt 1992; Quattro, Woods, and Powers 1993; Tsuji et
al. 1994; Quattro et al. 1995). Some points that remain
controversial or poorly resolved are the phylogenetic position of the LDH-Cs, the relationship
of cyclostome
LDHs to gnathostome
LDHs, and the monophyly
of
gnathostome
LDH-As. The former two issues involve
the earliest duplication events leading to the multigene
family in vertebrates. Such duplications
would be further elucidated by an examination
of the relationship of
invertebrate
and protochordate
LDHs to those of vertebrates. To date, the only complete LDH sequence available from an invertebrate animal is that of the nematode
Caenorhabditis
elegans (Tsoi and Li 1994), a represen1273
1274
Stock et al.
tative of a group that is quite distant from vertebrates.
Among protochordates,
tunicates and amphioxus have
been reported to possess single LDH loci (Fisher et al.
1980; Baldwin, Mortimer, and Patak 1988). Immunochemical analyses and comparisons
of amino acid composition were interpreted as evidence that tunicate LDH
was more closely related to the LDH-C of a teleost fish
than to either LDH-A or LDH-B (Baldwin, Mortimer,
and Patak 1988), while Fisher et al. (1980) suggested
that the single-locus
condition of amphioxus and tunicates predated the divergence of these groups from vertebrates. We have attempted to provide additional resolution of the early duplication history of chordate LDHs
by cDNA sequence
determination
and phylogenetic
analysis of an LDH locus from the tunicate Sty& plicata.
Adult specimens of the tunicate Styelu plicata were
obtained from Gulf Specimen Company (Panacea, Fla.).
A section of the body wall was cut from a single specimen, and the inner and outer surfaces were removed
with a scalpel. The remaining portion was ground to a
fine powder in liquid N2 using a mortar and pestle. Total
RNA was extracted from 2 g of powdered tissue by
homogenization
in the presence of sodium tri-isopropylnaphthalene
sulfonate
followed
by phenol/chloroform extraction, ethanol precipitation,
and precipitation
from sodium acetate, as previously described (Stock et
al. 1991).
Transcription
Total RNA was reverse transcribed with MMLV reverse transcriptase and either random hexamers or a dT
adaptor primer (5’-GATGCTGCAGAGCTCAAGC(T)is3’) as described by Stock and Powers (1995).
RT-PCR Amplification
Styela Ldh
RACE Amplification
of Internal
Fragment
of
A number of degenerate PCR primers (designed
from an alignment of bacterial and vertebrate LDH protein sequences-Stock
and Whitt 1992; Stock and Powers 1995) were tested for their ability to amplify a product of the expected size from Styela first-strand cDNA.
Of these degenerate
primers,
only Ldhll3FhF
(5’GCGAATTCGTNGGNATGGCNTGYGCNRT-3’,
where the underlined sequences indicate restriction sites,
and ambiguous bases are indicated by the appropriate
IUPAC codes) and LdhB736FhR
(S’-GCCGCTGCAGCCCANBWNGTRTANCCYTT-3’)
amplified a fragment of Styela L+dh. The sequence obtained from this
fragment was used to design gene-specific
primers for
additional
RT-PCR
(S’-GCCGGAGCTCAAGGTGAAGTAATGGACT-3
‘, used in combination
with
primer LdhB736FhR)
and RACE amplifications
(see below). Conditions
for RT-PCR were as described by
Stock and Powers (1995). Weak bands were reamplified
in a similar PCR reaction using as a template either a
dilution of a band cut from a low-melting-point
agarose
gel (NuSieve-FMC)
or the DNA adhering to a syringe
of the 3’ End of Styela Ldh
The 3’ end of the Styelu LDH cDNA was amplified
by 3’ RACE, essentially as described by Frohman (1990).
A dT-adaptor reverse transcription was used as the template for an initial PCR with an adaptor primer (5’-GATGCTGCAGAGCTCAAGCTT-3’)
and the gene-specific
primer 5’-GCCGGAGCTCGRTGGMCCWGAAGGYTGGGA-3’. PCR conditions were as described by Stock
and Powers (1995). The initial PCR was then used as a
template for a second PCR with the adaptor primer 5’GATGCTGCAGAGCTCAAG-3’
and the gene-specific
primer 5’-GCCGGAGCTCACAYAARCARGTYRTYGATG-3’.
RACE Amplification
Materials and Methods
RNA Extraction
Reverse
needle stabbed into a band of the expected size (band
stab technique of Bjourson and Cooper 1992).
of the 5’ End of Styela Ldh
5’ RACE amplification
followed Frohman (1990).
RNA was reverse transcribed with the degenerate LDH
primer 5’-GCCGCTGCAGTCNACNACYTGYTTRTG3’. The first-strand cDNA was tailed with dATP using
terminal deoxynucleotidyl
transferase.
PCR amplification of the dA-tailed template was with the gene-specific
primer
5’-GCCGCTGCAGTGGCCGATACGGAGTAATCT-3’, the dT adaptor primer, and one of the adaptor primers used above. The PCR reaction was subjected
to electrophoresis
on an agarose gel, and the band stab
technique
(Bjourson
and Cooper 1992) was used to
reampufy a faint band of the expected size with the nested LDH primer 5’-GCCGCTGCAGCCRTGYTGNARRTCCAT-3’ and the same adaptor primer as in the first
reaction.
Cloning
PCR products were cloned into Ml3 mp18 and
mp 19 or pBluescript
KS+ and KS - (Stratagene) using
standard procedures (Sambrook, Fritsch, and Maniatis
1989). PCR products were either digested with restriction enzymes (recognizing
sites at the 5’ ends of the
primers or in the region between the primers), made
blunt-ended
with the Klenow fragment of DNA polymerase I, or cloned directly using the t-vector technique
(Marchuk et al. 1991).
Sequencing
Single-stranded
DNA for sequencing was either extracted from Ml 3 phage particles or obtained from
pBluescript-containing
cultures infected with VCSM13
helper phage (Stratagene)
according
to Sambrook,
Fritsch, and Maniatis (1989). DNA sequencing was by
dideoxy chain termination
using a modified T7 DNA
polymerase (Sequenase, version 2.0, United States Biochemicals). In order to minimize errors associated with
the sequencing of cloned PCR products, each nucleotide
was determined
from at least six clones, with at least
three sequences representing
each strand.
Sequence
Assembly
and Alignment
The cDNA sequence of the LDH of Styelu plicata
was assembled
from the consensus
of overlapping
clones using the SEQMAN contig assembly program in
Tunicate
the DNASTAR
package (DNASTAR,
Inc.). The sequence described in this paper has been deposited in
GenBank under accession number AF023168
(fig. 1).
Additional
lactate dehydrogenase
sequences were obtained from the GenBank (release 92) or PIR database.
D-LDHs (E.C. 1.1.1.28) were not included in the analyses, as they represent an, at best, distantly related protein family or families (Kochhar et al. 1992). The LLDH sequences
used and their accession
numbers
(GenBank, unless otherwise noted) are: lamprey (Petromyzon marinus) LDH (M74064); dogfish shark (Squalus acanthias)
LDH-A (U38893);
killifish (Fundulus
heteroclitus) LDH-A (I-43525), LDH-B (M33969), and
LDH-C
(L07336);
frog (Xenopus
Zaevis) LDH-A
(UO7179), LDH-B (UO7176), and LDH-C (U07175);
duck LDH-B (503869); chicken LDH-A (X53828) and
LDH-B
(PIR accession
A00346);
cattle
LDH-A
(D90143); pig LDH-A (U07178) and LDH-B (UO7180);
rabbit LDH-A (M22585);
human LDH-A (X02152),
LDH-B (YOO711), and LDH-C (502938); mouse LDHA (YOO309), LDH-B (X51905), and LDH-C (X04752);
rat LDH-A (X01964) and LDH-C (U07177); fox (Vulpes vulpes) LDH-C (U19868); nematode (C. elegans)
LDH (U15420); barley LDH-A (M55685) and LDH-B
(M55684); rice LDH (D13817); maize LDH (211754);
the LDHs of the apicomplexan
protists ToxopZusma gondii (U35 118) and Plasmodium falciparum
(M93720);
and the L-LDHs of the eubacteria Streptococcus mutuns
(M72545), S. thermophilus (D 13405), Luctococcus lactis (M88490), Luctobacillus
casei (M76708), Luctobacillus
sake
(U26688),
Lactobacillus
plantarum
(D90340), Pediococcus
acidilactici (X70927), Bacillus
stearothermophilus
(M19396),
B.
caldolyticus
(M19394), B. caldotenax (M19395), B. psychrosacchar118 and LDH-B-X55
119), B.
olyticus (LDH-A-X55
subtilis
(PIR accession
A25805),
B. megaterium
(M22305), Mycoplasma
hyopneumoniae
(X67286), M.
genitalium (U39733), Thermus aquaticus (D00585), T.
caldophilus
(X045 19), Deinococcus
radiodurans
(D63899), Thermotoga maritima (X74302), BiJidobacterium
longum
(M33585),
and Synechocystis
sp.
(D64003).
Nucleotide
sequences were translated into protein
sequences according to the authors’ feature specifications. An alignment of amino acid sequences was constructed using CLUSTAL W (Thompson, Higgins, and
Gibson 1994) and the default settings. Length variability
among chordate LDH sequences in this alignment was
limited to the amino terminal arm (positions l-43 in fig.
2, as defined by Li et al. 1983) and the extreme carboxy
terminus. No attempt was made to improve the alignment of the former region because of extensive sequence
variability, but the alignment of the later region was adjusted manually. Invertebrate
and plant LDH sequences
require the addition of only a few additional gaps to the
alignment of chordate LDHs, but the alignment of bacterial and protist sequences appeared less reliable. Rather than attempting additional alignment methods or parameters, we conducted phylogenetic
analyses with and
without the latter two groups of LDHs.
Phylogenetic
LDH
1275
Analyses
Positions l-44 in the alignment of LDH sequences
(fig. 2) were omitted from all analyses because of the
difficulty in obtaining a reliable alignment of this region.
Phylogenetic
analyses were conducted using both neighbor-joining
and parsimony methods. Amino acids were
used as characters in both types of analysis because of
the large amount of sequence divergence among some
of the taxa and the fact that we were interested in early
branching
events in LDH evolution (Kumar, Tamura,
and Nei 1993). Neighbor-joining
trees were constructed
from pairwise amino acid distances (gamma corrected
with a = 2.0) using the MEGA package of programs
(Kumar, Tamura, and Nei 1993). Parsimony
analyses
were conducted
with PAUP, version 3.0s (Swofford
1991), and in most analyses, amino acids were treated
as unordered characters with all possible transformations
weighted equally. Preliminary
analyses with amino acid
replacements
weighted according to the minimum number of nonsynonymous
nucleotide substitutions required
(PROTPARS
weighting-Felsenstein
1993) gave similar results but were not pursued extensively because of
the computational
time required. Parsimony
analyses
with equal weighting were performed with 100 random
addition sequences of taxa and TBR branch swapping
(Swofford 1991), while those with PROTPARS weighting used 20 random addition sequences.
The CONSTRAINTS feature of PAUP was used to determine the
lengths of the shortest trees matching a number of alternative phylogenetic
hypotheses, In the case of constrained
analyses
using PROTPARS
weighting,
the
PROTPARS program of the PHYLIP package (Felsenstein 1993) was used to test whether the length differences between constrained
trees and the best unconstrained tree were significant according to the method
of Templeton (1983). Bootstrapping
was used to assess
the degree of support for particular nodes. Neighborjoining analyses were conducted with 500-1,000 bootand bootstrap parsimony
analyses
strap replications,
were conducted with 10 replications
of a random addition sequence for each of 100 bootstrap replications.
Results
Features
of Styelu Ldh
The 1,168-nt cDNA sequence of S. plicatu LDH is
shown in figure 1. Three of 10 clones of the 3’ end had
an additional 10 nucleotides before the poly-A tail (not
shown). No consensus polyadenylation
signal (Sheets,
Ogg, and Wickens 1990) was found in either class of
clone, suggesting that the reverse transcription
primer
may have bound upstream of the true poly-A tail. Of
the sites at which nucleotide differences from the sequence shown in figure 1 were found in individual
clones of PCR products, only seven exhibited the same
difference in more than one clone. Two of these differences occurred in the 3’ untranslated
region (positions
116 1 and 1164), three were silent (positions 923, 97 1,
and 1037), and only two encoded different amino acids
(N and P at nucleotide positions 454 and 583, respectively). These latter two differences were only found in
1276
Stock et al.
GATTGGAGTATTTGGTTGTATTAAGAATTTTGATAATTAAACACAA
TCATGGCTGATCTGAAGGGATTCGATATAATGTCCGAGTTATTCA
MADLKGFDIMSELF
CGGAAGTCTGTCCAGACATTCCAAAATCTGGACCATCCAAAGTCA
TEVCPDIPKSGPSKV
CTGTAGTTGGAGTTGGGATGGTGGGGATGGCGTGTGGGATGAGTG
TVVGVGMVGMACGMS
TGGTGTTGAAGGGACTATGTACAGATCTTGTACTAGTCGATGTTG
VVLKGLCTDLVLVDV
TTCAGGACAAACTTCAAGGTGAAGTAATGGACTTGCAACATGGTA
VQDKLQGEVMDLQHG
GTTTGTTTCTAGAGAATATTCTATGGAGACAAAGATTACT
SLFLENIKVYGDKDY
CCGTATCGGCCAACAGTCGGATTGTCATAGTAACTGCTGGTGCTC
SVSANSRIVIVTAGA
GTCAACZUXCTGGGGAATCTAGATTGAGTTCAACGG
RQQPGESRLSLVQRN
TCAACATTTTCTTCCACAAATTGCGAAATATAGTC
VNIFKHIIPQIAKYS
CAAGn=CTATTCTTGTTATn=TTTCAAATCCAGTTGACTT
PSAILVIVSNPVDLM
CATATGTTGCCTGGAAATTGTCAAACTTTCCTCGTAATCGTGTGA
TYVAWKLSNFPRNRV
TTGGTTCTGGZUuZGAATCTTGATTCTGCJUiGATTTCGACATCTTA
IGSGTNLDSARFRHL
TCGCGGAAAAATIY3AATTTGTCACCTGTCAGTGTTCATG
IAEKLNLSPVSVHGW
TCATCGGCGAACATGGCGATTCAAGTGTTCCAATGTGGAG
IIGEHGDSSVPMWSG
TTAACGTGAGTGGGZUATGTCGATTCATCCAAGAATAG
VNVSGKCLNSIHPRI
GATATCCGGATGGACCAGAAGGTTGGGATAAAATACATACATAAACAAG
GYPDGPEGWDKIHKQ
TTGTCGATGGGGCATACGATGTAATAAGATTAAAAGGATACACAA
VVDGAYDVIRLKGYT
ACTGGGCTATTGGTCTGAGCTGTGCAGAATTGCTTGCA?iCTATTT
NWAIGLSCAELLATI
TACATCACAGACATAGAATCCATCCAGTTACTTGTTTTGTGAAGG
LHHRHRIHPVTCFVK
GCCGATATGGAATCACTGATGATGTATGCCTATCTCTACCTTGTG
GRYGITDDVCLSLPC
TTTTGZUXX'GTAATGGAGTCAACTCTATTGT~TGTTGATTTGA
VLNCNGVNSIVNVDL
CTGCTGAAGAAGAAGCAATTGCAATGACTATTG
TAEEEAMIKKSAMTI
CTGATGTG CAAAAAGGCCTGAAATGGTAAAATTTACCAGAATACT
ADVQKGLKW*
ACATAAAAAGCTTTCATTGTGCTCTTTCTCAAAGCGCTTTCAAGT
CCCTTTAATGTCATGGCCGATATGCTAAAAGACCCAGACTTGT
FIG. l.+DNA
cata L_dh.
and deduced
amino acid sequence
45
90
14
135
29
180
44
225
59
270
74
315
89
360
104
405
119
450
134
495
149
540
164
585
179
630
194
675
209
720
224
765
239
810
254
855
269
900
284
945
299
990
314
1035
329
1080
338
1125
1168
of Styela pli-
2 of 11 and 3 of 15 clones and may have been PCR
errors. Two of the silent differences (positions 923 and
971) may be the result of heterozygosity,
as two bands
of relatively equal intensity were seen at these positions
on autoradiographs
of directly sequenced (uncloned) independent PCR products (data not shown).
The Styelu LDH protein that we inferred from the
nucleotide sequence (assigning the start codon according
to the properties of vertebrate translation start sites listed
by Kozak 1996) is 338 amino acids long including the
initial M; vertebrate LDHs, in comparison,
range from
332 to 334 amino acids in length. The additional length
of the tunicate sequence is in the N-terminal arm (AbadZapatero et al. 1987 and included references), a region
that has previously been shown to also exhibit the greatest degree of sequence
variability
among vertebrate
LDHs and is reduced or absent in bacterial sequences
(Li et al. 1983). The only other region of length difference among chordate LDHs reported to date is near the
carboxy terminus (fig. 2); at the penultimate position, all
vertebrate LDH-Bs and the LDH-B-derived
teleost fish
LDH-C (Quattro, Woods and Powers 1993) exhibit an
extra amino acid that is missing in mammal LDH-Cs,
lamprey LDH, and all vertebrate LDH-As. Styelu LDH,
like that of the nematode and the less reliably alignable
plant LDHs is also missing an amino acid at this position. The tunicate sequence, with its greater similarity
to vertebrate sequences in this region, provides additional evidence that the absence of the amino acid is the
primitive condition and that the amino acid was added
in the common ancestor of LDH-Bs and teleost fish
LDH-C. Interestingly,
there are no reversals of this character evident in the vertebrate sequences examined to
date.
Summaries of the pairwise sequence identities of
LDHs are shown in table 1. Styelu LDH is approximately equally similar to all vertebrate LDHs, with the
highest similarities to certain LDH-As and the lowest to
mammalian LDH-Cs. The lowest within-vertebrate
similarity is lower than some of the similarities of vertebrate
and Styela LDH. Compared to other groups outside the
Vertebrata, Styela-to-vertebrate
similarities are approximately the same as those between nematode and vertebrate LDH, and greater than those between plants and
vertebrates, apicomplexans
and vertebrates, and bacteria
and vertebrates.
Nonorthologous
Cyanobacteria
LDHs of Apicomplexan
Protists
and
A neighbor-joining
analysis of 54 LDH sequences
is shown in figure 3. The tree has been rooted at the
midpoint; it cannot be rooted in a manner that makes
eukaryotic LDHs a monophyletic
group because of the
clustering of the cyanobacterial
Synechocystis LDH with
the Apicomplexan
protist LDHs of Plasmodium
and
Toxoplasma and the location of this group on a branch
LDH and all other bacterial
uniting Bifidobacterium
LDHs. The node uniting Synechocystis
with Plasmodiurn and Toxoplasma is very strongly supported (100%
bootstrap support). This result, along with the location
of the midpoint root, suggested to us that these three
LDHs might not be orthologous to the other LDHs in
the tree. A search of GenBank using BLASTP revealed
that the closest matches to these sequences were to the
malate dehydrogenases
(MDH, E.C. 1.1.1.37) of three
eubacteria (Bacillus subtilis [Jin, de Jesus-Berries,
and
Sonenshein
19961, Bacillus sp. [Wynne et al. 19961, and
Chlorojlexus aurantiacus [Synstad, Emmerhoff, and Sirevag 19961). The cytoplasmic and mitochondrial
MDHs
of eukaryotes and eubacterial MDHs have been shown
to be related to L-LDHs in both sequence and, especially, three-dimensional
structure (Birktoft et al. 1982). Sequence comparisons
suggested that LDHs and MDHs
represent two distinct groups whose members are more
closely related among themselves than to members of
the other group (Iwabe et al. 1989). Recently, an MDH
from the archaebacterium
HuZoarcuZa marismortui was
cloned that exhibits greater sequence similarity to LDHs
than to other MDHs (Cendrin et al. 1993). The MDHs
of the three eubacteria mentioned above appear to exhibit a similar relationship to LDHs (Jin, de Jesus-Berrios and Sonenshein
1996; Synstad, Emmerhoff,
and
Tunicate
10
Hsa
Hsa
Fhe
Sac
Pma
Hsa
Fhe
Fhe
Spl
Cel
Hvu
Blo
Taq
Mhy
Bst
Pfa
ssp
Hsa
Hsa
Fhe
Sac
Pma
Hsa
Fhe
Fhe
SPl
Cel
Hvu
Blo
Taq
MhY
Bst
Pfa
Ssp
A
C
A
A
B
B
C
A
A
C
A
A
B
B
C
A
Hsa
Hsa
Fhe
Sac
Pma
Hsa
Fhe
Fhe
SPl
Cel
Hvu
Blo
Taq
MhY
Bst
Pfa
Ssp
A
C
A
A
Hsa
Hsa
Fhe
Sac
Pma
Hsa
Fhe
Fhe
SPl
Cel
Hvu
Blo
Taq
MhY
Bst
Pfa
SSP
A
C
A
A
B
B
C
A
B
B
C
A
20
30
40
I
I
I
I
110
120
130
I
I
I
I
140
150
160
I
I
I
250
260
270
280
290
I
I
I
I
EEQTP----DDENS.....
..PVG.C...
SQEPRS....
SKERDPP...
..A.VP....
SSAE.P....
SSPE.P....
DIPKSG....
PVENS.....
ATP.SSAVPH
TTVK......
-----.....
IKLMK.... .
MKNNG.....
__m.****.
IACQS.....
VTANSKLVII
.S...RI..V
.... ..V .VV
.S.G....V.
. ..G.R ..W
..I .VV
........
.R ..VV
..S..RI .VV
.S...RI ..V
I..G...C W
..K..D . ..V
ICRDADM.V.
DLEGARV ..V
DLKDADFIV.
DCRDAD..V.
DL.GADV ..V
A..G.DV.V.
210
220
230
240
I
I
I
I
I
VHPLSCHGWV
. ..T.....I
L..S.....I
. .SS......
.NSA.....I
I..S.....I
I.AS.FN...
I.SS.FN..I
LS.V.V...I
IA.S.....I
.NAQDVQAYM
.NVKNV.AYI
.A.Q.V.AY.
.S.N.VQAY.
.A.QNV.AYI
.C.RDVNAHI
AC.SDINTL.
310
320
330
340
350
IKGLYGIKDV
.E.
:iIi::::::i V~:iiI:i..
.C........ V.DF....N.
. . . . . . . . .H C..QH.VH..
.S.I...... V..M...EN.
.S........ V.DMF..GE.
MN.I...... A..M...G..
RH.1.. .TCF V..R...T..
S.N.FAL..N V..FH..N..
.VL AS.FH..S.G
Q
Tiii::. S. L.DFH..S..
EKG.FT..LF TPNE.VEE.
TKNIMIAGAN LR.E..FHG.
ENAILT..AY LD....ER..
.KK.LIC..L LE.Q..HS..
QS.IL.AA.Y LD.A..L...
-DVFLSVPCI
.EL.. .I..V
.E.. ..I..V
. . . ...L..V
. . . ...L..V
.E....L...
.E....L..V
.E.Y..L..V
. . .C..L..V
. ..Y..L.W
HE....L.AR
.-ICM.. .TL
.-.A..L.R.
.-.NIG..W
.-.YIG..AV
.-1.GGT.W
.-I..G...R
LGQNGISDLV
..R..V..V.
..NS.LT.VI
.DNH...NI.
..SE..T.II
.NAR.LTSVI
.NGS.VGSV.
.NSG.VGSV.
.NC..VNSI.
..SA.LTHV.
..RG..LGVA
.NRQ.VNNTI
..AR.VEATL
..A.. .EKII
INR...RNI
..A..VEQVI
..CR.VE.IL
KVTLTSEEEA
.IN.N.....
HM..KP...K
.MK.KPD..Q
NQ..KK....
NQK.KDD.V.
NM...AA.V.
NM...D..V.
N.D..A....
.QQ..EA.VQ
EMD..EA.A.
NTPVSDK.L.
YPR.NE..RQ
EIS.NDK.KE
EIE.NDD.KN
ELQ.N...-E.Q..P..K.
FIG. 2.-Alignment
killifish, Sac-dogfish
I
of representatives
shark, Pma-lamprey,
I
VWSGMNVAGV
L...V.....
. . ..V.I...
..... .....
. . ..V.....
. . ..V.....
. . ..A.....
T
il:::V::&i
. . ..V.....
I..SIS.G.M
L.ESATIG..
. ..SAQ.G..
AY.NIKI..E
. ..QAYIGVM
LKRYIT.G.1
LPRYCT.S..
I
1277
I
I
I
180
190
200
--QNKITWG
VGAVGMACAI SILMKDLADE LALVDVI--E DKLKGEMMDL QHGSLFLRTP
...C ...I .. T .......... ..L .......... ..A ..L .............. ..FS .S
..R..V .... ..M....S .. .V.L...C ...... ..M ....... ..A .... ..A...K .H
V
.M
Y
.H.A
::Hh:;::
::b::::i:: :+:iR::::: .::::.v ......
:i:::::::: L:::::.K.A
..N .........
.G.S ........ ..L ..................
..Q ..
..R..V ...... E::::::; :: .LR..C ...... ..M ... .R ......... ..L...K .S
..R..V .... ..Q .... ..V T..LRE ........ ..V ... ..V ............ ..K ..
..PS.V .... ..M.....G M .VVL.G.CTD .V....V ..Q . ..Q..V ........ ..JZN
I
..HG.V .... ..Q .... ..Y . ..QQN..N. .C....V ..A ........... ..LA.T.H C
RRLT..S.I. A.N....I .Q T..TQN .... I....AL ..P . ..R..AL .. ..AAA..PR V
-T.LA.I. A . . ..STL .F AAAQRGI.R. IV.E.IA ..K ERVEA.VL.M .. ..S.YP .V
::-V.VGI . . S.F..S.T .Y ALVLQGV.R. VV .. .LD ..R KLAQAHAE.I L.ATP.AH-.
..PI..ALI. A.N..NSFL Y AAMNQG..S. YGII.IN ..P .FAD.NAF.F EDA.AS.PF.
..GARVV.I. A.F..ASYV F ALMNQGI ... IV.I.AN ... S.AI.DA ..F N..KV.APK.
. .KA..VL . . S.MI.GVM.T L.VQ.N.G-D VV.F.IV ..K NMPH.KAL.T S.TNVMAYSN
..-WQV .... A.N..RTL .Q RLVQQNV.-N VV.L.IV ..P GLPQ.IAL .. MAAQSVEEYD
FRYLMGERLG
. . . . I..K..
. .H . . . .KFH
. .. . . . . . . .
. .. . .S . . . .
. . . . .A.K..
.M.A....
::F..ADK..
..H.IA.K.N
..F.LS.K.N
..F.IA.H.D
L.F.IAQQT.
..A.LAQH.L
LQFAIAK.AK
..F.L..YFS
LK.YISQK.N
LKAFIAMK..
I
I
TAGARQQEG- ----ESRLNL VQRNVNIFKF IIPNVVKYSP
. . . . .. . . . . . . . ..T..A. ..... A.M.1 . ..AI.H ...
....................
.............. I .....
............. DI..H ..
....................
....................
.............. I .....
..V
......V ......
..
..V ................................
......v ..c . ..g::::
. ..V ......... ..R .... D ........ H ...LI.RH ..
..... ..P ........ ..S. .........
H . ..QIA ....
.... ..R ...... ..T..S. ..... E ... G . ..QL .....
..ALYR K .V.P.AEH ..
.... ..IP ..... ..T .... L
. ..P..KP .... ..Q...E. .iT .. .L.A .M..L..VA.
A..VA.RP ..... ..T..Q. LD..AQV.A D VV.KIL.AA.
. ..RP.KP ..... ..T..E. .AD.IR.IRE .ALK.KESGF
C...N.KP ..... ..T..D. .DK.IA..R S .VES.MASGF
. ..FTKAP .K SDKEWN.DD. LPL.NK.MIE .GGHIK.NC.
. ..LPRRP .... ..M..DD. LGK.A..VA Q GAREALR ...
KIVS-GKDYN
.VT......S
... A.D ...S
......... S
...A.D . ..S
.A.D ..S
:;I.A.D..
..A
.A.D ..S
:~NG.D ..
. .S
TVKA.DT ..S
R.S-..T .AA
S.DG.SD.PE
VW.R.SGW.E
IS .. .RYE.K
VDIW.HG ..D
CK..GSNT .D
SKIIGTNE.E
LGEHGDSSVP
I.........
V........A
I.........
I.........
. .. . . . . ..A
. . . . ..T...
. . . . ..T...
I.........
I........A
V....... .A
A......E..
V......E.L
M...... .FV
I.....TEL.
V.A..NKM.L
..G...LML.
80
I
_-____---_ -MATLK---- -DQLIYNLLK
.......... ..S.V ..... .E.. .EK.IE
.......... .-MSTQ .... .EK..SHVM.
....................
..K..GH.AT
ST
.GK..HEMVP
:::::::::: 1:: ............
.EK..APVAE
.......... ..SSVL .... .QK..TP.AS
........... ..SVL .... .HK..TP.AC
........... ..D..GFD I MSE.FTEVCP
.......... MAS.I ..... .-EVFAEIAA
MHKASSLSEL GFDAGGASSG FFRPVADGCP
.......... .----- .... .------MAE
.......... .----- .... .--------.......... .----- .... .MKKFNKKGE
.......... .----- .... .------MNA
.......... .----- .... .__--____.......... .----- .... .-MN.LEYAP
LRRVHPVSTM
70
LDH
SLKTLHPDLG
A....D.K..
..Q..N.NM.
. ..E...E..
G.QS.N..I.
..QE.N.EM.
..QK.N.EI.
N.Q..N.NI.
C.NSI..RI.
T.HEIK..I.
PAFKSLR.SH
PMCDWT.LP.
D.EAFAQARCFCAYSKLTPIRK.VESKP.QEFINN-PITE.I.P--
I
170
I
I
I
NCKLLIVSNP VDILTYVAWK ISGFPKNRVI GSGCNLDSAR
D..I.V ........ ..IV ... ..L.VT .............
..I..V ..............
L....RH .... ..T ......
D.1I.V .... ..V ....... L..L.MH.I. ..........
..I..V ..............
L..L..H .............
D.IIIV ......... ..T .. L..L..H .............
..TI.V .... ..V....T .. L..L..H .... ..T ......
D.IIIV .... ..V....T .. L..L.MH .... ..T ......
SA1.V ..... ..LM ...... L.N..R ..... ..T ......
DTCI.V .... ..V....T .. L..L.RE ..F ...T ......
DAL..V
..V ....... L....AS
..T....S.
.AIYMLIT
.........A.H..Q. LT.L.E.QIF
..... ..T ......
EAV...AT .. ..VM.Q..Y R L..L.PE ..V . ..TI..T ..
SGISI..A ... ..I.RAYR D A...SDQK ... ..TV..T ..
QGLF.VAT ...... ..AT .. F..L.HE .... ..TI..T ..
.AFIIV.T .. ..VMVQLLH Q H..V...KI. .L.GV..TS.
.AI.IV.T .. L.VM..L ... VT.L.SQ ..M .MAGV .....
TDKDKEQWK..S...H...
A.G.S.N...
. . . . ..N...
.P..G.D...
..N.S.N...
..G.......
..F.E.N...
YPDGP.G.D.
EKT.N.H.EA
RSF.EAALE.
H.PLDADKRE
GRALTPDDRL
GIDSS-NYEK
GEEAQ-KDLE
KL-ISDAELE
------.TIE
EVHKQVVESA
NI....IQ..
.L..K..DG.
KL..D..D..
S......D..
. ...M.....
AT..A..D..
.T..M..D..
KI.....DG.
.I..K..D..
GIRRA..GG.
.I.QE.KNA.
RIDEG.RRA.
.LEYP.SRR.
RIFVN.RDA.
AIFDRT.NT.
.LVERTRNGG
360
370
I
I
YNIKLK--G
..I.......
. . . .. . . . . .
.. . .. . . . . .
.. . . . . .. . .
. . . . . . . .. .
. . . .. . . . . .
. . . .. . . . . .
.D..R.....
..I.......
. ...G.....
.KI.NG....
.RI.EG....
..I.NR...R
.QI.EK....
L.IVN.H..A
A.IAA.LQT.
YTSWAIGLSV
. . .. .. . . . .
. . . . . ..M..
. . . . . . . . ..
. . . . . .. . . .
..N.......
..N....F..
..N.......
..N......C
.. ........
. . . . . ..Y..
A.NY...M.G
A.YYG..AGL
A.FYG..AAI
A.YYG.AMGL
SPYV.PAAAI
TAYY.PAS.A
300
I
ADLAESIMKN
M..VG..L..
. ..V...V..
.. . ..T....
. .. ..T.L..
. ..I..ML..
. ..T...V..
. ..T..L.R.
.E.LAT.LHH
.KI.QG.FS.
.S..A.LLRD
V.II.AVLHD
.R.TRA.LTD
.KIVSN.I.D
.RVTRA.LH.
IEM...YL.D
.VMV...LR.
I _--
RLKKSADTLW GIQKELQ-FLF....E... N...D.I...
Q.V...E... .V....T.L.
Q.Q...T... D...D.K...
QVQ...E... NV....T...
Q......... D...D.KDL.
Q......... . . ..D.KDL.
Q.QG..S... D...D.RDI.
MI....M.IA DV..G.K.W.
K.HN..KA.L N.NG1V.M.
Q.RR..K... ENCQL.D.L.
A..R..E..K ETAAQFG...
A.RR..EI.K .AASA.G...
KFA..VAIID K.YQDAIKNI
.FHH..A..K SVLARAFTR.
--.AKF.EiAI AET.RMKALA
A.HL..FiAVR LNIDVALAMV
...
...
...
...
of major groups
Spl-tunicate,
of LDH sequences. Abbreviations
of taxa are as follows: Hsa-human,
FheCel-nematode,
Hvu-barley,
Blo--B$dobacterium
Zongum, Taq-Z’hermus
aquaticus,
Mhy-Mycoplasma
hyopneumoniae,
Bst-Bacillus
stearothermophilus,
Pfa-Plasmodium
falciparum,
Ssp-Synechocystis
sp.
A, B, and C refer to the different isozymes within a species. Dashes indicate alignment gaps and periods indicate matches to the sequence
of human LDH-A.
Sirevgg 1996; Wynne et al. 1996). In addition, all of
these LDH-like MDHs are tetrameric like LDH, rather
than dimeric like other MDHs. Although the MDH activity of the cloned sequences from Haloarculu, BacilZus, and Chlorojkxus has been relatively well charac-
terized (Jin, de Jesus-Berries and Sonenshein 1996; Synstad, Emmerhoff, and Sirevag 1996; Wynne et al. 1996),
the LDH activity of Plasmodium LDH has not been as
conclusively
demonstrated
(Bzik, Fox, and Gonyer
1993), and the Toxoplasma and Synechocystis sequences
1278
Stock et al.
Table 1
Summary
of Pairwise Differences
Pairwise
Among LDH Sequences
Comparisons
Range of Distance@
Bacteria to vertebrate ..................
Apicomplexa
to vertebrate. .............
Plant to vertebrate. ....................
Nematode to vertebrate ................
Tunicate to vertebrate. .................
Within vertebrates. ....................
Tunicate to vertebrate LDH-A. ..........
Tunicate to lamprey ...................
Tunicate to vertebrate LDH-B ...........
Tunicate to mammal LDH-C ............
Tunicate to killifish LDH-C .............
0.59-0.73
0.68-0.73
0.46-0.54
0.35-0.46
0.36-0.44
SO.40
0.35-0.39
0.36
0.37-0.40
0.39-0.44
0.38
a Distances were calculated using the alignment in figure 2, with gaps in the
pairwise comparisons deleted and all other positions considered. Distances are
proportions of nonidentical residues.
have been identified as LDHs only by sequence similarity to Plasmodium LDH (Kaneko et al. 1995; Yang
and Parmley 1995). Regardless of whether the “LDHs”
of Plasmodium, Toxoplasma, and Synechocystis actually
function as LDHs or MDHs, we suggest that they are
not orthologous
to the other LDHs in figure 3. Strong
support for this hypothesis is provided by the presence
LDH (Fig. 3) in addition
in B. subtilis of a conventional
to the LDH-like MDH described above.
Phylogeny
of Eukaryotic
LDHs
The midpoint rooting of figure 3, which supports a
monophyletic
group of nonprotistan
eukaryotic
LDHs
distinct from bacterial LDHs, is consistent with the results of simultaneous
analysis of LDH and MDH (Iwabe
et al. 1989). This monophyletic
group of plant and animal LDHs was found in 100% of the bootstrap NJ trees.
Plant LDHs also form a monophyletic
group with 100%
bootstrap support, although this is not surprising given
that all of the species are members of the family Poaceae. The monophyly
of animal LDHs was found in
99% of the bootstrap NJ tree. All of the above clusters
were also found in parsimony analyses (not shown).
Phylogenetic
Position
of Styela LDH
The phylogeny
of the LDHs of the three animal
phyla matches that of the organisms. In the tree in figure
3, nematode LDH clusters outside of urochordate (Styela) and chordate LDHs, but with only moderate bootstrap support (60%). Figure 3 shows significant support
(97% of the bootstrap trees) for Styelu LDH being outside of a monophyletic
group of all of the vertebrate
LDHs. This result indicates that all of the gene duplications within the vertebrates examined occurred after
the divergence
of tunicates and vertebrates.
In an attempt to provide increased resolution
of the relationships of tunicate and vertebrate LDHs, we conducted a
neighbor-joining
analysis without the more distantly related and less reliably alignable bacterial and protistan
LDHs and have rooted the resulting tree between plant
and animal LDHs (fig. 4). Such an analysis shows higher
support for the theory that the tunicate LDH is more
closely related to vertebrate LDH than is nematode LDH
(92% bootstrap
support), but lower support for the
monophyly
of vertebrate LDHs (87% of the bootstrap
trees).
We used maximum-parsimony
analyses to evaluate
the relative support for alternative hypotheses on the
phylogenetic
relationships
of tunicate LDH (fig. 4 and
table 2). It has been suggested based on immunochemical comparisons
that tunicate LDH is most closely related to LDH-C and lamprey LDH (Baldwin, Mortimer,
and Patak 1988). These authors assumed that mammal
and fish LDH-Cs were orthologous,
although this relationship was strongly contradicted by a subsequent phylogenetic analysis of a teleost LDH-C sequence (Quattro, Woods and Powers 1993). Not surprisingly,
given
this latter result, the forced grouping of Styela LDH,
lamprey LDH, and mammal and fish LDH-Cs required
a large number of extra steps (31 for equal weighting,
46 for PROTPARS weighting) relative to the most parsimonious trees and in the case of PROTPARS weighting was significantly
worse as judged by the method of
Templeton (1983). Relaxing the requirement
that mammal LDH-C be a part of this cluster (Baldwin, Mortimer,
and Patak’s [1988] immunochemical
data were based on
fish LDH-C) still results in trees significantly worse than
unconstrained
trees. Clustering
of tunicate LDH with
mammalian
LDH-Cs could also be rejected, but a specific relationship with fish LDH-C could not. Additional
phylogenetic
positions of the tunicate LDH inside the
vertebrate LDH clade (not necessarily suggested by any
authors) were also evaluated with respect to the number
of extra steps. The clustering of the tunicate LDH with
gnathostome
LDH-B and teleost LDH-C required the
Styela
fewest additional
steps. Hypotheses
clustering
LDH with LDH-C remained among the least likely.
Phylogeny
of the Vertebrate
LDHs
The phylogenetic
relationships
among vertebrate
LDHs were investigated with plant and invertebrate outgroups as shown in figure 4. Parsimony and neighborjoining analyses gave similar topologies, although the
bootstrap values were generally higher with neighborjoining. The values discussed below are from this latter
analysis. A branch uniting all of the jawed vertebrate
(gnathostome)
LDH-Bs as well as teleost fish (killifish)
LDH-C, Xenopus LDH-A, and Xenopus LDH-C was
found in 90% of the bootstrap trees. The tree topology
supports a number of gene duplications
within this
clade. Ninety-three percent of the bootstrap trees support
a derivation of teleost LDH-C from teleost LDH-B after
this group diverged from tetrapods, as initially proposed
on immunochemical
grounds
(Whitt 1969; Marker-t,
Shaklee, and Whitt 1975) and subsequently
supported
at the protein sequence level (Quattro, Woods, and Powers 1993). Two duplications
are also indicated (with
100% bootstrap support) in the Xenopus lineage after it
diverged from amniotes. One is likely to be the result
of tetraploidy (Kobel and Du Pasquier 1986), while the
other may be a gene duplication postulated by Wolff and
Kobel (1982). Stock and Powers ( 1995) concluded from
similar results that LDH-A and LDH-C have been incorrectly designated in Xenopus, with LDH-A probably
remaining to be cloned. Aside from these likely dupli-
Tunicate
LDH
1279
_c Cattle LDH-A
45
51
Pig LDH-A
i Rabbit LDH-A
loo_ Mouse LDH-A
77_ ' Rat LDH-A
- Human LDH-A
81 Chicken LDH-A
Fox LDH-C
Human LDH-C
‘M-“i;wL;FC
-
Dogfish shark LDH-A
Lamprey LDH
Kiilifish LDH-A
Killifish LDH-B
Killifish LDH-C
,
100
58
Tunicate LDH
Nematode LDH
100
100
(
8. longum LDH
T. aquaticus LDH
T. caldophilus LDH
D. radiodurans LDH
T. maritima LDH
M. hyopneumoniae LDH
M. genitalium LDH
87
P. acidilactici LDH
95
S. mutans LDH
FIG. 3.-Neighbor-joining
tree of 54 LDH sequences. The tree is rooted at the midpoint of the longest path length. Amino acid distances
were calculated with a gamma correction (a = 2). Alignment positions l-44 were omitted from consideration,
and the pairwise deletion option
of MEGA was used to treat alignment gaps. Numbers at nodes indicate the results of a bootstrap analysis with 1,000 replications. Bootstrap
support within mammal LDH isozymes has been omitted for clarity. Numbers in a larger font indicate the support for the interrelationships
among the major groups of eukaryotic LDHs.
cations, the phylogeny of the LDH-B genes matches the
well-accepted
organismal
phylogeny
of (teleost fish,
(frogs, (birds, (mammals)))).
Although
LDH-Bs and their derivatives
form a
well-supported
clade in figure 4, the sister group of this
clade (lamprey LDH + gnathostome
LDH-A + mammal LDH-C) is found in only 47% of the bootstrap trees.
Within this latter group, there is strong support, however, for the theory that mammalian LDH-Cs are derived
from within LDH-A. The strongest contradiction
of the
proposals that LDH-C is the earliest divergence among
vertebrate LDHs (e.g., Li et al. 1983; Baldwin, Mortimer, and Patak 1988; Crawford, Constantino,
and Powers 1989; Baldwin, Patak, and Mortimer 1992; Tsoi and
Li 1994; Tsuji et al. 1994; Mannen et al. 1996) is pro-
vided by the node uniting dogfish shark LDH-A with
mammal LDH-C and tetrapod LDH-A (96% bootstrap
support). Further support for a derivation of mammalian
LDH-Cs from LDH-A is provided by the node uniting
them with chicken and mammal LDH-As (80% of the
bootstrap trees). Mammalian
LDH-Cs are specifically
placed as the sister group of bird and mammal LDH-As
(77% bootstrap support), a position that requires gene
losses in a number of amniote groups, as discussed by
Matson (1989). This grouping may be due to a rapid
rate of evolution of LDH-C, which is obvious from inspection of the within-mammal
divergences relative to
those of LDH-A and LDH-B (fig. 4; Whitt 1984). A
clustering
of mammal LDH-C and mammal LDH-A,
which does not require additional gene losses, is found
1280
Stock et al.
Cattle LDH-A
and 48% bootstrap support) and one strongly supported
one (96% bootstrap support). The additional gene duplications and losses required by the tree in figure 4 over
a tree matching the organismic
phylogeny
have been
discussed by Tsuji et al. (1994), Quattro et al. (1993,
and Stock and Powers (1995).
Pig LDH-A
IL
rt
99
Rabbit LDH-A
Mouse LDH-A
60
Rat LDH-A
77
27
Human LDH-A
1
8C)
I
-
Chicken LDH-A
I
,
I-
Fox LDH-C
-
Human LDH-C
97
Mouse LDH-C
I
Rat LDH-C
Dogfish shark LDH-A
I-
Killifish LDH-A
Xenopus LDH-A
88
60
~
96
Duck LDH-B
Chicken LDH-B
91
L Mouse LDH-B
Tunicate LDH
Nematode LDH
FIG. 4.-Neighbor-joining
and parsimony analyses of 24 vertebrate LDH sequences with plant and invertebrate outgroups. The topology indicated was obtained from a neighbor-joining
analysis conducted as described in figure 3. Numbers above the nodes indicate the
results of a bootstrap neighbor-joining
analysis with 1,000 replications.
Numbers below the branches indicate the support obtained in a bootstrap parsimony analysis. Some sequences with close relatives were
deleted from the parsimony analyses to reduce the amount of time
required; these were cattle LDH-A, pig LDH-A, rabbit LDH-A, rat
LDH-A, Xenopus LDH-B, duck LDH-B, pig LDH-B, and barley LDHB. Three most-parsimonious
trees were found with 100 replications of
a random addition sequence and TBR branch swapping (Swofford
1991). These differed from the topology shown by placing mammalian
LDH-Cs within or as the sister group of mammalian LDH-As (the only
aspect of the three trees in which they differed among each other) and
by grouping killifish LDH-A with lamprey LDH. The bootstrap parsimony results indicated were obtained with 100 bootstrap replications,
each with 10 replications
of a random addition sequence and TBR
branch swapping.
in the most parsimonious
trees and in 48% of the bootstrap trees from a maximum-parsimony
analysis (not
shown).
The phylogeny
of gnathostome
LDH-A and lamprey LDH (suggested to be orthologous to LDH-A by
Stock and Whitt [1992]) in figure 4 does not match the
generally accepted phylogeny of the organisms; moving
the LDH-A of the killifish to a sister group relationship
with tetrapod LDH-A would bring the two phylogenies
into agreement. Such a position of the killifish LDH-A
is contradicted
by two weakly supported nodes (47%
Discussion
Gene Duplications
Leading to Multiple Vertebrate
LDHs Occurred After the Divergence of Tunicates
Vertebrates
and
Our analyses strongly support a sister group relationship between the LDH of Styela plicata and a clade
consisting of all of the vertebrate LDHs that have been
sequenced to date (those of lamprey and jawed vertebrates). If it is assumed that the locus we sequenced is
the only one encoding LDH in the genome of Styela
(see below), then this phylogenetic
result suggests that
all of the LDH duplications
represented by the present
collection of vertebrate sequences occurred after the divergence of vertebrates and tunicates. This pattern of
duplication is consistent with the hypotheses of Markert,
Shaklee, and Whitt (1975) and Fisher et al. (1980),
which were formulated
largely in the absence of sequence data. Our phylogenetic
results strongly contradict the hypothesis
of Baldwin, Mortimer, and Patak
(1988) that tunicate LDH is more closely related to the
LDH of lampreys and even more so to the LDH-C of
mammals and teleost fish than it is to the LDH-A and
LDH-B of jawed vertebrates. This hypothesis was based
on immunochemical
comparisons of the LDH of the tunicate Pyura stolonifera (distantly related to S. plicata)
with the LDH-A, LDH-B, and LDH-C of a teleost fish,
as well as on comparisons
of amino acid compositions
of tunicate and other vertebrate LDH isozymes. The lack
of agreement of our phylogenetic
conclusions based on
sequence comparisons with earlier conclusions based on
cruder immunochemical
and amino acid composition
analyses suggests that hypotheses on the relationships of
echinoderm LDH (Baldwin, Patak, and Mortimer 1992)
and hagfish LDHs (Baldwin, Mortimer, and Patak 1988
and included references) to the LDHs of lamprey and
jawed vertebrates that are based on similar evidence are
no longer persuasive.
LDH Locus Number
Invertebrate Groups
in Tunicates
and Other
Our PCR amplifications
of Styela L.dh did not provide any evidence for the existence of more than one
locus in this species. A number of the primers we used
are coniplementary
to regions with amino acid sequences conserved among multiple vertebrate paralogs. This
suggests that if the Styela locus we amplified were orthologous to a specific type of vertebrate LDH, then the
primers would be capable of amplifying
Styela genes
orthologous to other vertebrate genes. However, amplification of additional loci may have failed for a number
of reasons, including
restricted expression
or unique
substitutions
along the Styela lineage. A more systematic determination
of the number of loci present is pos-
Tunicate
Table 2
Lengths of Constrained
Trees in Parsimony
128 1
Analyses
PROTPARSC
EQUAL WEIGHTS~
CONSTRAINTS
Nonee..................................................
(tunicate,
(tunicate,
(tunicate,
(tunicate,
(tunicate,
(tunicate,
(tunicate,
(tunicate,
LDH
killifish C, mammal C, lamprey) . . . . . . . . . . . . . . . . . . .
killifish C, lamprey). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
killifish C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mammal C). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
all A except Xenopus, lamprey). . . . . . . . . . . . . . . . . . . .
all A except Xenopus, mammal C, lamprey) . . . . . . . . .
all B, all Xenopus). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
all B, all Xenopus, killifish C) . . . . . . . . . . . . . . . . . . . . .
Length
Extra Steps
Length
928
0
1,103
959
948
936
935
936
933
939
931
31
20
8
7
8
5
11
3
1,149
1,128
1,119
1,120
1,118
1,113
1,122
1,111
Extra Stepsd
0
46”
25”
16
17”
15
10
19
8
a Constraints consisted of requiring the indicated group of sequences to be monophyletic with no other relationships specified inside or outside this group. See
figure 4 for the sequences included in the analyses.
b Lengths of trees calculated with all amino acid transformations weighted equally.
c Lengths of trees calculated with amino acid transformations weighted according to the number of nonsynonymous nucleotide substitutions required.
d Asterisks indicate topologies judged to be significantly worse than the most parsimonious tree by the test of Templeton (1983) as implemented in PROTPARS
(Felsenstein 1993).
’ eUnconstrained trees placed the tunicate outside of all vertebrate LDHs.
sible with the characterization
of LDH enzymatic activity; such investigations
should detect additional loci that
have diverged extensively in sequence but not in function. The most thorough study of this type in tunicates
is that of Baldwin, Mortimer, and Patak (1988) on P.
stoloniferu.
Nondenaturing
electrophoresis
and histochemical staining of crude extracts of whole animals and
a number of tissues revealed evidence for a single locus,
as did analyses of purified LDH activity. Similar electrophoretic investigations
of LDH activity suggest that
echinoderms
(Baldwin, Patak, and Mortimer 1992) and
amphioxus
(Fisher et al. 1980) also possess a single
LDH locus. Assuming the generally accepted phylogeny
(Peterson 1995) of (echinoderms,
(tunicates, (amphioxus, (vertebrates)))),
the simplest explanation of these locus numbers and our phylogenetic
results is that the ancestral state of deuterostomes
(a single LDH locus) has
been retained by echinoderms,
tunicates, and amphioxus, while LDH duplications
have occurred within vertebrates after their divergence from amphioxus. This hypothesis
was proposed
previously
by Fisher et al.
(1980).
’
If, in spite of evidence to the contrary, an additional
tunicate LDH locus did exist, the implications
for LDH
evolution in vertebrates would depend on the phylogenetic position of the new locus in the LDH tree. Clustering with the existing tunicate locus would imply a
duplication in tunicates after their divergence from vertebrates and would not alter hypotheses of the timing of
vertebrate gene duplications.
Because of the phylogenetic position of the known tunicate locus, any other
position of the new locus would require that one of the
two types of loci was lost in all vertebrates. Placement
of the new locus within the clade consisting of vertebrate LDHs would require the loss of at least one type
of locus in the tunicate as well. In other words, even if
an additional tunicate locus is discovered, it will not be
possible to postulate that particular vertebrate duplications occurred earlier than the divergence of tunicates
and vertebrates without also postulating a more complex
pattern of gains and losses.
The Location
of the Root in the Tree of LDHs
We described above the evidence that the LDHs of
Synechocystis, Plasmodium, and Toxoplasma are not orthologous to the other LDHs in the tree and may in fact
be more closely related to tetrameric MDHs. With the
exception of a weakly supported node placing Bijidobacterium LDH closer to plant and animal LDHs than
are other bacterial LDHs, the tree in figure 3 (rooted at
the midpoint)
depicts a phylogeny
of the remaining
LDHs corresponding
to the organismal
phylogeny
of
(bacteria,
(plants,
(nematodes,
(tunicates,
(vertebrates))))). This provides support for a root separating
monophyletic
groups of bacterial and eukaryotic LDHs
(joining an internal branch with 100% bootstrap support
in fig. 3), as any other root position would require a
greater number of gene duplication and loss events. The
clustering of bacterial LDH outside of vertebrate LDHs
has also been obtained in trees of LDHs rooted with
MDH sequences (Iwabe et al. 1989; Cendrin et al. 1993;
Wynne et al. 1996).
LDH Evolution
from Bacteria
to Vertebrates
Determining
whether large numbers of LDH gene
duplications
and losses have occurred outside of chordates requires more extensive collection
of LDH sequences. Our phylogenetic
results obtained with LDHs
from a relatively small number of nonchordate taxa imply an evolutionary
pattern of a single locus persisting
in most lineages with occasional
(and usually recent)
gene duplications.
Examples
of gene duplications
in
nonvertebrate
taxa include the appearance of LDH-A
and LDH-B of Bacillus psychrosaccharolyticus
after its
divergence from the other Bacillus species in the tree
and the LDH-A and LDH-B of barley, which resulted
from a duplication
after the divergence of barley from
the other two members of the family Poaceae (fig. 3).
Although the number of LDH loci in invertebrates
has
not been well characterized, Drosophila melanogaster is
believed to have a single locus for L-LDH (Alahiotis et
al. 1983), and the evidence for a single locus in an echinoderm (Baldwin, Patak, and Mortimer 1992) has been
1282
Stock et al.
discussed above. A number of insects and molluscs are
believed to lack LDH activity altogether, which would
be loss events according to our phylogenetic
trees (e.g.,
fig. 3). However, the biggest remaining question in the
evolution of invertebrate LDHs relates to the orthology
of the loci encoding the proteins or enzyme activities
that have been described (Long 1976; Siebenaller et al.
1983). All of the LDH sequences that we have analyzed
and all known chordate LDHs are specific for the Lstereoisomer
of lactate (Whitt 1984). A number of
groups of invertebrates
have D-LDHs, however, and the
presence of D- or L-LDH appears to be mutually exclusive for any given taxon (Long 1976). Sequence analyses have shown that bacterial D-LDHs form a separate
family from L-LDHs that is much more distantly related
to the latter than are MDHs (Kochhar et al. 1992). No
invertebrate
D-LDH sequences are available to allow a
determination
of their relationship
to L-LDH, although
Siebenaller et al. (1983) suggested that the two types of
LDH were not orthologous based on the sequence of a
few short peptide fragments from a D-LDH of a horseshoe crab. If this hypothesis
is correct, then L-LDH
would have to have been lost and replaced by D-LDH
at least four times in invertebrates
(Long 1976).
Relationships
Within
Vertebrate
LDHs
The relationships
of vertebrate LDHs that we obtained in our analyses are generally consistent with those
of Stock and Whitt (1992), Quattro, Woods, and Powers
(1993), Quattro et al. (1995), and Stock and Powers
(1995). There was strong support for the derivation of
teleost LDH-C from LDH-B after the divergence of teleosts and tetrapods
and a derivation
of mammalian
LDH-C from LDH-A. This contradicts other sequence
analyses that suggested that LDH-C represents the earliest divergence among vertebrate LDHs (Li et al. 1983;
Crawford, Constantino,
and Powers 1989; Tsoi and Li
1994; Tsuji et al. 1994; Mannen et al. 1996). The results
of these analyses differed from ours largely in the location of the root of the vertebrate LDH tree. We conducted a series of phylogenetic
analyses with varying
subsets of outgroup sequences to the vertebrate LDHs
(not shown) and obtained a root in or between mammalian LDH-Cs and other LDHs only with highly divergent sequences as outgroups. The analyses of Tsoi
and Li (1994) and Mannen et al. (1996), in which the
position of LDH-C differed from ours with respect to
topological relationships
as well, were conducted with
the unweighted
pair-group
with arithmetic
mean
(UPGMA) method, one that has been shown to be highly sensitive to unequal rates of evolution (Huelsenbeck
and Hillis 1993).
Our analyses also agreed with previous studies in
providing
strong support for recent duplications
of
LDH-B in Xenopus and the monophyly of a group consisting of LDH-B and its derivatives in teleosts and Xenopus. Although the lamprey LDH clusters with LDH-As
as in the analyses of Stock and Whitt (1992), this result
was not strongly supported, and at least one node strongly contradicted
the orthology of killifish LDH-A with
tetrapod and shark LDH-As. Such a result was also ob-
tained in other studies that included teleost LDH-A sequences (Tsoi and Li 1994; Tsuji et al. 1994; Quattro et
al 1995). This phylogenetic
result cannot be brought in
line with the orthology of LDH-As by rerooting the tree.
Whether or not it is artifactual may be addressed by
analyses of the LDH-As of other groups of primitive
bony fishes.
The interrelationships
of four major lineages of
LDHs (shark LDH-A + tetrapod LDH-A + mammal
LDH-C; teleost LDH-A, lamprey LDH, and jawed vertebrate LDH-Bs and derivatives) were not well resolved
in our analyses, as indicated by low bootstrap support.
The persistence of at least some of these low bootstrap
values in the absence of an outgroup
(analysis not
shown) suggests that the sequence of more closely related outgroups (amphioxus is the only candidate invertebrate) will not by itself resolve all aspects of the phylogeny of vertebrate LDHs. The collection of additional
vertebrate sequences may decrease or eliminate support
for potentially
artifactual groupings but is unlikely to
result in their replacement
with strongly supported alternatives. The resolution of the phylogeny of vertebrate
LDHs may require analyses more sophisticated than the
rather simple methods we have used, such as the assignment of different expected rate categories to different sites and the consideration
of structural or functional
information
in weighting changes.
Gene Duplications
in Early Vertebrate
Evolution
Regardless of the exact pattern of gene duplication
leading to multiple LDH loci in vertebrates, our analyses
suggest that all duplications
occurred after the divergence of tunicates and vertebrates and that at least some
of them occurred early in the diversification
of vertebrates. This evolutionary
pattern is the same as that suggested by Markert, Shaklee, and Whitt (1975) and Fisher
et al. (1980). In the latter study, it was specifically proposed that the earliest LDH duplications
occurred in
jawless vertebrates after the divergence
of vertebrates
from cephalochordates
(amphioxus).
Ohno (1970) proposed that extensive gene duplications had occurred at two or more points in chordate phylogeny, based largely on evidence from genome size estimates. The first of these episodes of large-scale gene
duplication was proposed to have occurred in the lineage
leading to amphioxus and vertebrates after the divergence
of tunicates, and the second in the fish or amphibian ancestors of amniotes. Ohno suggested that the latter duplication was due to polyploidy but that the first may
have been due either to polyploidization
or extensive tandem duplication. Fisher et al. (1980) interpreted the evolution of four isozyme systems (including LDH) as indicating one or more rounds of polyplodization
in the
early vertebrate or prevertebrate lineage. Holland et al.
(1994) and Sharman and Holland (1996) surveyed the
phylogenetic relationships and number of loci encoding
genes involved in the regulation of development and also
concluded that widespread gene duplications occurred at
two periods in chordate evolution. These authors proposed a slightly different timing for the duplication episodes than Ohno (1970), however. The first phase was
Tunicate LDH
proposed to have occurred close to the origin of vertebrates after the divergence of this lineage from amphioxus, and the second near the origin of gnathostomes.
Our results indicating that the duplication of LDH loci in
vertebrates occurred after the divergence of tunicates is
consistent with both the hypotheses of Ohno (1970) and
of Holland et al. (1994) and Sharman and Holland (1996).
A more precise understanding
of the timing of the duplications leading to multiple LDH loci and their relationship to the duplications of other genes awaits the sequencing of the LDHs of amphioxus and hagfish.
That the duplications leading to LDH-A and LDHB involved large genomic regions is suggested by the
linkage relationship
of these two genes in mammals;
mouse and human Ldh-Bs are located on chromsomes 6
and 12p, respectively, and mouse and human Ldh-As are
located on chromosomes
7 and llp (Debry and Seldin
1996). In both species, the chromosome segments containing Ldh-A and Ldh-B contain additional loci with
paralogous relationships between the two regions, and it
has been proposed that these chromosomal segments are
ancestrally related, having arisen by a process such as
polyploidization
(Lundin 1993). A relatively recent origin
of mammalian Ldh-C from Ldh-A, as we have suggested
based on phylogenetic analyses of LDH genes, is consistent with the close linkage of these two loci (perhaps
within 100 kb-Stubbs
et al. 1994). Ldh-C and Ldh-A
have also been shown to be linked in some fish species
(Morizot 1990) despite evidence from sequence analyses
that the former gene is closely related to Ldh-B. This
latter result suggests that linkage arrangements in modern
species may bear a complex relationship to gene duplication histories. Nevertheless, it would be interesting to
compare phylogenies of chordate gene families containing paralogs linked to mammalian or fish LDH loci with
the duplication
history that we have proposed for the
LDH gene family. Phylogenetic
congruence
between
LDH and other linked gene families would provide additional evidence for our hypothesis.
BALDWIN, J., A. PATAK,
(Holothuria
1283
and K. MORTIMER.1992. Echinoderm
atra) lactate dehydrogenase: affinities with the
putative ancestral chordate enzyme. Biochem. Syst. Ecol.
20535-539.
BIRKTOFT,J. J., R. T FERNLEY,R. A. BRADSHAW,and L. J.
BANASZAK.1982. Amino acid sequence homology among
the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase. Proc. Natl. Acad. Sci. USA
79:6166-6170.
BJOURSON,A. J., and J. E. COOPER. 1992. Band-stab PCR: a
simple technique for the purification of individual PCR
products. Nucleic Acids Res. 20:4675.
BZIK, D. J., B. A. Fox, and K. GONYER.1993. Expression of
Plasmodium falciparum lactate dehydrogenase in Escherichia coli. Mol. Biochem. Parasitol. 59: 155-166.
CENDRIN,E, J. CHROBOCZEK,G. ZACCAI, H. EISENBERG,and
M. MEVARECH.1993. Cloning, sequencing, and expression
in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium HaZoarcula marismortui. Biochemistry 32:4308-43 13.
CRAWFORD,D. L., H. R. CONSTANTINO,and D. A. POWERS.
1989. Lactate dehydrogenase-B cDNA from the teleost
Fundulus heteroclitus:
evolutionary implications. Mol.
Biol. Evol. 6:369-383.
DEBRY,R. W., and M. E SELDIN.1996. Human/mouse homology relationships. Genomics 33:337-35 1.
FELSENSTEIN,J. 1993. PHYLIP phylogeny inference package.
Version 3.5~. University of Washington, Seattle.
FISHER, S. E., J. B. SHAKLEE,S. D. FERRIS,and G. S. WHIT-T.
1980. Evolution of five multilocus isozyme systems in the
chordates. Genetica 52/53:73-85.
FROHMAN,M. A. 1990. RACE: rapid amplification of complementary DNA ends. Pp. 28-38 in M. A. INNIS,D. H. GELFAND,J. J. SNINSKY,and T J. WHITE, eds. PCR protocols: a
guide to methods and applications. Academic Press, New
York.
HENDRIKS,W., J. W. M. MULDERS,M. A. BIBBY,C. SLINGSBY,
H. BLOEMENDAL,
and W. W. DE JONG. 1988. Duck lens Ecrystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions. Proc.
Natl. Acad. Sci. USA 85:7114-7118.
HOLLAND,I? W. H. 1992. Homeobox genes in vertebrate evolution. BioEssays 14:267-273.
Acknowledgments
HOLLAND,I? W. H., J. GARCIA-FERNANDEZ,
N. A. WILLIAMS,
and A. SIDOW. 1994. Gene duplications and the origins of
We wish to thank Patricia Schulte for the gift of
vertebrate development. Development (Suppl.): 125-l 33.
competent bacterial cells. This study was supported by
HUELSENBECK,
J. P, and D. M. HILLIS. 1993. Success of phyNSF grants BSR-87-17417
(to G.S.W.), BSR-87-18425
logenetic methods in the four-taxon case. Syst. Biol. 42:
(to D.A.P.), BSR-88-15362
(to G.S.W. and D.W.S.),
247-264
BSR-90-22648
(to D.A.P.), and DEB 96-29572
(to
IWABE, N., K.-I. KUMA, M. HASEGAWA,S. OSAWA, and T.
D.A.P.), an NSF Predoctoral Fellowship in Genetics (to
MIYATA. 1989. Evolutionary relationship of archaebacteria,
D.W.S.), and an Alfred F? Sloan Postdoctoral Fellowship
eubacteria, and eukaryotes inferred from phylogenetic trees
of duplicated genes. Proc. Natl. Acad. Sci. USA 86:9355in Studies of Molecular Evolution (to J.M.Q.).
9359.
JIN, S., M. DE JESCJS-BERR~OS,
and A. L. SONENSHEIN. 1996.
LITERATURE CITED
A Bacillus subtilis malate dehydrogenase gene. J. Bacterial.
178:560-563.
ABAD-ZAPATERO, C., J. I? GRIFFITH,J. L. SUSSMANN,and M.
KANEKO,T., A. TANAKA,S. SATO, H. KOTANI,T. SAZUKA,N.
G. ROSSMANN.1987. Refined crystal structure of dogfish
MIYAJIMA,M. SUGIURA,and S. TABATA. 1995. Sequence
M4 apo-lactate dehydrogenase. J. Mol. Biol. 198:445-467.
analysis of the genome of the unicellular cyanobacterium
ALAHIOTIS,S. N., A. ONOUFRIOU, M. FOTAKI, and M. PELESynechocystis sp. strain PCC6803. I. Sequence features in
CANOS. 1983. Drosophila lactate dehydrogenase: developthe 1 Mb region from map positions 64% to 92% of the
mental aspects. Biochem. Genet. 21:199-211.
genome. DNA Res. 2:153-166.
BALDWIN,J., K. MORTIMER,and A. PATAK. 1988. Do ascidians
KOBEL, H. R. and L. Du PASQUIER.1986. Genetics of polypossess the ancestral subunit type of vertebrate lactate dehydrogenase? J. Exp. Zool. 246: 109-l 14.
ploid Xenopus. Trends Genet. 2: 3 10-3 15.
1284
Stock et al.
KOCHHAR, S., I? E. HUNZIKER, I? LEONG-MORGENTHALER,and
H. HJOTTINGER. 1992. Evolutionary relationship of NAD+dependent D-lactate dehydrogenase:
comparison of primary
structure of 2-hydroxy acid dehydrogenases.
Biochem. Biophys. Res. Commun. 184:60-66.
KOZAK, M. 1996. Interpreting cDNA sequences: some insights
from studies on translation. Mamm. Genome 7563-574.
KUMAR, S., K. TAMURA, and M. NEI. 1993. MEGA: molecular
evolutionary genetics analysis. Version 1.Ol . Pennsylvania
State University, University Park.
LI, S. S.-L., W. M. FITCH, Y.-C. E. PAN, and E S. SHARIEF.
1983. Evolutionary
relationships
of vertebrate lactate dehydrogenase isozymes A4 (muscle), B, (heart), and C4 (testis). J. Biol. Chem. 258:7029-7032.
LONG, G. L. 1976. The stereospecific
distribution and evolutionary significance of invertebrate lactate dehydrogenases.
Comp. Biochem. Physiol. 55B:77-83.
LUNDIN, L. G. 1993. Evolution of the vertebrate genome as
reflected in paralogous chromosomal
regions in man and
the house mouse. Genomics 16:1-19.
MANNEN, H., S. C.-M. TSOI, D. B. PICKFORD, J. A. DONALD,
L. J. GUILLETTE, and S. S.-L. LI. 1996. Sequences of the
lizard cDNAs encoding lactate dehydrogenase
(LDH) isozymes A (muscle) and B (heart). Gene 171:303-304.
MARCHUK, D., M. DRUMM, A. SAULINO, and E S. COLLINS.
199 1. Construction of t-vectors: a rapid and general system
for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1154.
MARKERT, C. L. 1984. Biochemistry
and function of lactate
dehydrogenase.
Cell Biochem. Funct. 2:131-134.
MARKERT, C. L., J. B. SHAKLEE, and G. S. WHITT. 1975. Evolution of a gene. Science 189: 102-l 14.
MATSON, R. H. 1989. Distribution of the testis-specific LDHX among avian taxa with comments on the evolution of the
LDH gene family. Syst. Zool. 38:106-l 15.
MORIZOT, D. C. 1990. Use of fish gene maps to predict ancestral
vertebrate genome organization. Pp. 207-234 in Z.-I. OGITA
and C. L. MARKERT, eds. Isozymes: structure, function, and
use in biology and medicine. Wiley-Liss, New York.
OHNO, S. 1970. Evolution by gene duplication. Springer, New
York.
PETERSON, K. J. 1995. A phylogenetic test of the calcichordate
scenario. Lethaia 28:25-38.
QUATTRO, J. M., D. D. POLLOCK, M. POWELL, H. A. WOODS,
and D. A. POWERS. 1995. Evolutionary
relations among
vertebrate muscle-type
lactate dehydrogenases.
Mol. Mar.
Biol. Biotechnol. 4:224-23 1.
QUATTRO, J. M., H. A. WOODS, and D. A. POWERS. 1993.
Sequence analysis of teleost retina-specific lactate dehydrogenase C: evolutionary implications for the vertebrate lactate dehydrogenase
gene family. Proc. Natl. Acad. Sci. USA
90: 242-246.
SAMBROOK, J., E. E FRITSCH, and T. MANIATIS. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.
SHARMAN, A. C., and I? W. H. HOLLAND. 1996. Conservation,
duplication, and divergence of developmental
genes during
chordate evolution. Neth. J. Zool. 46:47-67.
SHEETS, M. D., S. C. OGG, and M. I? WICKENS. 1990. Point
mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. Nucleic Acids Res. 18:5799-5805.
SIEBENALLER, J. E, T. L. ORR, B. B. OLWIN, and S. S. TAYLOR.
1983. Comparison of the D-lactate stereospecific dehydrogenase of kmulus polyphemus with active-site regions of
L-lactate dehydrogenases.
Biochim. Biophys. Acta 749:
153-162.
STOCK, D. W., K. D. MOBERG, L. R. MAXSON, and G. S.
analysis of the 18s ribosomal
WHI’IT. 199 1. A phylogenetic
RNA sequence of the coelacanth Latimeria chalumnae. Environ. Biol. Fish. 32:99-l 17.
STOCK, D. W., and D. A. POWERS. 1995. The cDNA sequence
of the lactate dehydrogenase-A
of the spiny dogfish (SquaZus acanthias): corrections to the amino acid sequence and
an anlysis of the phylogeny of vertebrate lactate dehydrogenases. Mol. Mar. Biol. Biotechnol. 4:284-294.
STOCK, D. W., and G. S. WHITT. 1992. Evolutionary implications of the cDNA sequence of the single lactate dehydrogenase of a lamprey. Proc. Natl. Acad. Sci. USA 89:17991803.
STUBBS, L., E. M. RINCHIK, E. GOLDBERG, B. RUDY, M. A.
HANDEL, and D. JOHNSON. 1994. Clustering of six human
11~15 gene homologs within a 500-kb interval of proximal
mouse chromosome 7. Genomics 24:324-332.
SWOFFORD,D. L. 1991. PAUP phylogenetic analysis using parsimony. Version 3.0s. Illinois Natural History Survey,
Champaign.
SYNSTAD, B., 0. EMMERHOFF, and R. SIREVAG. 1996. Malate
dehydrogenase
from the green gliding bacterium Chlorojlexus uuruntiucus is phylogenetically
related to lactic dehydrogenases.
Arch. Microbial. 165:346-353.
TEMPLETON, A. R. 1983. Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and the apes. Evolution
371221-244.
THOMPSON, J. D., D. HIGGINS, and T. J. GIBSON. 1994. CLUSTAL W improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic
Acids Res. 22:4673-4680.
TSOI, S. C.-M., and S. S.-L. LI. 1994. The nucleotide and deduced amino-acid sequences of a cDNA encoding lactate
dehydrogenase from Cuenorhubditis eleguns: the evolutionary relationships of lactate dehydrogenases
from mammals,
birds, amphibian, fish, nematode, plants, bacteria, mycoplasma, and plasmodium.
Biochem. Biophys. Res. Commun. 205:558-564.
TSUJI, S., M. A. QURESHI, E. W. Hou, W. M. FITCH, and S. S.L. LI. 1994. Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish,
barley, and bacteria: LDH cDNA sequences from Xenopus,
pig and rat. Proc. Natl. Acad. Sci. USA 91:9392-9396.
WHITT, G. S. 1969. Homology of lactate dehydrogenase genes:
E gene function in the teleost nervous system. Science 166:
1156-l 158.
1984. Genetic, developmental
and evolutionary
aspects of the lactate dehydrogenase
isozyme system. Cell
Biochem. Funct. 2: 134-139.
WOLFF, J., and H. R. KOBEL. 1982. Lactate dehydrogenase
of
Xenopus luevis luevis and Xenopus borealis depends on a
multiple gene system. J. Exp. Zool. 223:203-210.
WYNNE, S. A., D. J. NICHOLLS, M. D. SCAWEN, and T. K.
SUNDARAM. 1996. Tetrameric malate dehydrogenase from a
thermophilic
Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and
characterization
of the recombinant enzyme. Biochem. J.
3171235-245.
YANG, S., and S. E PARMLEY. 1995. A bradyzoite stage-specifically expressed gene of Toxoplasma gondii encodes a
polypeptide
homologous
to lactate dehydrogenase.
Mol.
Biochem. Parasitol. 73:291-294.
SHOZO YOKOYAMA,
Accepted
August
reviewing
29, 1997
editor