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Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters
The paper “Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters” by Wetten et al. [1] presents data on Atlantic cod globin genes, the expression pattern of some of these genes (i.e., of hemoglobin genes), and discuss the genomic context of globin genes.
A closer inspection of this paper reveals that a significant amount of data is presented in a way that makes the distinction between new data, and already published data, extremely difficult for most readers.
To substantiate this observation this comment will be focused on:
1. The fact that the 9 Atlantic cod hemoglobin (Hb) genes, reported and discussed by Wetten et al. [1], have been previously characterized. Wetten et al.’s [1] new data is restricted to the flanking regions and the finding that these genes are organized in two clusters (though Hb genes are organized in two clusters in most vertebrates).
2. The fact that the expression of Hb genes in adult fish, reported and discussed by Wetten et al. [1], represents data already published. Oddly, Wetten et al. [1] fail to acknowledge this though they indicate that for their qPCR analyses they used the primers designed by the group who published this data. Wetten et al.’s [1] qPCR new data is restricted to the expression pattern during development.
1. In the introduction (Background) section the reader learns that: “A variable number of cod hemoglobin genes and allelic variants have been reported in Norwegian, Icelandic and Canadian populations [27,28,29]. Here, we screened the draft cod genome [30] and identified nine α- and β-globin genes, which are organized in two unlinked clusters flanked by highly conserved syntenic regions.”
and in the discussion section that: “The Atlantic cod genome was shown to harbor altogether nine α- and β-globin genes organized in two unlinked clusters similar to the other teleost genomes available. The expression of many hemoglobin genes in adult cod is consistent with the multiple tetrameric hemoglobin types and subtypes identified by gel electrophoresis of blood proteins [33,34]. The cod hemoglobin repertoire is further extended by the polymorphic α1, β1, β3 and β4 globins [27,29] of which the functionally different variants of β1 are differentially distributed in cod populations [27,35,36].”
An unadvised reader might think that at least some of these hemoglobin (Hb) genes in Atlantic cod have been identified by Wetten et al. [1]. However, all these genes have been identified prior to this study by the efforts of different groups [2-6] and the genomic sequence data for all these nine Hb genes has been reported in 2009 [2].
In the results section Wetten et al. [1] indicates that “The presented sequence information therefore represents the north-east Arctic population of Atlantic cod.” Se same information is provided in “Figure 2. Sequence alignment of the Atlantic cod α-β globins, myoglobin, neuroglobin globin-X and cytoglobin 2. The sequences are based on the draft genome of the northeast Arctic population of Atlantic cod. Human β-globin is included for comparison. The alignment was optimized by omitting the N-and/or C-terminal sequences of the non-hemoglobins, and numbers refer to the residues presented. The consensus sequence shows residues with >80 % identity. Putative residues required for Root effect are boxed. GenBank accession numbers: α1 (ACJ66341), α2 (ACJ66342), α3 (ACV69832), α4 (ACV69835), β1 (ACV69840), β2 (ACJ66344), β3 (ACJ66345), β4 (ACJ66346), β5 (ACV69854).”
However, none of the GenBank listed here are novel or come from the draft genome. GenBank accession numbers for Hb genes α1, α2, β2- β4 are related to Andersen et al. [4] and correspond to mRNA data rather than genomic data while GenBank accession numbers for Hb genes α3, α4, β1, β5 are from Borza et al. [2] which analysed cod from West Atlantic populations.
Also, it seems that there are no GenBank accession numbers listed for myoglobin, neuroglobin, globin-X and cytoglobin 2 or for the two hemoglobin clusters and the flanking regions reported in this paper [1].
The feeling that the information about the sequence and structure of Hb genes represents new data is reinforced in the results section where Wetten et al. [1] talks about genomic data related to cod MC locus “The hemoglobin genes show the characteristic structure of three exons and two introns encoding the predicted α- and β-globins of 143 and 147 amino acids (aa), respectively (Fig. 2).” and cod LA locus “The second cod globin cluster was shown to contain five hemoglobin genes in the order β3-β4-α2-α3-β2 positioned within a region of about 12 kb in a scaffold spanning 381 kb (Fig. 3). The three exons encode the 147-aa long β-globins, while the predicted α3 contains only 141 aa compared to the other α-globins of 143 aa (Fig. 2).”
Again, the reader might think that the information related to the structure (length, exons & introns) of these Hb genes represents a novelty. However, similar information can be found in [2], page 3, where among other things one can read among that: “Atlantic cod Hb genes show an archetypal structure for Hbs, having three exons and two introns. All introns display a standard GT-AC structure at the exon-intron junction and their position in the coding regions is rather similar; however, the length of introns varies from gene to gene. Exceptionally long introns were found in the α3 Hb gene (568 nt and 794 nt for intron 1 and 2, respectively) while intron 2 of the β1 Hb gene contains a GT module repeated up to 60 times.” and “...α3 Hb gene ... has other peculiar features: it encodes a polypeptide that is only 141 amino acids long while most α Hb genes in fish and other vertebrates encode products consisting of 143 amino acids”
Extensive genomic data on Hb gene α2 and β3 was also reported by other groups [5, 6].
2. In the results section (subsection Globin gene mapping and expression) Wetten et al. [1] indicate that: “The nine α-β globin genes were shown to be transcriptionally active by quantifying the mRNA levels throughout the life cycle of Atlantic cod using real-time qPCR (Fig. 4).“ and “Abundant expression of α1, α2, β1 and β2 was measured in the adult fish, while the other genes showed low mRNA levels (Fig. 5).” And in the discussion section that: “The dominant expression of α1, α2, β1 and β2 in adult fish is in agreement with the isolation of three major tetramers designated Hb1, Hb2 and Hb3, which comprise different combinations of these four subunits [37].” In the methods section it is specified that: “Primers used for real-time qPCR were adopted from Borza et al. [29] for the globins, while ubiquitin primers were taken from Olsvik et al. [61] (Table 2)”.
However, in spite of the fact that Wetten et al. [4] indicate the origin of the primers used for their study they fail to acknowledge in the results or discussion section that qPCR was previously performed on all Hb genes using adult fish samples. Information such as “The diverged cod hemoglobin genes displayed different expression levels in adult fish” (abstract) or “The dominant expression of α1, α2, β1 and β2 in adult fish is in agreement ... (the discussion section)” gives the impression that it represents new data. However, if we put side by side table 2 from Wetten et al. [4] and additional file 4 from Borza et al. [2], and compare the results reported in figure 5 by Wetten et al. [4] to those described in figure 1 by Borza et al. [2], the emerging picture is that Wetten et al. [4] just replicated the experiment reported by Borza et al. [2].
Considerable information regarding the expression of these genes can be found just by checking the abstract of Borza et al.’s paper [2]: “RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly” (Abstract, results) and “This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes” (Abstract, conclusion).
1. Wetten OF, Nederbragt AJ, Wilson RC, Jacobsen KS, Edvardsen RB, Andersen O: Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters. BMC Evolutionary Biology 2010, 10:315. 2. Borza T, Stone C, Gamperl AK, Bowman S: Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism. BMC Genet 2009, 10:51. 3. Verde C, Balestrieri M, de Pascale D, Pagnozzi D, Lecointre G, di Prisco G: The oxygen transport system in three species of the boreal fish family Gadidae. Molecular phylogeny of hemoglobin. J Biol Chem 2006, 281(31):22073-22084. 4. Andersen O, Wetten OF, De Rosa MC, Andre C, Carelli Alinovi C, Colafranceschi M, Brix O, Colosimo A: Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations. P Roy Soc B - Biol Sci 2009, 276(1658):833-841. 5. Halldorsdottir K, Arnason E: Organization of a beta and alpha globin gene set in the teleost Atlantic cod, Gadus morhua. Biochem Genet 2009, 47(11-12):817-830. 6. Halldorsdottir K, Arnason E: Multiple linked beta and alpha globin genes in Atlantic cod: a PCR based strategy of genomic exploration. Marine Genomics 2009, 2:169-181.
Difficult to distinguish between new and old data
27 October 2010
The paper “Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters” by Wetten et al. [1] presents data on Atlantic cod globin genes, the expression pattern of some of these genes (i.e., of hemoglobin genes), and discuss the genomic context of globin genes.
A closer inspection of this paper reveals that a significant amount of data is presented in a way that makes the distinction between new data, and already published data, extremely difficult for most readers.
To substantiate this observation this comment will be focused on:
1. The fact that the 9 Atlantic cod hemoglobin (Hb) genes, reported and discussed by Wetten et al. [1], have been previously characterized. Wetten et al.’s [1] new data is restricted to the flanking regions and the finding that these genes are organized in two clusters (though Hb genes are organized in two clusters in most vertebrates).
2. The fact that the expression of Hb genes in adult fish, reported and discussed by Wetten et al. [1], represents data already published. Oddly, Wetten et al. [1] fail to acknowledge this though they indicate that for their qPCR analyses they used the primers designed by the group who published this data. Wetten et al.’s [1] qPCR new data is restricted to the expression pattern during development.
1. In the introduction (Background) section the reader learns that: “A variable number of cod hemoglobin genes and allelic variants have been reported in Norwegian, Icelandic and Canadian populations [27,28,29]. Here, we screened the draft cod genome [30] and identified nine α- and β-globin genes, which are organized in two unlinked clusters flanked by highly conserved syntenic regions.”
and in the discussion section that:
“The Atlantic cod genome was shown to harbor altogether nine α- and β-globin genes organized in two unlinked clusters similar to the other teleost genomes available. The expression of many hemoglobin genes in adult cod is consistent with the multiple tetrameric hemoglobin types and subtypes identified by gel electrophoresis of blood proteins [33,34]. The cod hemoglobin repertoire is further extended by the polymorphic α1, β1, β3 and β4 globins [27,29] of which the functionally different variants of β1 are differentially distributed in cod populations [27,35,36].”
An unadvised reader might think that at least some of these hemoglobin (Hb) genes in Atlantic cod have been identified by Wetten et al. [1]. However, all these genes have been identified prior to this study by the efforts of different groups [2-6] and the genomic sequence data for all these nine Hb genes has been reported in 2009 [2].
In the results section Wetten et al. [1] indicates that “The presented sequence information therefore represents the north-east Arctic population of Atlantic cod.” Se same information is provided in “Figure 2. Sequence alignment of the Atlantic cod α-β globins, myoglobin, neuroglobin globin-X and cytoglobin 2. The sequences are based on the draft genome of the northeast Arctic population of Atlantic cod. Human β-globin is included for comparison. The alignment was optimized by omitting the N-and/or C-terminal sequences of the non-hemoglobins, and numbers refer to the residues presented. The consensus sequence shows residues with >80 % identity. Putative residues required for Root effect are boxed. GenBank accession numbers: α1 (ACJ66341), α2 (ACJ66342), α3 (ACV69832), α4 (ACV69835), β1 (ACV69840), β2 (ACJ66344), β3 (ACJ66345), β4 (ACJ66346), β5 (ACV69854).”
However, none of the GenBank listed here are novel or come from the draft genome. GenBank accession numbers for Hb genes α1, α2, β2- β4 are related to Andersen et al. [4] and correspond to mRNA data rather than genomic data while GenBank accession numbers for Hb genes α3, α4, β1, β5 are from Borza et al. [2] which analysed cod from West Atlantic populations.
Also, it seems that there are no GenBank accession numbers listed for myoglobin, neuroglobin, globin-X and cytoglobin 2 or for the two hemoglobin clusters and the flanking regions reported in this paper [1].
The feeling that the information about the sequence and structure of Hb genes represents new data is reinforced in the results section where Wetten et al. [1] talks about genomic data related to cod MC locus “The hemoglobin genes show the characteristic structure of three exons and two introns encoding the predicted α- and β-globins of 143 and 147 amino acids (aa), respectively (Fig. 2).” and cod LA locus “The second cod globin cluster was shown to contain five hemoglobin genes in the order β3-β4-α2-α3-β2 positioned within a region of about 12 kb in a scaffold spanning 381 kb (Fig. 3). The three exons encode the 147-aa long β-globins, while the predicted α3 contains only 141 aa compared to the other α-globins of 143 aa (Fig. 2).”
Again, the reader might think that the information related to the structure (length, exons & introns) of these Hb genes represents a novelty. However, similar information can be found in [2], page 3, where among other things one can read among that:
“Atlantic cod Hb genes show an archetypal structure for Hbs, having three exons and two introns. All introns display a standard GT-AC structure at the exon-intron junction and their position in the coding regions is rather similar; however, the length of introns varies from gene to gene. Exceptionally long introns were found in the α3 Hb gene (568 nt and 794 nt for intron 1 and 2, respectively) while intron 2 of the β1 Hb gene contains a GT module repeated up to 60 times.”
and
“...α3 Hb gene ... has other peculiar features: it encodes a polypeptide that is only 141 amino acids long while most α Hb genes in fish and other vertebrates encode products consisting of 143 amino acids”
Extensive genomic data on Hb gene α2 and β3 was also reported by other groups [5, 6].
2. In the results section (subsection Globin gene mapping and expression) Wetten et al. [1] indicate that: “The nine α-β globin genes were shown to be transcriptionally active by quantifying the mRNA levels throughout the life cycle of Atlantic cod using real-time qPCR (Fig. 4).“ and “Abundant expression of α1, α2, β1 and β2 was measured in the adult fish, while the other genes showed low mRNA levels (Fig. 5).”
And in the discussion section that:
“The dominant expression of α1, α2, β1 and β2 in adult fish is in agreement with the isolation of three major tetramers designated Hb1, Hb2 and Hb3, which comprise different combinations of these four subunits [37].”
In the methods section it is specified that: “Primers used for real-time qPCR were adopted from Borza et al. [29] for the globins, while ubiquitin primers were taken from Olsvik et al. [61] (Table 2)”.
However, in spite of the fact that Wetten et al. [4] indicate the origin of the primers used for their study they fail to acknowledge in the results or discussion section that qPCR was previously performed on all Hb genes using adult fish samples. Information such as “The diverged cod hemoglobin genes displayed different expression levels in adult fish” (abstract) or “The dominant expression of α1, α2, β1 and β2 in adult fish is in agreement ... (the discussion section)” gives the impression that it represents new data.
However, if we put side by side table 2 from Wetten et al. [4] and additional file 4 from Borza et al. [2], and compare the results reported in figure 5 by Wetten et al. [4] to those described in figure 1 by Borza et al. [2], the emerging picture is that Wetten et al. [4] just replicated the experiment reported by Borza et al. [2].
Considerable information regarding the expression of these genes can be found just by checking the abstract of Borza et al.’s paper [2]:
“RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly” (Abstract, results) and “This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes” (Abstract, conclusion).
1. Wetten OF, Nederbragt AJ, Wilson RC, Jacobsen KS, Edvardsen RB, Andersen O: Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters. BMC Evolutionary Biology 2010, 10:315.
2. Borza T, Stone C, Gamperl AK, Bowman S: Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism. BMC Genet 2009, 10:51.
3. Verde C, Balestrieri M, de Pascale D, Pagnozzi D, Lecointre G, di Prisco G: The oxygen transport system in three species of the boreal fish family Gadidae. Molecular phylogeny of hemoglobin. J Biol Chem 2006, 281(31):22073-22084.
4. Andersen O, Wetten OF, De Rosa MC, Andre C, Carelli Alinovi C, Colafranceschi M, Brix O, Colosimo A: Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations. P Roy Soc B - Biol Sci 2009, 276(1658):833-841.
5. Halldorsdottir K, Arnason E: Organization of a beta and alpha globin gene set in the teleost Atlantic cod, Gadus morhua. Biochem Genet 2009, 47(11-12):817-830.
6. Halldorsdottir K, Arnason E: Multiple linked beta and alpha globin genes in Atlantic cod: a PCR based strategy of genomic exploration. Marine Genomics 2009, 2:169-181.
Competing interests
None declared