*Institute of Zoology;
Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University
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Abstract |
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Introduction |
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Materials and Methods |
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Expression Analysis of Human CYGB
An RNA Master BlotTM (Clontech) containing normalized amounts of polyA+ RNA from 50 human tissues was hybridized to a 32P-labelled (Feinberg and Vogelstein 1983
) subcloned CYGB cDNA probe containing the complete coding sequence (accession number AJ315162). Washing was performed at 68°C in 0.1 x standard saline citrate (SSC) solution. Signals were quantified on a Fuji BAS-1800 phosphorimager. No nonspecific binding of the probe was observed to yeast total RNA and tRNA, E. coli rRNA, poly rA, and human Cot 1 repetitive DNA.
Sequence and Phylogenetic Analyses
Sequence analyses were carried out with the programs provided by the Software Package 9.0 from the Genetics Computer Group (GCG), Wisconsin, and the ExPASy web server (http://www.expasy.ch). The genomic organization of the human CYGB gene was depicted using the PIPMAKER program (http://nog.cse.psu.edu/pipmaker/; Schwartz et al. 2000
). Amino acid sequences of selected vertebrate globins were aligned with ClustalX (Thompson et al. 1997
) and corrected using published alignments (Burmester and Hankeln 1999
; Burmester et al. 2000
) and globin structural data. The software packages PHYLIP 3.6 (Felsenstein 2000
) and TREE-PUZZLE 5.0 (Strimmer and von Haeseler 1996
) were applied for phylogenetic inference. Gamma-corrected distances were calculated using the PAM250 model with eight rate categories (Dayhoff, Schwartz, and Orcutt 1978
). Tree constructions were performed using the neighbor-joining method. The reliability of the trees was tested by bootstrap analysis (Felsenstein 1985
) with 100 replications using PUZZLEBOOT (shell script by M. Holder and A. Roger). Synonymous and nonsynonymous nucleotide substitution rates were calculated using the method of Nei and Gojobori (1986)
.
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Results |
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Discussion |
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Protein Structure and Genomic Organization of CYGBs
Within the conserved globin fold, which covers the standard alpha helices A through H, the key residues important for the function of CYGB as a typical oxygen-binding protein are strictly conserved. The proximal and distal histidines in the positions E7 and F8 as well as the phenylalanine at the CD1 corner are present in the CYGBs (fig. 1
). The lengths of the mammalian (190 amino acids) and fish (174 amino acids) CYGBs exceed those of vertebrate myo- and hemoglobins. The length differences are exclusively because of N- and C-terminal protein extensions, which occasionally have been observed previously in invertebrate globins (e.g., in Caenorhabditis elegans; Neuwald et al. 1997
) but whose functional relevance is unclear. Whereas the C-terminal extension of CYGBs may be caused by the recruitment of an additional exon (see subsequent discussion), the N-terminal extension of murine and human CYGBs seems to have partly resulted from a direct duplication of 21 nucleotides (with 7/21 mismatches) at the 5' end of the coding region (data not shown).
The antiquity of introns within globin genes and their positional stability during evolution have been a matter of intense debate (e.g., Hankeln et al. 1997
; Logsdon, Stoltzfus, and Doolittle 1998
). The human CYGB gene displays the B12-2 and G7-0 introns which are typically found in many globins, including the vertebrate hemo-, myo- and neuroglobins, and which must therefore be considered phylogenetically ancient (Dixon and Pohajdak 1992
; Burmester et al. 2000
). However, the additional intron in the 3'-most region of the murine and human CYGB coding sequences (corresponding to the C-terminal position HC11-2) is unprecedented. The origin of the small exon 4 sequence, which only encodes the 10 mostC-terminal amino acid residues, is unclear. The C-terminus of zebrafish CYGB is shorter and lacks the additional exon 4 sequence (fig. 1 ). In the murine and human CYGBs, the HC11-2 intron occurs just downstream of the C-terminal end of other globin sequences, and we therefore consider that exon 4 might have been acquired only during the evolution of the tetrapod CYGBs.
Molecular Evolution of CYGBs
Mouse and human CYGB share 92.8% of the nucleotides and 95.3% of the amino acids in the coding region. The zebrafish CYGB shows 49% amino acid identity to the mammalian proteins. CYGBs display the highest degree of amino acid sequence similarity to the hemoglobins of the Agnatha (26% to 33% identity). Somewhat lower scores were observed when the CYGBs were compared with the myoglobins and hemoglobins (30% identity). Assuming that mice and humans diverged about 80 MYA (Kimura 1987
), an amino acid substitution rate of about 0.3 x 10-9 replacements per site per year was inferred for the mammalian CYGBs. This is much lower than calculated for the orthologous mammalian hemoglobins (0.9 x 10-9 to 1.2 x 10-9) and myoglobins (0.8 x 10-9 to 1.2 x 10-9) but lies in the range of the neuroglobins (0.4 x 10-9). These values are in agreement with our calculations of very low nonsynonymous nucleotide substitution rates in human and mouse cyto- and neuroglobin (dn = 0.02 and 0.03 nonsynonymous substitutions per site, respectively), compared with human and mouse hemoglobins (dn = 0.09 for
globin, 0.21 for ß globin) and myoglobin (dn = 0.09). In the case of CYGB, the low nonsynonymous substitution rate is correlated to an unusually low substitution rate at synonymous codon positions (ds = 0.28 synonymous substitutions per site), the reason for which is unclear. The ds:dn ratio (Nei and Gojobori 1986
) of >>1, however, clearly demonstrates that mammalian CYGBs evolve under strong purifying selection.
A Model of Globin Evolution in Vertebrates
Phylogenetic analyses suggest that the CYGBs share a common clade with the vertebrate myoglobins (fig. 4
). An independent confirmation of the common ancestry of cyto- and myoglobins may come from data suggesting that the chromosomal regions encompassing CYGB (17q25) and myoglobin (22q12) represent long, paralogous stretches of genomic DNA, which are thought to have originated by an ancient duplication event (A. McLysaght, K. Hokamp, and K. H. Wolfe, personal communication).
Taking into account the antiquity of the neuroglobins, the last common ancestor of all vertebrates most likely possessed two different types of globins (fig. 5
). Neuroglobin maintained its function in the nervous system, which it had acquired early in the evolution of the Bilateria (Burmester et al. 2000
). The other globin likely differentiated into a cellular globin, which later gave rise to the myoglobins and CYGBs on the one hand and to hemoglobin on the other. The hemoglobins obtained their function in the circulatory system of the gnathostomian vertebrates after their divergence from the lineage leading to the myoglobins and CYGBs, probably as early as 500 to 600 MYA (Goodman et al. 1987
). This event was probably correlated with an increase in body size and the evolution of an efficient circulatory system. Myoglobin and CYGB separated later, but before the divergence of the Chondrichthyes and the other gnathostomes more than 450 MYA (Benton 1990
, p. 44). It is conceivable that myoglobin, which is present in high concentrations in skeletal and smooth muscle (Wittenberg and Wittenberg 1989
; Qiu, Sutton, and Riggs 1998
), and which supplies the cells with high amounts of oxygen, is in fact an offspring of a more general tissue-globin of similar or other function.
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Acknowledgements |
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Footnotes |
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Abbreviations: CYGB, cytoglobin; RT-PCR, reverse transcriptionpolymerase chain reaction; SSC, standard saline citrate.
Keywords: globin
myoglobin
cytoglobin
evolution
gene duplication
Address for correspondence and reprints: Dr. Thomas Hankeln, Johannes Gutenberg University Mainz, Institut für Molekulargenetik, Becherweg 32, D-55099 Mainz, Germany. hankeln{at}molgen.biologie.uni-mainz.de
.
Correspondence and requests for material should be addressed to T.B. (burmeste{at}mail.uni-mainz.de
) or T.H. (hankeln{at}molgen.biologie.uni-mainz.de
)
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