From the Department of Biology, Northeastern
University, Boston, Massachusetts 02115 and the ¶ Istituto di
Biochimica delle Proteine ed Enzimologia, Consiglio Nazionale delle
Ricerche, 80125 Naples, Italy
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ABSTRACT |
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The icefishes of the Southern Ocean (family
Channichthyidae, suborder Notothenioidei) are unique among vertebrates
in their inability to synthesize hemoglobin. We have shown previously
(Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella,
L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995)
Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that
icefishes retain inactive genomic remnants of adult notothenioid
-globin genes but have lost the gene that encodes adult
-globin.
Here we demonstrate that loss of expression of the major adult
-globin,
1, in two species of icefish (Chaenocephalus
aceratus and Chionodraco rastrospinosus) results from
truncation of the 5' end of the notothenioid
1-globin gene. The
wild-type, functional
1-globin gene of the Antarctic yellowbelly
rockcod, Notothenia coriiceps, contains three exons and two
A + T-rich introns, and its expression may be controlled by two or
three distinct promoters. Retained in both icefish genomes are a
portion of intron 2, exon 3, and the 3'-untranslated region of the
notothenioid
1-globin gene. The residual, nonfunctional
-globin
gene, no longer under positive selection pressure for expression, has
apparently undergone random mutational drift at an estimated rate of
0.12-0.33%/million years. We propose that abrogation of hemoglobin
synthesis in icefishes most likely resulted from a single mutational
event in the ancestral channichthyid that deleted the entire
-globin
gene and the 5' end of the linked
1-globin gene.
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INTRODUCTION |
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Alone among vertebrate taxa, the 15 species of Antarctic icefishes
(family Channichthyidae, suborder Notothenioidei) are unique in their
failure to synthesize the respiratory oxygen transport protein
hemoglobin (1). Although icefish blood contains "erythrocyte-like" cells in small numbers (2, 3), these cells are devoid of hemoglobin,
and icefishes transport oxygen to their tissues solely in physical
solution. In the cold (1.86 to +1 °C), stable, and oxygen-rich
environment experienced by these organisms, reduction of the hematocrit
to near zero may have been selectively advantageous because it
significantly diminishes the energetic cost associated with circulation
of a highly viscous, corpuscular blood fluid (4-7). Indeed,
hematocrit, mean cellular hemoglobin concentration, and hemoglobin
chain multiplicity all decrease with increasing phylogenetic divergence
among the red-blooded Antarctic notothenioid fishes (8), and the
Bathydraconidae (the sister group to the channichthyids) approach the
hematological extreme displayed by the white-blooded icefishes.
Furthermore, di Prisco et al. (9) have shown that a
red-blooded nototheniid fish, Pagothenia bernacchii, survives under resting conditions when its hemoglobin is converted to
the carbon monoxide form in vivo. Apparently, red-blooded
Antarctic fishes can sustain their basal metabolism using
plasma-dissolved oxygen, and they draw on their hemoglobin stores
primarily when respiratory demand increases. Nevertheless, the
development in icefishes of compensatory physiological and circulatory
adaptations that reduce tissue oxygen demand and enhance oxygen
delivery (e.g. modest suppression of metabolic rates,
enhanced gas exchange by large, well-perfused gills and through a
scaleless skin, and large increases in cardiac output and blood volume)
argues that loss of hemoglobin and erythrocytes was maladaptive under
conditions of physiological stress. Thus, the most plausible
evolutionary scenario is that the phylogenetic trend to reduced
hematocrits and decreased hemoglobin synthesis in notothenioid fishes
developed concurrently with enhancements to their respiratory and
circulatory systems, leading ultimately to the acorpuscular,
hemoglobinless condition of the icefishes.
The channichthyids diverged from other Antarctic notothenioids
approximately 7-15 million years ago, but radiation of species within
the icefish clade (i.e. lineage branch) appears to have been
confined to the last one million years (10). Recently, we demonstrated
that icefish species belonging to both primitive and advanced genera
retain in their genomes inactive remnants of the major adult
notothenioid -globin gene but have lost the gene that encodes adult
-globin (11). Thus, the hemoglobinless phenotype appears to be a
primitive channichthyid character that was established by deletion or
rapid mutation of the gene encoding
-globin before diversification
of the clade. Our present objective is to determine the evolutionary
fate of the channichthyid
1-globin gene. In this report we describe
the structures of the functional
1-globin gene of the red-blooded
Antarctic rockcod Notothenia coriiceps (family
Nototheniidae) and of the
-globin gene remnants of two icefishes,
Chaenocephalus aceratus and Chionodraco
rastrospinosus. To our surprise, we find that the icefish
-globin gene is a truncated version of the
1-globin gene of
red-blooded notothenioids. These remnants, which contain the 3' portion
of intron 2, all of exon 3, and the 3'-untranslated region of the
1-globin gene, appear to be mutating randomly. Using transversion
substitutions, we estimate that these nonfunctional nuclear gene
fragments are diverging at the rate of 0.12-0.33%/million years.
Because the notothenioid adult globin genes are tightly linked in 5' to
5' orientation,1 we now
propose that the hemoglobinless phenotype was established by a single
deletional event in the ancestral channichthyid that eliminated the
entire
-globin gene and the 5' half of the
1-globin gene.
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EXPERIMENTAL PROCEDURES |
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Collection of Animals and Storage of Tissues--
Specimens of
N. coriiceps, C. aceratus, and C. rastrospinosus were collected by bottom-trawling from the
R/V Hero or from the R/V Polar Duke south
of Low Island (Antarctic Treaty Protected Area System Marine Site of
Special Scientific Interest (MSSSI) 35, Western Bransfield Strait) or
west of Brabant Island (MSSSI 36, East Dallmann Bay). Fishes were
transported alive to Palmer Station, Antarctica, where they were
maintained in seawater aquaria at 1 to +1 °C. Blood from mature
adults was collected into heparinized syringes by caudal venipuncture
of unanesthetized specimens, and erythrocytes, isolated by differential
centrifugation (12), were either used immediately or frozen and stored
at
70 °C. Testes were dissected immediately after sacrifice of
mature males, frozen in liquid nitrogen, and maintained at
70 °C
until use.
Construction and Screening of Genomic Libraries--
Genomic DNA
of high molecular weight (>50 kilobase pairs) was purified from a
single testis of each fish species by the method of Blin and Stafford
(13). The N. coriiceps library was constructed in the vector Charon 35 (14). DNA was digested partially with MboI,
and fragments of 15-20 kilobase pairs, obtained by sucrose gradient
centrifugation, were ligated to the BamHI sites of the vector arms. Recombinant phage DNA was packaged in vitro
(Packagene; Promega), and phage were amplified in Escherichia
coli K803
to yield a stock at 8 × 107 plaque-forming units/ml. The C. aceratus
library was constructed in LambdaGEM-11 (Promega). DNA was digested
partially with MboI, fragments were ligated to phage arms
containing XhoI half-sites, and recombinant phage DNA was
packaged in vitro as described above. The latter library was
propagated in E. coli KW251 to give an amplified stock with
a titer of 2.4 × 107 plaque-forming units/ml.
Subcloning and DNA Sequencing--
Tertiary isolates were grown
and DNA was extracted by the plate-lysis method (16). Genomic DNA
inserts were excised from the recombinant phage by digestion with
appropriate restriction endonucleases, the DNA digests were
electrophoresed on 1% (w/v) agarose gels in 1× TBE (0.089 M Tris borate, 2 mM EDTA, pH 8.0), and DNA
fragments containing -globin gene sequences were identified by
transfer to nylon membranes (17) followed by hybridization of the gel
replicas to 32P-labeled NcHb
1-1 (see "Construction
and Screening of Genomic Libraries"). Positive fragments were excised
from duplicate gels, DNA was recovered from low-melt agarose or by
SPIN-X column (Costar Corp.) centrifugation, and the fragments were
ligated into the multicloning sites of pUC19 or pBluescript
(Stratagene). Recombinant plasmids were transformed into competent
E. coli XL-1 Blue cells, the cells were grown on
LB/ampicillin plates containing
isopropyl-1-thio-
-D-galactopyranoside and
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside, and
white colonies were picked for further analysis. Plasmid DNA was
purified by alkaline-lysis plasmid mini-prep (16).
DNA Sequence Analysis--
Genomic -globin sequences were
aligned (k-tuple = 3, gap penalty = 3, largest gap = 20)
by use of the algorithm of Wilbur and Lipman (19), and sequence
similarities were calculated by the method of Dayhoff (20), as
implemented by DNASTAR Align.
GenBank Accession Numbers--
The Antarctic notothenioid
-globin gene sequences reported in this paper have been deposited in
the GenBank data base under the following accession numbers: N. coriiceps, AF049916; C. aceratus, AF049914; C. rastrospinosus, AF049915. These sequences have been scanned
against the GenBank data base, and representative
-globin genes from
other vertebrates are reported in Table I.
Erythrocyte RNA Isolation-- Total RNA from erythrocytes of adult N. coriiceps was purified from red blood cells by a modification (21) of the acid guanidinium isothiocyanate/phenol/chloroform method (22).
Mapping of 1-Globin Transcription Start Sites by Primer
Extension--
Three antisense oligonucleotide primers derived from
the 5' end of the N. coriiceps
1-globin gene (Fig. 2)
were used to map potential transcription start sites. Primers were
labeled at their 5' termini by reaction with [
-32P]ATP
and T4 polynucleotide kinase (23). Labeled primers were annealed to
total RNA (30 µg/reaction) from erythrocytes of N. coriiceps, the extension reactions were performed for 60 min using 25 units/µl avian myeloblastosis virus reverse transcriptase and 10 mM concentrations of the four dNTPs, and single-stranded
RNA was degraded by treatment with RNase A. The products of each primer extension reaction were co-electrophoresed on a 9% polyacrylamide,7 M urea DNA sequencing gel with sequencing ladders derived
from the same primer.
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RESULTS |
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Structure of the 1-Globin Gene of N. coriiceps--
To
establish a framework for genetic comparison, we isolated and
characterized the adult
1-globin gene from the hemoglobin-expressing notothenioid N. coriiceps. After restriction digestion with
EcoRI, three independent N. coriiceps genomic
phage clones each gave a 5.2-kilobase pair
1-globin-positive DNA
fragment. This fragment was subcloned into pUC19 to yield the clone
NcHbG
1. Fig. 1 presents the structural
diagram (panel A) of and the strategy employed to sequence
(panel B) the
-globin gene of NcHbG
1. Fig.
2 shows its nucleotide sequence and
translation. The coding sequence and 5'- and 3'-untranslated regions of
the NcHbG
1
-globin gene exactly match the corresponding regions
of the N. coriiceps
1-globin cDNA (11), thus
demonstrating that this gene encodes the major adult
-globin
expressed by nototheniid fishes. The
432-bp2 coding sequence is
interrupted by two introns, the first (I1) splitting codon 32 and the
second (I2) separating codons 101 and 102. These positions are
conserved within a few nucleotides among all vertebrate
-globin
genes studied to date (24). Both introns are substantially longer than
their counterparts in the adult
-globin genes of other fishes,
amphibians, or mammals (24-27) and are extremely rich in A + T
residues. Intron 1 is 450 bp in length and 70% A + T, whereas intron 2 is 278 bp long and 64% A + T. Intron 1 also contains a 24-bp tandem
repeat of the dinucleotide TA. The splice junctions of both introns
conform to the GT/AG rule, containing GT at their 5' donor junctions
and CAG preceded by pyrimidine-rich tracts at their 3' acceptor sites
(28-30). The 5'-upstream and 3'-downstream sequences of the
1-globin gene are also A + T-rich (58 and 61%, respectively), but
the coding sequence is A + T-poor (44% A+T). Thus, the adult
-globin gene of N. coriiceps shares the intron/exon
organization of other vertebrate
-globin genes, but its content of A + T nucleotides in noncoding regions (introns, 5'-upstream and
3'-downstream sequences) is high (see "Discussion").
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-Globin Gene Remnants from Two Hemoglobinless Antarctic
Icefishes--
In contrast to the 2-3
-globin-hybridizing DNA
fragments observed in the genomes of red-blooded notothenioid fishes,
the genomes of three icefishes, C. aceratus, C. rastrospinosus, and Champsocephalus gunnari, contain
single DNA fragments that hybridize at reduced intensity to a
notothenioid adult
1-globin cDNA probe (11). One plausible
interpretation of this observation is that the icefishes have lost,
probably by deletion, a portion of the ancestral notothenioid adult
-globin gene. To investigate this possibility, we isolated and
sequenced two independent
1-globin-positive genomic clones from
C. aceratus and one clone from C. rastrospinosus. Fig. 3 compares the structure of the
icefish
-globin fragments to that of the N. coriiceps
1-globin gene. The
1-globin genes of the two icefishes contain a
portion of intron 2, the entirety of exon 3, and all of the
3'-untranslated region. The apparent 5' chromosomal breakpoint within
intron 2 (between positions 931 and 932; see also Fig.
4) is identical in the two icefish genes, and the 5'-flanking sequences preceding the breakpoint bear no relation
to any portion of the N. coriiceps
1-globin gene. Beyond the second polyadenylation signal, the icefish and rockcod genes share
sequence similarity for at least 180 bp (Fig. 4). These observations
are consistent with deletional loss of 5'-upstream
1-globin
sequences (5'-untranslated region, exons 1 and 2, intron 1 and part of
intron 2) before divergence of these two relatively advanced icefish
species. Determination of the status of the
1-globin gene in the
ancestral channichthyid will require analysis of more primitive icefish
species.
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Calibration of the Mutational Clock for Notothenioid Nuclear Genes
in the Absence of Selective Pressure--
The results presented above
strongly suggest that failure of the icefishes to synthesize
1-globin is due to deletional loss of the 5' end of the notothenioid
1-globin gene. The residual icefish
1-globin gene, no longer
under positive selection pressure, subsequently experienced random
mutational drift. Using the mitochondrial DNA-based age of radiation of
the notothenioid fishes, 7-15 million years ago (10), we can estimate
the rate of icefish nuclear gene divergence from the frequency of
transversion substitutions in the
1-globin gene remnant (35, 36).
C. aceratus and the more advanced C. rastrospinosus have accumulated 11 and 14 transversions, respectively, in this fragment. Therefore, the estimated rate of
icefish nuclear gene divergence in the absence of selective pressure
falls in the range 0.12-0.33%/million years. This slow rate of
nuclear gene divergence is consistent with the low specific metabolic
rates of the cold-adapted notothenioids (4, 36).
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DISCUSSION |
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In this report we have demonstrated that the -globin genes of
two icefishes are partial 5'-truncated variants of the
1-globin gene
of red-blooded notothenioid fishes. Furthermore, the residual icefish
gene fragments have accumulated deletions, insertions, and nucleotide
substitutions with respect to the functional globin gene. The striking
similarity of the icefish remnants to each other, both in the apparent
chromosomal breakpoint and in other mutations, strongly suggests that
most of the changes evolved in the ancestral channichthyid
approximately 7-15 million years ago. The relatively minor differences
between the gene remnants of C. aceratus and C. rastrospinosus probably evolved by mutational drift during the
radiation of the icefishes over the past one million years. The
apparent random mutation of the icefish
1 remnant should provide a
useful tool for development of a molecular phylogeny of icefishes based
on nuclear gene divergence.
Structure of a Functional Notothenioid -Globin Gene--
The
structure of the functional
1-globin gene of N. coriiceps
is remarkably similar to the
-globin genes of other vertebrates. Intron positions have been maintained, and splice junctions conform to
the GT/AG rule (28-30). Thus, the basic splicing mechanism is likely
to have been conserved in the cold-adapted Antarctic fishes. The
possibility that compensatory adaptations are required to permit
efficient splicing in their cold thermal regime remains open.
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Globin Gene Organization and Expression in Fishes--
In contrast
to the distinct - and
-globin gene clusters of higher
vertebrates, the adult
- and
-globin genes of N. coriiceps, its temperate relative, N. angustata (the
New Zealand black cod), and presumably other red-blooded notothenioids
are tightly linked in 5' to 5' orientation.1 Head-to-head
linkage of
/
gene pairs has also been observed in the Atlantic
salmon (44), carp (45), and the zebrafish (27). Thus, this gene
organization probably represents the ancestral condition of gnathostome
fishes and is likely to have arisen by duplication, inversion, and
divergence of the primordial globin gene of primitive jawless fishes
(e.g. the lamprey) to give an
/
gene pair (27). One
plausible advantage of head-to-head linkage is coordinate regulation of
globin gene transcription mediated by shared promoter and/or enhancer
elements located in the intergenic sequences (27).
Mechanism of Globin Gene Loss in Antarctic Icefishes--
With the
demonstration that icefishes have undergone deletion of the adult
-globin gene (11) and 5' truncation of the major adult
-globin
gene (this work), we can now propose a simple mechanism for globin gene
loss. Fig. 5 shows that a single
deletional event (scenario X) in the ancestral channichthyid, with
chromosomal breakpoints located within intron 2 of the
1-globin gene
and downstream of the 3'-untranslated region of the
-globin gene, would abrogate expression of adult globin polypeptides. This mechanism is supported strongly by the observation that the residual globin loci
of C. aceratus and C. rastrospinosus share common
5' breakpoints with respect to the N. coriiceps
1-globin
gene. Multiple deletions (Y1, Y2) occurring before diversification of
the icefish clade that together yield the disrupted icefish globin
locus are a formal but less likely possibility. The evolutionary fate
of embryonic and juvenile globin genes in icefish genomes is unknown,
but the linkage of embryonic and adult globin genes in the zebrafish
(27) raises the possibility that the single deletional event postulated here (Fig. 5, scenario Z), or perhaps multiple events, may have removed
almost the entire notothenioid globin gene complex.
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Divergence of the Icefish -Globin Gene Remnants and the
Mutational Clock--
Martin and Palumbi (36), summarizing nucleotide
divergence rates among diverse taxonomic groups, have suggested that
specific metabolic rate is the major parameter controlling the
mutational clock. The rate of mutational change appears to be mediated
by reactive oxygen species, generated metabolically, that can damage DNA either directly or indirectly. The data presented here indicate that rates of nuclear gene divergence in notothenioid fishes
(0.12-0.33%/million years) in the absence of selective pressure are
among the smallest observed in poikilotherms, in agreement with the
their low specific metabolic rates (4). Thus, the "nucleotide
generation time" (average interval for a nucleotide to be copied
through replication or repair) of the nuclear genes of Antarctic
teleosts is likely to be long (36). The low mutational rates that we
have estimated for the
-globin gene remnants of C. aceratus and C. rastrospinosus should be verified by
analysis of these fragments in other icefish species and by examination
of additional gene families in other notothenioid fishes. Nevertheless,
our results suggest that the chronology of evolution of antifreeze
glycoprotein genes from the notothenioid trypsinogen gene, estimated at
5-14 million years ago (47) based on mitochondrial divergence rates
(0.5-0.9%/million years) of the salmon (36), may require
reappraisal.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the excellent logistic support provided to our field research team at Palmer Station and on board the R/V Polar Duke by the personnel of Antarctic Support Associates, by the captains and crews of the R/V Polar Duke, and by the staff of the Office of Polar Programs of the National Science Foundation. We also acknowledge Patricia Singer (University of Maine DNA Sequencing Facility) for her excellent technical assistance in automated DNA sequencing.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grants OPP-9120311 and OPP-9420712 (to H. W. D.) and by the Italian National Programme for Antarctic Research (to E. C., L. C., and G. d. P).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF049916, AF049914, and AF049915.
§ These individuals contributed equally to the work described here and should be considered joint first authors.
To whom correspondence should be addressed: Dept. of Biology,
Northeastern University, 414 Mugar Hall, 360 Huntington Ave., Boston, MA 02115. Tel: 617-373-4495; Fax: 617-373-3724; E-mail: iceman{at}neu.edu.
1
A. Saeed, D. Lau, and H. W. Detrich III,
manuscript in preparation. Linkage of - and
-globin genes on the
same chromosome is common in fishes and amphibians. In higher
vertebrates (e.g. birds and mammals), the
- and
-globins are encoded by distinct gene clusters on separate
chromosomes (see "Discussion").
2 The abbreviation used is: bp, base pairs.
3 S. K. Parker and H. W. Detrich III, unpublished results.
4 E. Cocca, L. Camardella, H. W. Detrich III, and G. di Prisco, unpublished results.
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REFERENCES |
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