The myoglobin gene of the Antarctic icefish, Chaenocephalus aceratus, contains a duplicated TATAAAA sequence that interferes with transcription
1
School of Marine Sciences, University of Maine, Orono, ME 04469,
USA
2
Department of Biochemistry, Microbiology and Molecular Biology, University
of Maine, Orono, ME 04469, USA
*
Present address: Maine Medical Center Research Institute, Center for Molecular
Medicine, 81 Research Drive, Scarborough, ME 04074, USA
Present address: Marine Education and Research Center, c/o Biological Sciences
Department, California Polytechnic State University, San Luis Obispo, CA
93407, USA
Author for correspondence at address 1 (e-mail:
bsidell{at}maine.edu)
Accepted 10 October 2002
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Summary |
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Key words: myoglobin, Antarctic fish, heart muscle, gene expression, promoter regulation, oxygen transport, icefish, Chaenocephalus aceratus, Channichthyidae
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Introduction |
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Icefish are the only known vertebrates whose adult forms do not express the
circulating oxygen-binding protein, hemoglobin
(Ruud, 1954;
Hemmingsen, 1991
). All icefish
exhibit dramatic cardiovascular adaptations to compensate for the lack of
hemoglobin in the circulation (Hemmingsen,
1991
; Johnston et al.,
1993
). The loss of hemoglobin expression, however, is not the only
unusual feature of oxygen delivery systems in these fish. At least six species
of icefish have lost the ability to express the intracellular oxygen-binding
protein, myoglobin (Mb), within their cardiac muscle; oxidative skeletal
muscle of all species in this family is devoid of Mb
(Sidell et al., 1997
;
Vayda et al., 1997
). Mb
functions as an intracellular oxygen reservoir and facilitates delivery of
oxygen to mitochondria in tissues that rely heavily on oxidative
phosphorylation to meet their energetic needs
(Wittenberg and Wittenberg,
1989
).
Acierno et al. (1997)
demonstrated that hearts of icefish that do express Mb are capable of
maintaining cardiac output at greater afterload challenges than are hearts of
closely related species that do not express the protein. Enhanced performance
of the Mb-containing hearts is erased in the presence of sodium nitrite, a
specific inhibitor of Mb function, demonstrating that loss of Mb has
significant physiological consequences. Ventricular tissues from icefish
species that lack Mb also contain significantly higher densities of
mitochondria and are spongier, with more spaces filled by luminal blood, than
tissue from channichthyids that do express Mb
(O'Brien and Sidell, 2000
;
O'Brien et al., 2000
). Both of
these features are thought to reduce the diffusion path length for oxygen and
ensure its efficient delivery from blood to mitochondria.
In some species of icefish, the reason for loss of Mb expression is clear.
For example, Champsocephalus gunnari expresses a non-functional Mb
mRNA containing a five-nucleotide duplication that causes a frameshift
resulting in premature termination of the Mb polypeptide
(Vayda et al., 1997). We
recently have found an identical 5-bp insertion in the Mb-coding sequence of
congeneric Champsocephalus esox (T. J. Grove, J. Hendrickson and B.
D. Sidell, unpublished observations). Mb mRNA is found in trace amounts in
Pagetopsis macropterus cardiac muscle, its scarcity presumably due to
an aberrant polyadenylation site (Vayda et
al., 1997
). The loss of Mb expression in Chaenocephalus
aceratus, however, appears to be distinct because Mb mRNA cannot be
detected in the heart ventricle of this species
(Sidell et al., 1997
) nor can
Mb transcripts be detected by nuclear run-on assays (D. J. Small, M. E. Vayda
and B. D. Sidell, unpublished results).
The C. aceratus Mb genomic sequence does not reveal aberrant
splice junctions or other lesions in the transcriptional unit that can explain
the absence of Mb expression. In fact, the C. aceratus Mb genomic
sequence is 98% identical to the functional Mb genomic sequence of
Chionodraco rastrospinosus, an Mb-expressing species
(Small et al., 1998). The only
differences between Mb genes of these icefish species are insertions/deletions
within intron sequences, variations in the number of a simple sequence repeat
ATCT located 141 bp upstream of transcription start relative to the C.
rastrospinosus Mb gene sequence, and a 15-bp insertion in the C.
aceratus Mb gene relative to that of C. rastrospinosus, which is
located between residues -647 and -648 of the reference C.
rastrospinosus sequence (Small et
al., 1998
). This insertion contains the sequence TATAAAA, which is
identical to the muscle-specific transcription factor IID (TFIID) target
sequence that is found 25 bp upstream of the C. rastrospinosus Mb
transcription start site. This sequence is typical of the `TATA' sequences of
muscle-specific genes (Basel-Duby et al., 1993). The number of ATCT repeats
varied between Mb promoters of C. rastrospinosus (13 repeats) and
C. gunnari (25 repeats), both of which are transcribed, suggesting
that repeat length of this microsatellite is unlikely to explain the failure
of C. aceratus Mb (22 repeats) to be transcribed
(Small et al., 1998
). These
observations prompted us to determine whether the duplicated TATAAAA sequence
is responsible for the loss of Mb expression in C. aceratus. Results
of our experiments show that the duplicated TATAAAA sequence binds TFIID and
binds factors of icefish heart nuclear extracts and that constructs containing
the duplicated TATAAAA sequence are not expressed. These results substantiate
a third discrete molecular mechanism that has led to the loss of Mb expression
within this unique family of fishes.
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Materials and methods |
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Isolation of nuclear extracts
Intact nuclei were isolated from 4-10 g of heart ventricular tissue from
freshly killed animals for each preparation. Tissue was rinsed with 50 ml
ice-cold Ringer's solution (260 mmol l-1 NaCl, 5 mmol
l-1 KCl, 2.5 mmol l-1 MgCl2, 3 mmol
l-1 CaCl2, 2.5 mmol l-1 NaHCO3 and
2 mmol l-1 NaH2PO4) and then diced and
homogenized in 30 ml of homogenization buffer [300 mmol l-1
sucrose, 10 mmol l-1 Hepes pH 7.5, 5 mmol l-1 KCl, 0.75
mmol l-1 spermidine, 0.15 mmol l-1 spermine, 0.1 mmol
l-1 EDTA, 0.1 mmol l-1 EGTA, 0.5 mmol l-1
phenylmethanesulfonyl fluoride (PMSF; Sigma, St Louis, MO, USA) and 2 µg
ml-1 final concentration each of aprotinin, leupeptin and
pepstatin] in a 40 ml dounce homogenizer by five strokes with pestle B (loose
fitting) and three strokes with pestle A (tight fitting). The crude homogenate
was then filtered through two layers of cheesecloth pre-wetted with the
homogenization buffer and mixed with an equal volume of cushion buffer [9
volumes of 80% ultrapure sucrose solution mixed with 1 volume of 10x
salts: 100 mmol l-1 Hepes pH 7.5, 50 mmol l-1 KCl, 7.5
mmol l-1 spermidine, 1.5 mmol l-1 spermine, 1 mmol
l-1 EDTA, 1 mmol l-1 EGTA and 10 mmol l-1
dithiothreitol (DTT)]. The mixture was divided evenly and overlayed over 10 ml
of cushion buffer in three Beckman SW25.1 ultracentrifuge tubes. Nuclei were
collected by centrifugation at 59 750 g at 4°C for 60 min
in a Beckman SW25.1 swinging bucket rotor. Nuclear pellets were resuspended in
a 1 ml nuclei storage buffer (25% glycerol, 50 mmol l-1 Hepes pH
7.5, 3 mmol l-1 MgCl2, 0.1 mmol l-1 EDTA, 1
mmol l-1 DTT, 0.1 mmol l-1 PMSF, 2 µg ml-1
each of aprotinin, leupeptin, pepstatin). 200 µl samples were placed in
cryovials and flash-frozen in liquid nitrogen. Nuclei were stored in liquid
nitrogen or at -70°C until use. Nuclear proteins used in the gel mobility
shift assays were extracted from frozen nuclei isolated from C.
rastrospinosus heart ventricular tissue by incubating 150 µl of nuclei
in nuclear extraction buffer (0.5 mmol l-1 PMSF, 2 µg
ml-1 each of aprotinin, leupeptin and pepstatin, 0.5 mol
l-1 NaCl, 0.7 mmol l-1 spermidine, 0.13 mmol
l-1 spermine and 0.17 mmol l-1 EGTA) with constant
rotation for 90 min at 4°C. Soluble nuclear proteins were separated from
chromatin by centrifugation at 16 000 g in an Eppendorf
microcentrifuge for 20 min. The supernatant containing the soluble nuclear
proteins was removed, concentrated using a Micron 3 protein concentrator
(Millipore, Bedford, MA, USA) and dialyzed against 1x GMS buffer [20
mmol l-1 Hepes pH 7.9, 25 mmol l-1 KCl, 2 mmol
l-1 spermidine; 0.1 mmol l-1 EDTA, 10% glycerol, 0.5
mmol l-1 DTT and 100 µg ml-1 bovine serum albumin
(BSA)].
Gel mobility shift assays
Sense and anti-sense oligonucleotides (purchased from Integrated DNA
Technologies, Coralville, IA, USA) representing the DNA targets depicted in
Fig. 1A used for the gel
mobility shift assays were end-labeled using T4 polynucleotide kinase and
[-32P]ATP as described in Sambrook et al.
(1989
). After labeling,
complementary sense and antisense oligonucleotides were mixed together, heated
to 96°C for 5 min and then slowly cooled to room temperature. The annealed
oligonucleotides were then purified by electrophoresis through native 12%
acrylamide gels cast in 50 mmol l-1 Tris-borate, 0.5 mmol
l-1 EDTA, pH 8.3. After electrophoresis, the labeled
double-stranded oligonucleotides were excised from the gel and eluted
overnight in TE buffer. 2 pmol of the indicated oligonucleotide template was
mixed with 5 µg of a nuclear extract or 2 ng of purified TFIID (Promega,
Madison, WI, USA), 2 µg of poly(dI-dC) and 1x final concentration GMS
buffer for a total volume of 25 µl. For competition assays, 100-fold excess
(200 pmol) of unlabeled oligonucleotide was added to each sample. The reaction
was incubated at room temperature for 30 min then mixed with 3 µl 10x
gel-loading buffer (Promega), loaded onto 6% native acrylamide gels cast in 50
mmol l-1 Tris-borate, 0.5 mmol l-1 EDTA, pH 8.3 and
subjected to electrophoresis at 25 V for 2 h. Gels were dried and exposed to
film overnight.
|
Construction of reporter plasmids
Reporter plasmids were generated by PCR amplification of icefish Mb
promoter sequences using DNA isolated from Mb genomic clones and PCR primers
specific to conserved regions of the icefish promoter
(Small et al., 1998). Each
primer also contained short DNA sequences at the 5' end that
corresponded to either the SacI (forward primer) or BglII
(reverse primer) restriction site for cloning purposes. The forward primer
used for constructs pR1L and pA1L was
5'-gccgagctcCTGCAGCCCTCGAGTCGGTTTCTTC-3' (position -1525 to -1501
of the C. rastrospinosus Mb sequence; -1575 to -1551 of the C.
aceratus Mb sequence); the forward primer used for constructs pR2L and
pA2L was 5'-gccgagctcGGTGTTTTCCGGGTGTTGA-3' (position -599 to -580
of the C. rastrospinosus Mb sequence; -634 to -403 of the C.
aceratus Mb sequence); the forward primer used for constructs pR3L and
pA3L was 5'-gccgagctcGGACAAGAAGAGGAAACATAGGATAGTG-3' (position
-398 to -369 of the C. rastrospinosus Mb sequence; -434 to -403 of
the C. aceratus Mb sequence). The reverse primer used to generate all
six constructs was 5'-gccagatctGATGTTGTACAAAATTCTTCTTGACCTGAC-3'
(complementary to positions +32 to +3 of both the C. rastrospinosus
and C. aceratus Mb sequences). After PCR amplification, products were
column purified (Qiagen, Valencia, CA, USA) and digested with SacI
and BglII. Digested PCR products were ligated to the pGL2 basic
vector (Promega) at the SacI and BglII sites. All constructs
were sequenced at the University of Maine DNA sequencing core facility prior
to use.
Transient expression assay in vivo
Specimens of C. aceratus and C. rastrospinosus were
maintained in flowing seawater tanks at Palmer Station, Antarctica at an
ambient temperature of -1°C. Individual fish were injected at multiple
sites into their pectoral abductor muscle; each site was injected with a 1-5
µl solution containing 1 µg of a test plasmid promoter construct (pR1L,
pA1L, pR2L, pA2L, pR3L, pA3L or pSV40Luc) and 0.5 µg of the
Renilla luciferase internal reference construct driven by the
cytomegalovirus (CMV) promoter (Promega). Each injection site was marked by a
suture. Fish were returned to flowing seawater tanks and maintained until
0.3-0.6 g blocks of tissue surrounding each injection site were harvested 3-7
days post-injection (Schulte et al.,
1998; Friedenreich and
Schartl, 1990
) and immediately frozen in liquid nitrogen.
Luciferase assays were conducted using the dual-luciferase reporter assay
system (Promega) according to the supplier. Light units emitted were measured
using an MJ Research lumenometer (MJ Research, Waltham, MA, USA).
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Results |
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Oligonucleotides corresponding to this segment of the C. aceratus
and C. rastrospinosus Mb promoters, and the authentic TATAAAA
sequence common to both Mb genes (Small et
al., 1998), were synthesized, labeled with 32P using T4
polynucleotide kinase and used for gel-retardation assays
(Fig. 1A). The C.
aceratus upstream Mb sequence (-715/-674) bound purified human TFIID in
gel-retardation assays (Fig.
1B, lane 10) as evident by the presence of two prominent
retardation complexes. The authentic TATAAAA oligonucleotide (-45/-4) formed
similar retardation complexes (Fig.
1B, lane 2). These two oligonucleotides competed for the same
factor when one was present in excess, as indicated by a nearly complete
elimination of labeled retardation complexes in
Fig. 1B, lanes 3, 4, 7 and 8.
The C. rastrospinosus upstream Mb sequence (-666/-640) did not form a
retardation complex with TFIID (Fig.
1B, lane 12), nor was it effective in preventing the formation of
retardation complexes with either of the TATAAAA-containing oligonucleotides
(Fig. 1B, lanes 5 and 9). The
C. aceratus upstream Mb sequence (-715/-674) binds to factors present
in heart ventricle nuclear extracts from C. rastrospinosus to form
several complexes (Fig. 2A,
lanes 3 and 6). These complexes had similar mobility to those generated by
incubation with HeLa TFIID (Fig.
2A, lane 1). Formation of labeled retardation complex was
prevented by both an excess of the (-45/-4) TATAAAA oligonucleotide
(Fig. 2A, lane 5) and by an
excess of non-labeled C. aceratus (-715/-674) oligonucleotide
(Fig. 2A, lane 4). The C.
aceratus upstream (-715/-674) Mb oligonucleotide also prevented the
formation of complexes between factors present in the icefish nuclear extract
and labeled (-45/-4) TATAAAA oligonucleotide (shown by arrows in
Fig. 2B, lane 3). These results
suggest that the upstream insertion in the C. aceratus Mb promoter
binds TFIID in an inappropriate context, which may explain the aberration of
this promoter.
|
The presence of the -695 bp TATA sequences interferes with transient
expression in C. aceratus muscle in vivo. In order to
determine whether the -695 TATAAAA sequence inhibited transcription, segments
of the C. rastrospinosus and C. aceratus Mb promoters were
selectively amplified and cloned. The promoter segments were amplified using
sense primers MP8 (-1525 of C. rastrospinosus Mb and -1575 of C.
aceratus Mb), MP7F (-599 of C. rastrospinosus Mb and -634 of
C. aceratus Mb) or MP6F (-398 of C. rastrospinosus Mb and
-434 of C. aceratus Mb) and antisense primer Myo3B (+32)
(Vayda et al., 1997;
Small et al., 1998
). Each of
these amplified segments was linked to the firefly luciferase coding sequence
reporter gene (Fig. 3A) and
introduced into C. aceratus aerobic pectoral adductor muscle for
transient expression assay. This tissue was competent to express the
full-length (-1525) C. rastrospinosus Mb promoter construct
(Fig. 3B). By contrast, the
corresponding C. aceratus full-length (-1575) promoter construct
containing the TATAAAA duplication was not expressed above the level of
promoter-less controls. The C. rastrospinosus construct truncated at
position -599 and the C. aceratus constructs truncated at position
-634 both exhibited expression higher than the full-length C.
aceratus construct containing the TATAAAA duplication
(Fig. 3B). However, the low
levels of expression provided by these truncated constructs indicate that an
element upstream of -599 is essential for efficient transient expression
in vivo. Further truncation of either the C. rastrospinosus
or C. aceratus Mb genes reduced transient expression to that of
promoter-less controls (Fig.
3B). This striking difference between expressions of the
full-length C. rastrospinosus Mb construct pR1L and the full-length
C. aceratus Mb construct pA1L suggests that the presence of the
duplicated TATAAAA element severely interferes with transcription.
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Discussion |
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In previous communications, we have described two of the specific mutations
leading to the loss of Mb expression in other icefish species,
Champsocephalus gunnari (5-nucleotide insertion leading to premature
termination) and Pagetopsis macropterus (aberrant polyadenylation
signal) (Vayda et al., 1997).
In the present study, we report a third distinct mechanism by which Mb
expression has been lost among channichthyid fishes. We can also conclude that
the mutation we describe here must be very recent indeed. Compared with the
gene from closely related Mb-expressing Chionodraco rastrospinosus,
the now-silent Mb gene in Chaenocephalus aceratus does not exhibit
any lesions within the normally transcribed sequence that would lead to
inactivation of the gene product. Apparently, the relatively subtle event of
duplicating a normal promoter element (the muscle-specific TATAAAA sequence)
has resulted in the loss of Mb expression in this species. Numerous examples
are known where removal of a negative regulatory element (thus eliminating
binding of negative regulatory factor) results in the elevated expression of a
promoter construct (e.g. Yan et al.,
2001
). However, we are unaware of any other examples where
duplication of a TATAAAA element results in silencing of gene expression.
Lack of activity in transient expression assays of the `full-length'
promoter construct (pA1L) from C. aceratus indicates that this region
of the gene contains a mutation that prevents its transcription. The only
significant difference between this Mb promoter from C. aceratus and
that from C. rastrospinosus, which is transcribed normally, is the
15-bp insertion containing a duplicated TATAAAA sequence at -695 bp. This
insertion is located only 10 bp downstream from a putative `E-box', a site
that binds the basic helix-loop-helix (bHLH) class of transcription factors,
such as MyoD, and the ubiquitously expressed E class factors, such as E12
(Small et al., 1998;
Blackwell and Weintraub, 1990
;
Weintraub et al., 1991
). Our
gel mobility shift assays demonstrate that the transcription factor, TFIID,
binds in vitro to the upstream insertion in C. aceratus and
that this region also binds factors that are present in nuclear extracts from
icefish. On the strength of these data, it is reasonable to conclude that the
failure to observe Mb transcription in vivo is probably due to
inappropriate recruitment of TFIID to the duplication at this site. TFIID
bound to this duplicated TATAAAA sequence may sterically hinder the binding of
MyoD or E-box class factors to putative binding sites at -575 and -663,
respectively (Small et al.,
1998
). Binding of the TATA-binding protein (TBP) is known to
induce a sharp 80° bend in DNA, distorting the backbone to force open the
minor groove at the TATA sequence (Kim et al.,
1993a
,b
).
The introduction of such a conformational distortion between the potential
MyoD-binding site at -575 or the E2A site at -663 could preclude binding of
these skeletal muscle transcriptional activators. Although well-documented as
skeletal muscle-specific transcriptional activators
(Weintraub et al., 1991
),
E-box elements upstream of the start site are not essential for expression of
Mb in mammalian cardiac muscle
(Navankasattusas et al., 1992
)
or aerobic skeletal muscle (Yan et al.,
2001
). However, an intragenic E-box in close proximity to the
transcriptional start site (+5 to +10) is known to act as a negative regulator
element that probably contributes to myofiber type-specific Mb expression
(Yan et al., 2001
). We suggest
that inappropriate positioning of the TATAAAA duplication near the E2A site
may seed assembly of an inhibitory complex that silences Mb expression in
C. aceratus.
Results from our in vivo transient expression assays with
oxidative skeletal muscle of C. aceratus
(Fig. 3) permit two additional
conclusions. First, the minimal promoter required for C.
rastrospinosus Mb expression is significantly longer than that required
for mammalian Mb (Bassel-Duby et al.,
1993) and includes at least one element distal to position -634.
Second, despite the lack of endogenous Mb expression in aerobic skeletal
muscle from any notothenioid fish that we have examined to date
(Moylan and Sidell, 2000
),
this tissue apparently contains all the necessary transcription factors
required for transient expression of the Mb promoter constructs. Thus,
silencing of Mb expression in oxidative skeletal muscle, even in those species
that normally express the protein in heart ventricle, must be the result of
some type of developmental patterning, possibly rendering the gene
inaccessible due to higher order DNA/chromosomal structure in the
differentiated tissue.
Both the highly derived position of C. aceratus in channichthyid
phylogeny and the punctuated loss of Mb expression prior to significant
mutation in the coding sequence of the gene strongly indicate that the genetic
lesion in this species has occurred quite recently. Our relatively extensive
sequence analysis indicates that a single duplication event (duplication of
the TATAAAA promoter element) apparently has led to the loss of Mb expression
in C. aceratus. This observation is not unique among the
channichthyid icefish. In fact, loss of Mb expression in C. gunnari
and congeneric C. esox is also due to a single 5-bp duplication
within the coding sequence of the gene
(Vayda et al., 1997; T. J.
Grove et al., unpublished data). We have identified a different mutation in
the polyadenylation signal of the Mb gene in another icefish species, P.
macropterus, which also has led to loss of Mb production
(Vayda et al., 1997
). Taken
together, loss of myoglobin expression via both multiple events and
multiple mechanisms during evolution of the icefish family seems to suggest
that Mb function may not be of physiological relevance in the oxygen-rich and
severely cold environment of the Southern Ocean. Compelling physiological
evidence, however, suggests otherwise.
When isolated, perfused hearts from Mb-expressing and Mb-lacking icefish
species are challenged with increasing afterload, hearts whose ventricles
contain Mb are capable of greater pressure-work
(Acierno et al., 1997).
Furthermore, specific poisoning of Mb function in these preparations causes
performance of Mb-expressing hearts to decrement below that of normally
Mb-lacking hearts, indicating that the latter display mechanisms that
partially compensate for the absence of this oxygen-binding protein. In fact,
one of our laboratories has documented several structural differences in
Mb-lacking icefish hearts that probably contribute to this compensation. These
include dense populations of mitochondria, which reduce intracellular
diffusion distance for oxygen (O'Brien and
Sidell, 2000
), and a very spongy characteristic of the
endocardium, which diminishes the diffusion path length for oxygen from
blood-filled lumen of the heart to the interior of muscle cells
(O'Brien et al., 2000
). Some
of these characteristics are analogous to those seen with Mb `knockout' mouse
strains. Murine Mb-knockouts were originally thought to show no significant
functional disadvantage compared with Mb-expressing wild-type strains
(Garry et al., 1998
). However,
more recent work has established that a large fraction of embryos from the
Mb-deficient strain do not survive gestation. Furthermore, those Mb-deficient
offspring that do survive display adaptive responses in their heart muscle
that apparently compensate for lack of the oxygen-binding protein and permit
normal function (Meeson et al., 2000). Notable among these is a significantly
increased vascularization of the tissue, which reduces diffusion path length
for oxygen, a response apparently mediated by induction of hypoxia-inducible
transcription factors (HIF 1
and HIF 2). This observation opens up the
intriguing possibility that a constitutive upregulation of the HIF
transduction pathway might underlie the differences in mitochondrial densities
and tissue architecture in Mb-deficient icefish hearts.
Results of experiments with isolated, perfused hearts from icefish that are
described above, in combination with measurements of oxygen-binding kinetics
of icefish Mb (Cashon et al.,
1997), clearly indicate that Mbs from icefish are functional at
cold body temperature and enhance cardiac performance. The conundrum presented
by these observations is that modern population genetics theory dictates that
loss of Mb expression, an apparently deleterious trait, should be subject to
negative selective pressures and that such genetic mutation(s) ultimately
should be eliminated from the population. Yet, Mb-lacking icefish species have
arisen at least four times in the radiation of this family and continue to
persist. We have argued previously that the uniquely cold and oxygen-rich
waters of the Southern Ocean and relatively low niche competition in this
marine habitat of relatively low species diversity during evolution of the
icefish family have permitted icefish species lacking Mb to persist
(Moylan and Sidell, 2000
). A
recent review by Montgomery and Clements
(2000
), in fact, cited the
pattern of Mb loss in icefish as one example of a pattern of disaptation and
recovery (i.e. detrimental genetic change followed by compensatory
adaptation). As our knowledge of notothenioid phylogeny and the timing of
events during the radiation of these fishes becomes more complete, we can
anticipate that this group will offer even more lessons in evolutionary
biology.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acierno, R., Agnisola, C., Tota, B. and Sidell, B. D.
(1997). Myoglobin enhances cardiac performance in Antarctic fish
species that express the protein. Am. J. Physiol.
273, R100
-R106.
Bargelloni, L. and Lecointre, G. (1998). Four years of notothenioid systematics: a molecular perspective. In Fishes of Antarctica (ed. G. di Prisco, E. Pisano and A. Clarke), pp. 259-273. Milan: Springer-Verlag Italia.
Bargelloni, L., Ritchie, P. S., Partarnello, T., Battagia, B., Lambert, D. M. and Meyer, A. (1994). Molecular evolution at sub-zero temperatures: mitochondrial and nuclear phylogenies of fishes from Antarctica (suborder Notothenoidei) and the evolution of antifreeze. Mol. Biol. Evol. 6, 854 -863.
Bassel-Duby, R., Grohe, C. M., Jessen, M. E., Parsons, W. J., Richardson, J. A., Chao, R., Grayson, J., Ring, W. S. and Williams, R. S. (1993). Sequence elements required for transcriptional activity of the human myoglobin promoter in intact myocardium. Circ. Res. 73, 360 -366.[Abstract]
Blackwell, T. K. and Weintraub, H. (1990). Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250, 1104 -1110.[Medline]
Cashon, R. E., Vayda, M. E. and Sidell, B. D. (1997). Kinetic characterization of myoglobins from vertebrates with vastly different body temperatures. Comp. Biochem. Physiol. 117B, 613 -620.
Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella,
L., Ciaramella, M., diPrisco, G. and Detrich, H. W., III
(1995). Genomic remnants of -globin genes in the
hemoglobinless Antarctic icefishes. Proc. Natl. Acad. Sci.
USA 92, 1817
-1821.[Abstract]
DeWitt, H. H. (1971). Folio 15. In Antarctic Map Folio Series (ed. V. C. Bushnell), pp. 1 -10. New York: American Geographical Society.
Eastman, J. T. (1993). Antarctic Fish Biology: Evolution in a Unique Environment. New York: Academic Press.
Friedenreich, H. and Schartl, M. (1990). Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells. Nucleic Acids Res. 18, 3299 -3305.[Abstract]
Garry, D. J., Ordway, G. A., Lorenz, J. N., Radford, N. B., Chin, E. R., Grange, R. W., Bassel-Duby, R. and Williams, R. S. (1998). Mice without myoglobin. Nature 395, 905 -908.[CrossRef][Medline]
Hemmingsen, E. A. (1991). Respiratory and cardiovascular adaptation in hemoglobin-free fish: resolved and unresolved problems. In Biology of Antarctic Fish (ed. G. di Prisco, B. Maresca and B. Tota), pp. 191-203. New York: Springer-Verlag.
Hofmann, G. E., Buckley, B. A., Airaksinen, S., Keen, J. E. and
Somero, G. N. (2000). Heat-shock protein expression is absent
in the Antarctic fish, Trematomus bernacchii (Family Nototheniidae).
J. Exp. Biol. 203, 2331
-2339.
Johnston, I. A., Fitch, N., Zummo, G., Wood, R. E., Harrison, P. and Tota, B. (1993). Morphometric and ultrastructural features of the ventricular myocardium of the haemoglobinless icefish, Chaenocephalus aceratus. Comp. Biochem. Physiol. 76A, 475 -480.
Kennett, J. P. (1980). Paleoceanographic and biogeographic evolution of the Southern Ocean during the Cenozoic, and Cenozoic microfossil datums. Paleogeogr. Paleoclimatol. Paleoecol. 31, 123 -152.
Kim, J. L., Nikolov, D. B. and Burley, S. K. (1993a). Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365, 520 -527.[CrossRef][Medline]
Kim, Y., Geiger, J. H., Hahn, S. and Sigler, P. B. (1993b). Crystal structure of a yeast TBP/TATA-box complex. Nature 365, 512 -520.[CrossRef][Medline]
Littlepage, J. L. (1965). Oceanographic investigation in McMurdo Sound, Antarctica. In Biology of the Antarctic Seas, vol. II (ed. G. A. Llano), pp. 1-37. Washington, DC: American Geophysical Union.
Meeson, A. P., Radford, N., Shelton, J. M., Mammen, P. P. A.,
DiMaio, J. M., Hutcheson, K., Kong, Y., Elterman, J., Williams, R. S. and
Garry, D. J. (2001). Adaptive mechanisms that preserve
cardiac function in mice without myoglobin. Circ. Res.
88, 713
-720.
Montgomery, J. and Clements, K. (2000). Disaptation and recovery in the evolution of Antarctic fishes. Trends Ecol. Evol. 15, 267 -271.[CrossRef][Medline]
Moylan, T. J. and Sidell, B. D. (2000).
Concentrations of myoglobin and myoglobin mRNA in heart ventricles from
Antarctic fishes. J. Exp. Biol.
203, 1277
-1286.
Navankasattusas, S., Zhu, H., Garcia, A. V., Evans, S. M. and Chien, K. R. (1992). A ubiquitous factor (HF-1a) and a distinct muscle factor (H1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression. Mol. Cell. Biol. 12, 1469 -1479.[Abstract]
O'Brien, K. M. and Sidell, B. D. (2000). The
interplay among cardiac ultrastructure, metabolism and the expression of
oxygen-binding proteins in Antarctic fishes. J. Exp.
Biol. 203, 1287
-1297.
O'Brien, K. M., Xue, H. and Sidell, B. D. (2000). Quantification of diffusion distance within the spongy myocardium of hearts from Antarctic fishes. Resp. Physiol. 122, 71 -80.[CrossRef][Medline]
Ruud, J. T. (1954). Vertebrates without erythrocytes and blood pigment. Nature 173, 848 -850.[Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd edn, Cold Spring Harbor: Cold Spring Harbor Press.
Schulte, P. M., Powers, D. A. and Schartl, M. (1998). Efficient gene transfer into Xipophorus muscle and melanoma by injection of supercoiled plasmid DNA. Mol. Mar. Biol. Biotechnol. 7, 241 -247.[Medline]
Sidell, B. D., Vayda, M. E., Small, D. J., Moylan, T. J.,
Londraville, R. L., Yan, M., Rodnick, K. J., Eppley, Z. A. and Costello,
L. (1997). Variable expression of myoglobin among the
hemoglobinless Antarctic icefishes. Proc. Natl. Acad. Sci.
USA 94, 3420
-3424.
Small, D. J., Vayda, M. E. and Sidell, B. D. (1998). A novel vertebrate myoglobin gene containing three A+T-rich introns is conserved among Antarctic teleost species, which differ in myoglobin expression. J. Mol. Evol. 47, 156 -166.[Medline]
Vayda, M. E., Small, D. J., Yuan, M., Costello, L. and Sidell, B. D. (1997). Conservation of the myoglobin gene among Antarctic notothenioid fishes. Mol. Mar. Biol. Biotechnol. 6, 207 -216.[Medline]
Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Yuan, Z. and Lassar, A. (1991). The MyoD gene family: nodal point during specification of the muscle cell lineage. Science 251, 761 -766.[Medline]
Wittenberg, B. A. and Wittenberg, J. B. (1989). Transport of oxygen in muscle. Ann. Rev. Physiol. 51, 857 -878.[CrossRef][Medline]
Yan, Z., Serrano, A. L., Schiaffino, S., Bassel-Duby, R. and
Williams, R. S. (2001). Regulatory elements governing
transcription in specialized myofiber subtypes. J. Biol.
Chem. 276, 17361
-17366.