Advertisement

The Major Adult α-Globin Gene of Antarctic Teleosts and Its Remnants in the Hemoglobinless Icefishes

CALIBRATION OF THE MUTATIONAL CLOCK FOR NUCLEAR GENES*
Open AccessPublished:June 12, 1998DOI:https://doi.org/10.1074/jbc.273.24.14745
      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 α-globin genes but have lost the gene that encodes adult β-globin. Here we demonstrate that loss of expression of the major adult α-globin, α1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus ) results from truncation of the 5′ end of the notothenioid α1-globin gene. The wild-type, functional α1-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 α1-globin gene. The residual, nonfunctional α-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 β-globin gene and the 5′ end of the linked α1-globin gene.
      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 (
      • Ruud J.T.
      ). Although icefish blood contains “erythrocyte-like” cells in small numbers (
      • Hureau J.C.
      • Petit D.
      • Fine J.M.
      • Marneux M.
      ,
      • Barber D.L.
      • Mills Westermann J.E.
      • White M.G.
      ), 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 (
      • Eastman J.T.
      Antarctic Fish Biology: Evolution in a Unique Environment.
      ,
      • D'Avino R.
      • Caruso C.
      • Camardella L.
      • Schininà M.E.
      • Rutigliano B.
      • Romano M.
      • Carratore V.
      • Barra D.
      • di Prisco G.
      ,
      • di Prisco G.
      • D'Avino R.
      • Caruso C.
      • Tamburini M.
      • Camardella L.
      • Rutigliano B.
      • Carratore V.
      • Romano M.
      ,
      • Macdonald J.A.
      • Montgomery J.C.
      • Wells R.M.G.
      ). Indeed, hematocrit, mean cellular hemoglobin concentration, and hemoglobin chain multiplicity all decrease with increasing phylogenetic divergence among the red-blooded Antarctic notothenioid fishes (
      • di Prisco G.
      ), and the Bathydraconidae (the sister group to the channichthyids) approach the hematological extreme displayed by the white-blooded icefishes. Furthermore, di Prisco et al. (
      • di Prisco G.
      • Macdonald J.A.
      • Brunori M.
      ) 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 (
      • Bargelloni L.
      • Ritchie P.A.
      • Patarnello T.
      • Battaglia B.
      • Lambert D.M.
      • Meyer A.
      ). Recently, we demonstrated that icefish species belonging to both primitive and advanced genera retain in their genomes inactive remnants of the major adult notothenioid α-globin gene but have lost the gene that encodes adult β-globin (
      • Cocca E.
      • Ratnayake-Lecamwasam M.
      • Parker S.K.
      • Camardella L.
      • Ciaramella M.
      • di Prisco G.
      • Detrich III, H.W.
      ). Thus, the hemoglobinless phenotype appears to be a primitive channichthyid character that was established by deletion or rapid mutation of the gene encoding β-globin before diversification of the clade. Our present objective is to determine the evolutionary fate of the channichthyid α1-globin gene. In this report we describe the structures of the functional α1-globin gene of the red-blooded Antarctic rockcod Notothenia coriiceps (family Nototheniidae) and of the α-globin gene remnants of two icefishes,Chaenocephalus aceratus and Chionodraco rastrospinosus . To our surprise, we find that the icefish α-globin gene is a truncated version of the α1-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 α1-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,
      A. Saeed, D. Lau, and H. W. Detrich III, manuscript in preparation. Linkage of α- and β-globin genes on the same chromosome is common in fishes and amphibians. In higher vertebrates (e.g. birds and mammals), the α- and β-globins are encoded by distinct gene clusters on separate chromosomes (see “Discussion”).
      1A. Saeed, D. Lau, and H. W. Detrich III, manuscript in preparation. Linkage of α- and β-globin genes on the same chromosome is common in fishes and amphibians. In higher vertebrates (e.g. birds and mammals), the α- and β-globins are encoded by distinct gene clusters on separate chromosomes (see “Discussion”).
      we now propose that the hemoglobinless phenotype was established by a single deletional event in the ancestral channichthyid that eliminated the entire β-globin gene and the 5′ half of the α1-globin gene.

      DISCUSSION

      In this report we have demonstrated that the α-globin genes of two icefishes are partial 5′-truncated variants of the α1-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 α1 remnant should provide a useful tool for development of a molecular phylogeny of icefishes based on nuclear gene divergence.

      Structure of a Functional Notothenioid α-Globin Gene

      The structure of the functional α1-globin gene of N. coriiceps is remarkably similar to the α-globin genes of other vertebrates. Intron positions have been maintained, and splice junctions conform to the GT/AG rule (
      • Breathnach R.
      • Chambon P.
      ,
      • Keller E.B.
      • Noon W.A.
      ,
      • Padgett R.A.
      • Grabowski P.J.
      • Konarska M.M.
      • Seiler S.
      • Sharp P.A.
      ). 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.
      Surprisingly, three potential transcription start sites were detected for the α1-globin gene. The two cap sites most proximal to the α1-coding sequence (CAP 1, CAP 2) are associated both with basal TATA and CCAAT boxes and with hematopoietic CACCC and GATA promoter elements. A single NF-E2 site precedes CAP 1. The third, most distal site (CAP 3) contains a putative TATA box but lacks the CACCC element. Like many other globin genes (
      • Liebhaber S.A.
      • Gootsens M.J.
      • Kan Y.W.
      ,
      • Myers R.M.
      • Tilly K.
      • Maniatis T.
      ), the sequences of the TATA boxes of the N. coriiceps promoters deviate from the vertebrate canon, TATAAA (
      • Bucher P.
      ). The use of alternative promoters and multiple transcription initiation sites to regulate gene expression in eukaryotes is now recognized to be a common phenomenon (
      • Ayoubi T.A.Y.
      • van de Ven W.J.M.
      ). For example, Hu et al. (
      • Hu Z.
      • Zhuang L.
      • Dufau M.L.
      ) have shown that tissue-specific expression of prolactin receptors in the rat is controlled by three promoters, two of which contain noncanonical TATA elements. We propose that the 5′-upstream sequence of the N. coriiceps α1-globin gene has evolved multiple promoters to offset slow rates of pol II transcription at psychrophilic temperatures by simultaneous recruitment of multiple transcriptional complexes. Indeed, the adult α1/β-globin intergenic region of N. coriiceps stimulates strongly, in either orientation, the transcription of a luciferase reporter construct in differentiated MEL cells.1
      Other departures of the notothenioid α-globin gene from the norm in higher vertebrates entail the length and nucleotide composition of the two introns. Both are longer and very rich in A + T residues. The 5′- and 3′-noncoding sequences of the gene are similarly A + T-rich. TableI presents a comparison of the nucleotide compositions of these subregions in α-globin genes from cold-living, temperate, and warm-bodied vertebrates. It is immediately evident that the noncoding regions of the cold-bodied and temperate vertebrates are significantly richer in A + T residues than are the corresponding regions in warm-bodied organisms. By contrast, the coding sequences, which are presumably constrained by functional requirements of the encoded globin polypeptides, are relatively A + T-poor and quite similar in composition in all taxa. The introns of several other notothenioid genes (myoglobin (
      • Small D.J.
      • Vayda M.E.
      • Sidell B.D.
      ), the dynein heavy chain (
      • King S.M.
      • Marchese-Ragona S.P.
      • Parker S.K.
      • Detrich III, H.W.
      ), and three α-tubulins
      S. K. Parker and H. W. Detrich III, unpublished results.
      are also rich in A + T residues (61–71%). What functional significance can we attribute to the biased nucleotide compositions of genes from cold- and warm-bodied vertebrates?
      Bernardi et al. (
      • Bernardi G.
      • Olofsson B.
      • Filipski J.
      • Zerial M.
      • Salinas J.
      • Cuny G.
      • Meunier-Rotival M.
      • Rodier F.
      ,
      • Bernardi G.
      • Bernardi Gi
      ,
      • Bernardi G.
      • Mouchiroud D.
      • Gautier C.
      • Bernardi Gi
      ) have demonstrated that the genomes of warm-bodied homeotherms (mammals, birds, etc.) are organized heterogeneously, with G + C- and gene-rich “isochores” interspersed with regions of lower G + C content. Among poikilotherms, warm-bodied (37–40 °C) Tilapia species also contain G + C-rich isochores, whereas their temperate congeners lack them (
      • Bernardi G.
      • Bernardi Gi
      ). High GC isochores may have evolved to maintain genome stability, to protect genes against DNA “breathing” and mutability (
      • Bernardi G.
      • Olofsson B.
      • Filipski J.
      • Zerial M.
      • Salinas J.
      • Cuny G.
      • Meunier-Rotival M.
      • Rodier F.
      ), and to enhance mRNA and protein stability (
      • Bernardi G.
      • Bernardi Gi
      ) at relatively high body temperatures. Conversely, the A + T-rich genomes of cold-living and temperate poikilotherms may facilitate DNA strand separation during transcription and replication in low temperature regimes (
      • Small D.J.
      • Vayda M.E.
      • Sidell B.D.
      ). At the extreme temperatures experienced by Antarctic fishes, the latter advantage may be particularly important.

      Globin Gene Organization and Expression in Fishes

      In contrast to the distinct α- and β-globin gene clusters of higher vertebrates, the adult α- and β-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 α/β gene pairs has also been observed in the Atlantic salmon (
      • McMorrow T.
      • Wagner A.
      • Deryckere F.
      • Gannon F.
      ), carp (
      • Miyata M.
      • Aoki T.
      ), and the zebrafish (
      • Chan F.-Y.
      • Robinson J.
      • Brownlie A.
      • Shivdasani R.A.
      • Donovan A.
      • Brugnara C.
      • Kim J.
      • Lau B.-C.
      • Witkowska E.
      • Zon L.I.
      ). 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 α/β gene pair (
      • Chan F.-Y.
      • Robinson J.
      • Brownlie A.
      • Shivdasani R.A.
      • Donovan A.
      • Brugnara C.
      • Kim J.
      • Lau B.-C.
      • Witkowska E.
      • Zon L.I.
      ). 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 (
      • Chan F.-Y.
      • Robinson J.
      • Brownlie A.
      • Shivdasani R.A.
      • Donovan A.
      • Brugnara C.
      • Kim J.
      • Lau B.-C.
      • Witkowska E.
      • Zon L.I.
      ).
      In higher vertebrates, expression of the distinct α- and β-globin gene complexes is regulated during development by locus control regions that govern the sequential, stage-specific expression of embryonic, fetal, and adult globin genes (
      • Orkin S.H.
      ). Although locus control regions have not yet been described for fish globin genes, the developmental switching of globin genes in the phylogenetically ancient lamprey suggests that such elements exist throughout the piscine taxon (
      • Lanfranchi G.
      • Pallavicini A.
      • Laveder P.
      • Valle G.
      ). In zebrafish, paired embryonic globin genes are linked to the adult α/β pairs (
      • Chan F.-Y.
      • Robinson J.
      • Brownlie A.
      • Shivdasani R.A.
      • Donovan A.
      • Brugnara C.
      • Kim J.
      • Lau B.-C.
      • Witkowska E.
      • Zon L.I.
      ). Recently, we isolated an N. coriiceps genomic clone that contains two non-adult α/β-globin gene pairs,
      E. Cocca, L. Camardella, H. W. Detrich III, and G. di Prisco, unpublished results.
      with each α/β pair linked 5′ to 5′. (We do not, as yet, know whether this presumptive embryonic/juvenile globin complex is contiguous to the adult α1/β gene pair.) The common organization of embryonic, juvenile, and adult globin genes in several teleost fishes suggests that a single, primordial locus control region may have evolved to regulate developmental switching of pairs of globin genes before the development of distinct α- and β-globin gene complexes, each with its own locus control region, in higher vertebrates.

      Mechanism of Globin Gene Loss in Antarctic Icefishes

      With the demonstration that icefishes have undergone deletion of the adult β-globin gene (
      • Cocca E.
      • Ratnayake-Lecamwasam M.
      • Parker S.K.
      • Camardella L.
      • Ciaramella M.
      • di Prisco G.
      • Detrich III, H.W.
      ) and 5′ truncation of the major adult α-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 α1-globin gene and downstream of the 3′-untranslated region of the β-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 α1-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 (
      • Chan F.-Y.
      • Robinson J.
      • Brownlie A.
      • Shivdasani R.A.
      • Donovan A.
      • Brugnara C.
      • Kim J.
      • Lau B.-C.
      • Witkowska E.
      • Zon L.I.
      ) 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.
      Figure thumbnail gr5
      Figure 5Possible mechanisms of globin gene deletion in the Antarctic icefishes. Mechanism 1, simultaneous deletion of the β-globin gene and the linked 5′-portion of the α1-globin gene (X ), followed by random mutation of the α1-globin gene remnant. Mechanism 2, independent deletion of the β-globin gene (Y1 ) and the 5′ portion of the linked α1-globin gene (Y2 ). If embryonic and juvenile α/β-globin gene pairs are linked in the notothenioid genome to the adult globin gene pair, then either model (e.g. single deletion scenario Z ) can be extended easily to encompass their probable loss in icefish genomes.

      Divergence of the Icefish α-Globin Gene Remnants and the Mutational Clock

      Martin and Palumbi (
      • Martin A.P.
      • Palumbi S.R.
      ), 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 (
      • Eastman J.T.
      Antarctic Fish Biology: Evolution in a Unique Environment.
      ). 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 (
      • Martin A.P.
      • Palumbi S.R.
      ). The low mutational rates that we have estimated for the α-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 (
      • Chen L.
      • DeVries A.L.
      • Cheng C.-H.C.
      ) based on mitochondrial divergence rates (0.5–0.9%/million years) of the salmon (
      • Martin A.P.
      • Palumbi S.R.
      ), may require reappraisal.

      Acknowledgments

      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 theR/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.

      REFERENCES

        • Ruud J.T.
        Nature. 1954; 173: 848-850
        • Hureau J.C.
        • Petit D.
        • Fine J.M.
        • Marneux M.
        Llano G.A. Adaptations Within Antarctic Ecosystems. Smithsonian Institution, Washington, D. C.1977: 459-477
        • Barber D.L.
        • Mills Westermann J.E.
        • White M.G.
        J. Fish Biol. 1981; 19: 11-28
        • Eastman J.T.
        Antarctic Fish Biology: Evolution in a Unique Environment.
        Academic Press, San Diego, CA1993
        • D'Avino R.
        • Caruso C.
        • Camardella L.
        • Schininà M.E.
        • Rutigliano B.
        • Romano M.
        • Carratore V.
        • Barra D.
        • di Prisco G.
        di Prisco G. Life Under Extreme Conditions. Biochemical Adaptation. Springer-Verlag, Berlin1991: 15-33
        • di Prisco G.
        • D'Avino R.
        • Caruso C.
        • Tamburini M.
        • Camardella L.
        • Rutigliano B.
        • Carratore V.
        • Romano M.
        di Prisco G. Maresca B. Tota B. Biology of Antarctic Fish. Springer-Verlag, Berlin1991: 263-281
        • Macdonald J.A.
        • Montgomery J.C.
        • Wells R.M.G.
        Adv. Mar. Biol. 1987; 24: 321-388
        • di Prisco G.
        di Prisco G. Pisano E. Clarke A. Fishes of Antarctica. A Biological Overview. Springer-Verlag, Berlin1998 (in press)
        • di Prisco G.
        • Macdonald J.A.
        • Brunori M.
        Experientia (Basel). 1992; 48: 473-475
        • Bargelloni L.
        • Ritchie P.A.
        • Patarnello T.
        • Battaglia B.
        • Lambert D.M.
        • Meyer A.
        Mol. Biol. Evol. 1994; 11: 854-863
        • Cocca E.
        • Ratnayake-Lecamwasam M.
        • Parker S.K.
        • Camardella L.
        • Ciaramella M.
        • di Prisco G.
        • Detrich III, H.W.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1817-1821
        • D'Avino R.
        • di Prisco G.
        Eur. J. Biochem. 1989; 179: 699-705
        • Blin N.
        • Stafford D.W.
        Nucleic Acids Res. 1976; 3: 2303-2308
        • Loenen W.A.M.
        • Blattner F.R.
        Gene. 1983; 26: 171-179
        • Feinberg A.P.
        • Vogelstein B.
        Anal. Biochem. 1983; 132: 6-13
        • Sambrook J.
        • Fritsch E.F.
        • Maniatis T.
        Molecular Cloning: A Laboratory Manual.
        Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989
        • Southern E.M.
        J. Mol. Biol. 1975; 98: 503-517
        • Sanger F.
        • Nicklen S.
        • Coulson A.R.
        Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467
        • Wilbur W.J.
        • Lipman D.J.
        Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 726-730
        • Dayhoff M.O.
        Atlas of Protein Sequence and Structure.
        National Biomedical Research Foundation, Washington, D. C.1979
        • Puissant C.
        • Houdebine L.-M.
        Biotechniques. 1990; 8: 148-149
        • Chomczynski P.
        • Sacchi N.
        Anal. Biochem. 1987; 162: 156-159
        • Triezenberg S.J.
        Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, New York1995: 4.8.1-4.8.5
        • Liebhaber S.A.
        • Gootsens M.J.
        • Kan Y.W.
        Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7054-7058
        • Patient R.K.
        • Elkington J.A.
        • Kay R.M.
        • Williams J.G.
        Cell. 1980; 20: 565-573
        • Miyata M.
        • Hirono I.
        • Aoki T.
        Bull. Jpn. Soc. Sci. Fish. 1993; 59: 1077-1083
        • Chan F.-Y.
        • Robinson J.
        • Brownlie A.
        • Shivdasani R.A.
        • Donovan A.
        • Brugnara C.
        • Kim J.
        • Lau B.-C.
        • Witkowska E.
        • Zon L.I.
        Blood. 1997; 89: 688-700
        • Breathnach R.
        • Chambon P.
        Annu. Rev. Biochem. 1981; 50: 349-383
        • Keller E.B.
        • Noon W.A.
        Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7417-7420
        • Padgett R.A.
        • Grabowski P.J.
        • Konarska M.M.
        • Seiler S.
        • Sharp P.A.
        Annu. Rev. Biochem. 1986; 55: 1119-1150
        • Myers R.M.
        • Tilly K.
        • Maniatis T.
        Science. 1986; 232: 613-618
        • Orkin S.H.
        Cell. 1990; 63: 665-672
        • Bucher P.
        J. Mol. Biol. 1990; 212: 563-578
        • Strauss E.C.
        • Orkin S.H.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5809-5813
        • Martin A.P.
        • Naylor G.J.P.
        • Palumbi S.R.
        Nature. 1992; 357: 153-155
        • Martin A.P.
        • Palumbi S.R.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4087-4091
        • Ayoubi T.A.Y.
        • van de Ven W.J.M.
        FASEB J. 1996; 10: 453-460
        • Hu Z.
        • Zhuang L.
        • Dufau M.L.
        J. Biol. Chem. 1996; 271: 10242-10246
        • Small D.J.
        • Vayda M.E.
        • Sidell B.D.
        J. Mol. Evol. 1998; (in press)
        • King S.M.
        • Marchese-Ragona S.P.
        • Parker S.K.
        • Detrich III, H.W.
        Biochemistry. 1997; 36: 1306-1314
        • Bernardi G.
        • Olofsson B.
        • Filipski J.
        • Zerial M.
        • Salinas J.
        • Cuny G.
        • Meunier-Rotival M.
        • Rodier F.
        Science. 1985; 228: 953-958
        • Bernardi G.
        • Bernardi Gi
        J. Mol. Evol. 1986; 24: 1-11
        • Bernardi G.
        • Mouchiroud D.
        • Gautier C.
        • Bernardi Gi
        J. Mol. Evol. 1988; 28: 7-18
        • McMorrow T.
        • Wagner A.
        • Deryckere F.
        • Gannon F.
        DNA Cell Biol. 1996; 15: 407-414
        • Miyata M.
        • Aoki T.
        Biochim. Biophys. Acta. 1997; 1354: 127-133
        • Lanfranchi G.
        • Pallavicini A.
        • Laveder P.
        • Valle G.
        Dev. Biol. 1994; 164: 402-408
        • Chen L.
        • DeVries A.L.
        • Cheng C.-H.C.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3811-3816
        • Kozak M.
        J. Cell Biol. 1991; 115: 887-903