Phosphoglucose Isomerases of Hagfish, Zebrafish, Gray Mullet, Toad, and Snake, with Reference to the Evolution of the Genes in Vertebrates

Hsiao-wei Kao and Sin-Che Lee

Institute of Zoology, Academia Sinica, Taipei, Taiwan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Phosphoglucose isomerase (PGI) is a protein with multiple functions. To infer its structure changes and evolution in vertebrates, we cloned cDNAs encoding PGI genes from hagfish (Paramyxine yangi), gray mullet (Mugil cephalus), zebrafish (Danio rerio), toad (Bufo melanosticus), and snake (Boiga kraepelini). Only one PGI gene was cloned in each of hagfish, toad, and snake, but two PGI genes were found in zebrafish and gray mullet, respectively. The PGI of hagfish encodes 554 amino acids, in contrast to the PGIs of bonyfishes, toad, and snake which encode 553 amino acids and the PGIs of mammals which encode 558 amino acids. Among 558 aligned amino acid sites, there are 314 sites (56.27%) totally conserved. To see if diversifying selection acts on PGI amino acids of vertebrates, we calculated the pairwise ratio of nonsynonymous versus synonymous substitution per site (Ka/Ks) and the ratio of radical amino acid changes versus conservative amino acid changes per sites (dR/dC) between PGI sequences. The average pairwise ratio between nonsynonymous substitutions per nucleotide (Ka) and synonymous substitutions per nucleotide (Ks) among vertebrate PGI sequences equals 0.047 ± 0.019. The average pairwise ratio between radical amino acid changes and conservative amino acid changes (dR/dC) among the vertebrate PGIs equal 0.938 ± 0.158 for charge changes, 0.558 ± 0.085 for polarity changes, and 0.465 ± 0.0714 when both polarity and volume are considered. There is no amino acid within the vertebrate PGIs under diversifying selection as analyzed by the method of Yang et al. (2000b)Citation . The results suggest that the present vertebrate PGIs are at evolutionary stasis and are being subjected to intense purifying selection. The purifying selection is to maintain polarity and volume of the protein but not the charge groups of amino acids. Phylogenetic analysis reveals that vertebrate PGIs can be classified into three major groups: the mammalian, amphibian-reptilian, and teleostean PGIs. The gene tree suggests that the gene duplication event of PGI in bonyfishes occurred before diversification of Acanthopterygii but after the split of bonyfishes and tetrapods. The evolution of multiple functions of PGI is discussed.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Phosphoglucose isomerase (PGI: E.C. 5.3.1.9) is a multifunctional protein. It is also called neuroleukin (NLK), autocrine mobility factor (AMF), or differentiation and maturation mediator (DMM) depending on its functions. PGI catalyzed the interconversion of d-glucose-phosphate and d-fructose-6-phosphate at the second step of the glycolytic pathway. NLK is secreted by T cells and acts as a neurotrophic factor to promote the survival of spinal and sensory neurons (Gurney, 1984Citation ; Chaput et al. 1988Citation ; Faik et al. 1988Citation ). AMF is a tumor-secreted cytokine that stimulates cancer cell migration (Liotta et al. 1986Citation ; Watanabe et al. 1996Citation ). DMM is also secreted by T cell and is capable of inducing differentiation of human myeloid leukemia HL-60 cells to terminal monocytic cells in vitro (Xu et al. 1996Citation ). Other previously unknown functions, such as a sperm antigen involved in sperm agglutination from mouse sperm and a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus) were also reported recently (Cao et al. 2000Citation ; Yakirevich and Naot 2000)Citation .

Jeffery et al. (2000)Citation proposed that the multiple functions of mammalian PGIs result from the gradual modification of its amino acid compositions through evolutionary lineages. To examine such changes, we analyzed the pairwise ratio between nonsynonymous substitutions per site (Ka) and synonymous substitutions per site (Ks) of vertebrate PGIs. Ka is the rate of DNA substitution that affects amino acid compositions, and Ks is the rate of DNA substitutions that does not change amino acid compositions. When the ratio of Ka/Ks is larger than one, the protein is under diversifying selection (Kimura 1980Citation ; Kimura 1983Citation ; Gillespie 1991Citation ; Ohta 1995Citation ; Yang and Nielsen 2000Citation ; Yang, Nielsen, and Hasegawa 2000aCitation ). This method has been applied to many areas of research, such as the evolution of duplicate genes (Zhang, Rosenberg, and Nei 1998Citation ; Lynch and Conery 2000Citation ), the diversifying selection of abalone sperm lysin (Lee, Ota, and Vacquier 1995Citation ; Yang, Swanson, and Vacquier 2000cCitation ), and the evolution of reproductive genes (Wycoff, Wang, and Wu 2000Citation ). In addition to the ratio of Ka/Ks for examining the evolution of a gene, a new model has recently been developed that can detect amino acid sites within a gene under diversifying selection (Yang et al. 2000bCitation ).

To gain further insight into what types of amino acids are more likely to be under selection, we examined the pairwise ratio between radical amino acid changes (dR) per site and conserved amino acids per sites (dC) of vertebrate PGIs. The 20 amino acids can be grouped according to their physiochemical properties such as charge, polarity, and volume. Amino acid substitutions within groups are called conservative substitutions, whereas those between groups are radical ones. A significantly higher rate of radical nonsynonymous substitutions than conservative substitutions has been taken as evidence for positive Darwinian selection on radical substitutions even without a significantly higher rate of nonsynonymous than synonymous substitutions being observed (Hughes 1992, 1994Citation ; Hughes and Hughes 1993Citation ; Zhang 2000Citation ).

Fish have two PGI loci in contrast to only one in terrestrial vertebrates as detected by isozyme electrophoresis (Dando 1980Citation ; Fisher et al. 1980Citation ). It has been suggested that two rounds of gene duplication can account for the multilocus isozymes in fish (Holland et al. 1996Citation ), occurring, respectively, before and after the divergence of ray-finned and lobe-finned fishes. Because two PGI loci were observed in hagfish (Paramyxine yangi), shark, and bonyfish, Fisher et al. (1980)Citation postulated that duplication of PGI had probably taken place at the origin of the agnatha (jawless fishes).

Although numbers of PGI loci can be detected by isozyme electrophoresis, this method is unable to infer the PGI genealogy. Furthermore, PGI has not been cloned in any fish or terrestrial vertebrate other than mammals. In this paper, we described cloning of PGIs from hagfish (agnatha), zebrafish (Danio rerio), gray mullet (Mugil cephalus), toad (Bufo melanosticus), and snake (Boiga kraepelini). We analyzed pairwise ratios of Ka/Ks and dR/dC of vertebrate PGIs, in order to identify their structural changes. We finally constructed gene tree of vertebrate PGIs to infer the PGI genealogy and to identify the gene duplication events in bonyfishes.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals, RNA Extraction, and RT-PCR
Hagfish were collected from Tachi harbor, northeastern Taiwan. Gray mullet were collected from the Tanshui Estuary in northeastern Taiwan. Zebrafish were provided by the zebrafish aquarium at the Institute of Zoology, Academia Sinica, Taipei, Taiwan. The snake and toad were collected on the campus of Academia Sinica, Taipei. Total RNA was extracted from whole fishes or muscle of the animals using TRIZOL reagent (GIBCO-BRL) following the TRIZOL protocol. Five micrograms of total RNA was reverse transcribed into first strand cDNA in a 20-µl reaction containing 10 mM oligo(dT)12–18, MMLVRT 1x buffer, 0.4 mM dNTP, 16 units RNasin, and 120 units MMLVRTase (GIBCO-BRL) at 39°C for 60 min, then heated to 95°C for 10 min, and quenched on ice.

Cloning of PGI cDNA
A pair of degenerate primers (PGI-5', TTYGAGTTCTGGGAYTGGGTKGGWGGC and PGI-3', CCCAGCTCMACWCCCCACTGRTCAWA) was designed on the basis of conserved regions of published mouse, pig, and human PGI sequences for amplification of a core sequence of PGI. PCR amplification was assembled in a 100-µl reaction mixture containing 2 µl of RT reaction product, 2.5 units of Taq polymerase (TaKaRa Ex Taq TM), 1x PCR buffer, 0.2 mM of each dNTP, and 20 pmole each of the PGI-5' and PGI-3' degenerate primers. The thermal reaction consisted of 1 cycle at 95°C for 4 min, 35 cycles at 94°C for 4 min, 50°C for 1 min, and 72°C for 1 min, followed by 1 cycle at 72°C for 10 min. The PCR products were analyzed on 1.2% agarose gels. The bands of about 750 base pairs (bp) in length were cut and purified by glassmilk powder elution (Gene Clean II, BIO 101). The elutions were ligated into pGEM-T Easy T vector (Promega), transformed into E. coli JM109, and sequenced with an autosequencer using T7 or SP6 primers. After sequencing, the sequences were used to design gene-specific primers for amplification of the 5' and 3' ends of PGI using 5' and 3' RACE kits (GIBCO, BML), respectively.

Sequence Analysis
Nucleotide sequence homology searches of a nonredundant database in GenBank (National Center for Biotechnology Information) were performed using the Blast program. The 5', core, and 3' fragments of the cloned PGI sequences were connected using the SeqMan program of the Lasergene software. Determinations of open reading frames (ORFs), translation of the putative amino acids, prediction of molecular weight, isolectric point, and charge were carried out using the Lasergene software package (Hein 1990Citation ; DNASTAR 1994Citation ). The cloned PGI sequences will appear in EMBL/GenBank nucleotide sequence databases with the accession numbers of AJ306391AJ306397. To infer the evolution of vertebrate PGIs, five mammalian PGI sequences from the GenBank were included in the analysis. These sequences are human PGI (accession number K03515), pig PGI (accession number X07382), rabbit PGI (accession number AF199601), mouse PGI (accession number M14220), and Chinese hamster PGI (accession number Z37977). Sequence alignment was performed initially using the Lasergene software and Clustal W program and later modified manually. To examine the extent of sequence divergence, we computed the number of synonymous (Ks) and nonsynonymous (Ka) substitutions per nucleotide for all pairs of PGI sequences between PGIs in all species following the method of Yang and Nielson (2000)Citation . Amino acid sites within vertebrate PGIs under diversifying selection were detected using the method of Yang et al. (2000b)Citation . Rates of conservative and radical nonsynonymous nucleotide substitutions were calculated using the method of Zhang (2000)Citation . Phylogenetic trees for protein sequences were constructed using the Neighbor-Joining (NJ) method (Saitou and Nei 1987Citation ) or protein sequence parsimony method (Propars) implemented in the PHYLIP package (Felsenstein 1993Citation ). Majority-rule consensus trees were obtained from 100 bootstrap replicates with hagfish PGI as the outgroup.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Characteristics of PGI in Hagfish, Zebrafish, Gray Mullet, Toad, and Snake
One PGI gene was cloned in hagfish, toad, and snake, whereas two PGI genes were cloned in each of zebrafish and gray mullet. We denoted these duplicate PGI-1 and PGI-2 as zebrafish-1, zebrafish-2, mullet-1, and mullet-2. The lengths of the cDNA characterized in hagfish, zebrafish, gray mullet, toad, and snake ranged from 1,963 to 2,109 bp. The largest ORF determined by the Lasergene software encodes 554 putative amino acids for hagfish but 553 amino acids each for zebrafish, gray mullet, toad, and snake. Sequence alignment, together with published mammalian PGIs, indicated that there is one amino acid deletion in the 5' region of zebrafish, gray mullet, toad, and snake PGIs but three amino acid insertions in the 3' region of mammalian PGI relative to hagfish PGI (fig. 1 ). The identity of amino acids between sequences (including mammalian PGIs) ranges from 73.2% (mullet-1 vs. Chinese hamster) to 92.6% (human vs. pig or rabbit) (table 1 ).



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Fig. 1.—Amino acid alignment of vertebrate PGIs. Residues among sequences sharing 100% identity are framed

 

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Table 1 Amino Acid Identity Among Vertebrate Phosphoglucose Isomerases

 
Two regions of conserved amino acids, [LIVM]-G-G-R-[FY]-S-[LIVM]-x-[ST]-A-[LIVM]-G and [FY]-D-Q-x-G-V-E-x-x-K, documented as signature patterns for PGI (Bairoch, Bucher, and Hofmann 1996Citation ) are located, respectively, at positions of amino acids 270–280 and 510–520 in our alignment. We found a discrepancy in the signature pattern of zebrafish PGI. Instead of having a lysine (L) at the position of amino acid 276, the PGI of Zebrafish-1 has a proline (P) at that position (fig. 1 ).

The proposed active sites of human PGI for substrate binding include Lys211, Gln354, Glu358, Gln512, Lys519, and His389 (Read et al. 2001Citation ) which are all totally conserved in the alignment (fig. 1 ). The proposed active sites for rabbit PGI include Ser160, Ser210, Lys211, Thr215 (Jeffery et al. 2000)Citation which are all totally conserved. The proposed active sites for Bacillus PGI include Ile157, Gly159, Arg273, Gln354, Glu358, His389, Gln512, and Lys519 (Chou et al., 2000Citation ) which are still totally conserved (fig. 1 ).

The amino acid sequence of SNID was regarded as the recognition sequence for casein kinase II (CKII). Phosphorylation at the serine of SNID confers the ability for PGI to be secreted out of cells (Haga, Niinaka, and Raz 2000Citation ). The sequence is located at position 185–189 in our alignment (fig. 1 ). An inverted repeat (DINS) at position 506–510 was also found in all vertebrate PGIs, except those of zebrafish and gray mullet. The PGIs of Mullet-1 and Zebrafish-1 have the sequences of NINS, and those of mullet-2 and zebrafish-2 have the sequence of EINS (fig. 1 ).

The predicted molecular weights of vertebrate PGIs range from 61,763.84 Da (toad PGI) to 63,125.13 (Pig PGI) Da (table 2 ). Isolectric points range from 6.585 (PGI-1 of gray mullet) to 8.613 (snake PGI) (table 2 ). The charges at pH = 7 of PGIs range from -3.239 (PGI-1 of gray mullet PGI) to 6.331 (snake PGI) (table 2 ). Among them, only the PGI-1 forms of gray mullet and zebrafish carry negative charges at pH = 7 (table 2 ).


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Table 2 Predicted Number of Amino Acids, Molecular Weight, Isolectric Point, and Charge of Vertebrate Phosphoglucose Isomerases in this Study

 
Autapomorphic Sites of PGI in Specific Ecological Groups or Lineages of Vertebrates
We identified several amino acids that change in a unique and consistent pattern within a specific ecological group or evolutionary lineage, and these have been designated as autapomorphic sites of the PGI group (or as synapomorphic sites of the member) of PGI. In addition to sequence length, differences between homeotherms (mammals) and poikilotherms (hagfish, gray mullet, zebrafish, toad, and snake) occur at positions 373 and 400, respectively. Homeotherm PGIs have a histidine (positive charge) and lysine (positive charge), but poikilotherms have a tyrosine (nonpolar) and arginine (positive charge) at amino acid positions of 373 and 400 in our alignment, respectively (fig. 1 ). The amino acid at position 29 differentiates the bonyfish and tetrapod. Bonyfish PGI has a methionine (nonpolar) but that of tetrapods has a leucine (nonpolar). The amino acid position 60 differentiates aquatic vertebrates (hagfish, zebrafish, gray mullet, and toad) and terrestrial vertebrates (snake, mouse, Chinese hamster, rabbit, pig, and human), having an isoleucine (nonpolar) and a valine (nonpolar), respectively (fig. 1 ).

Substitution Patterns Among PGI Sequences
The pairwise values of Ks among PGI sequences range from 0.563 (mouse vs. Chinese hamster) to 4.410 (hagfish vs. mullet-1), with an equivalent average value of 3.17 ± 1.38. The pairwise values of Ka range from 0.034 (human vs. rabbit) to 0.185 (hagfish vs. pig) with an equivalent average value of 0.129 ± 0.040. The pairwise Ka/Ks ratios range from 0.0274 (snake vs. rabbit) to 0.104 (hagfish vs. toad), with an equivalent average value of 0.047 ± 0.019 (fig. 2 ). There are no amino acid sites within the vertebrate PGIs that are under diversifying selection, as analyzed by the method of Yang et al. (2000b)Citation .



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Fig. 2.—Nonsynonymous versus synonymous substitutions per site of vertebrate PGIs

 
Radical Amino Acid Change per Site (dR) and Conserved Amino Acid Changes per Sites (dS) Among Phosphoglucose Isomerases of Vertebrates
The pairwise ratios between radical amino acid changes and conservative amino acid changes per site (dR/dC) among vertebrate PGIs range from 0.497 (mouse vs. Chinese hamster) to 1.193 (pig vs. zebrafish-2) for charge change (fig. 3 ); 0.413 (human vs. Chinese hamster) to 0.819 (human vs. rabbit) for polarity changes (fig. 4 ); and 0.298 (human vs. Chinese hamster) to 0.559 (hagfish vs. zebrafish-1) for both polarity and volume (fig. 5 ). The average pairwise ratios equal 0.938 ± 0.158 for charge changes, 0.559 ± 0.085 for polarity changes, and 0.422 ± 0.051 both polarity and volume. In all cases, radical amino acid change increased linearly with conservative amino acid change. The equations describing the relationship between radical amino acid change per site and conservative amino acid change per site were y = 1.0271x - 0.0091 (r2 = 0.8285) for charge change (fig. 3 ), y = 0.4891x + 0.0085 (r2 = 0.7943) for polarity (fig. 4 ), and y = 0.4267x - 0.001 (r2 = 0.8047) for both polarity and volume (fig. 5 ).



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Fig. 3.—Radical versus conservative amino acid changes per site for charge of vertebrate PGIs

 


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Fig. 4.—Radical versus conservative amino acid changes per site for polarity of vertebrate PGIs

 


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Fig. 5.—Radical versus conservative amino acid changes per site for both polarity and volume of vertebrate PGIs

 
Genealogy of PGI in Vertebrates
Phylogenetic trees of PGI constructed from the predicted amino acids obtained in the present study and sequences of vertebrates published elsewhere using both NJ or protein-parsimony (Propars) methods indicate similar topology (figs. 6 and 7 ). The only difference between the two trees is the relationships among human, rabbit, and pig PGIs. The NJ tree grouped human and pig are clustered together and rabbit is the sister group (fig. 6 ); however, human and rabbit are clustered together and pig is the sister group in the Propars tree (fig. 7 ). Nevertheless, both trees supported that vertebrate PGIs can be classified into three major groups: mammalian PGIs, toad-snake PGIs, and teleostean PGIs. In teleostean PGIs, mullet-1 and zebrafish-1 are clustered together and mullet-2 and zebrafish-2 are clustered together (figs. 6 and 7 ).



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Fig. 6.—A Neighbor-Joining tree constructed from amino acid sequences of PGIs using hagfish as an outgroup. Bootstrap probability from 100 replications is shown on the corresponding branches

 


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Fig. 7.—A protein-parsimony tree constructed from amino acid sequences of PGI using hagfish as an outgroup. Bootstrap probability from 100 replications is shown on the corresponding branching

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We have cloned seven different PGI cDNAs from five species of lower vertebrates, including hagfish, zebrafish, gray mullet, toad, and snake. Although these animals (including mammals) represent five systematically different classes of vertebrates, the PGIs among the species are highly conserved both in size and composition of amino acids.

The average pairwise ratio of Ka/Ks equals 0.047 ± 0.019. According to the Ka/Ks values estimated from 108 nonessential and 67 essential genes in the mouse and rat, Hurst and Smith (1999)Citation found that the immune-nonessential genes have the highest Ka/Ks ratio with a mean value of 0.444 ± 0.048. On the contrary, the neuron-essential genes have the lowest Ka/Ks value of 0.096 ± 0.02 which is about twice the magnitude of that of PGIs among vertebrates on an average. The low ratio of Ka/Ks in our analysis might result from the high value of Ks when comparing divergent PGIs of vertebrates. However, we also observed a very small Ka/Ks value when the Ks value was less than 1. For example, a Ks value of 0.603 between human and pig PGIs results in a low Ka/Ks ratio of 0.0546, and a Ks value of 0.646 between rabbit and human PGIs results in a low Ka/Ks ratio of 0.0525. This is also true for the duplicate PGIs in gray mullet and zebrafish, indicating a rather low Ka/Ks ratio of 0.0475 between mullet-1 and mullet-2 and 0.0724 between zebrafish-1 and zebrafish-2. In addition to calculation of the pairwise Ka/Ks ratios among vertebrate PGIs, we also performed an analysis of amino acids within the gene under diversifying selection by using the method of Yang et al. (2000b)Citation . However, no amino acid within vertebrate PGIs was found to be under diversifying selection by this method. The results suggest that the present vertebrate PGIs are at evolutionary stasis and are being subjected to intense purifying selection.

The low ratios of Ka/Ks among vertebrate PGIs might reflect that the PGIs are being constrained by their multiple functions. Until now, more than five different functions have been found for PGI. In addition, one receptor responsible for the function has been cloned (Shimizu et al. 2000Citation ). Kisters-Woike, Vangierdegom, and Müller-Hill (2000)Citation proposed that the amino acid conservation of enzymes might be the result of the fact that they function as part of multienzyme complexes. The specific interactions between the proteins involved would hinder evolutionary change of their surfaces.

The predicted charges of the two PGI forms are -3.239 and 4.756 for gray mullet and -1.916 and 0.894 for zebrafish at pH 7.0. In contrast to other vertebrate PGIs, only mullet-1 and zebrafish-1 carry a negative charge. Riddoch (1993)Citation suggested that higher anodal allozyme-isozyme activity is favored under a suit of conditions of increased temperature, salinity and risk of desiccation, and reduced oxygen availability. However, the charge change of dR/dC = 0.73 between zebrafish-1 and zebrafish-2 and that of dR/dC = 0.87 between mullet-1 and mullet-2 suggest that the charge change of duplicate bonyfish PGIs may be selectively neutral. Nevertheless, we noticed that each vertebrate PGI contains two potential phosphorylation sites for casein kinase II with the exception of bonyfishes which contain only one in each duplicate PGI gene. It remains to be seen whether the secretion of bonyfish PGIs can be regulated by such a change.

Although it was proposed that the multiple functions of GPI were gained gradually by amino acid changes (Jeffery et al. 2000Citation ), an alternative hypothesis is that, instead, PGI might be recruited by other proteins for novel functions during evolution. Two lines of evidence support this hypothesis. First, the protein is highly constrained, as reflected by the low Ka/Ks and dR/dC ratios among vertebrates. It is true for both the duplicate PGI genes and those of PGIs with high Ks values. Second, Bacillus PGI is capable of acting as NLK and AMF mammalian PGI in mammalian cells (Sun et al. 1999Citation ; Chou et al. 2000Citation ). We propose that the multiple functions are innate characteristics of PGI at the origin of the protein. The novel functions might have evolved by cellular compartmentalization of the protein, dimerization, and evolution of its receptor.

Although Fisher et al. (1980)Citation observed two loci of PGIs in hagfish by isozyme electrophoresis, only one locus of hagfish PGI was cloned in this study. Thus, we are unable to justify whether a PGI duplication event occurred before the origin of the aganatha. However, PGIs of mullet-1 and zebrafish-1 clustering together and those of mullet-2 and zebrafish-2 clustering together suggest that the gene duplication event of bonyfish PGIs occurred before the divergence of the Acathoptergii. In addition, PGIs of gray mullet and zebrafish not clustering with PGIs of mammals, snake, and toad suggest that the PGIs in bonyfishes deverged after the split between bonyfishes and tetrapods. Taken together, our inference does not favor the hypothesis that the present two loci of PGIs in bonyfishes resulted from gene duplication before the origin of the agnatha. It probably occurred after the split between bonyfishes and tetrapods but before the divergence of the Acathopterygii.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
H.-W.K. was a recipient of a postdoctoral fellowship from Academia Sinica. This research was supported by the Institute of Zoology, Academia Sinica, Taiwan, ROC.


    Footnotes
 
Claudia Kappen, Reviewing Editor

Keywords: gene duplication nonsynonymous substitution purifying selection radical amino acid change synonymous substitution Back

Address for correspondence and reprints: Sin-Che Lee, Institute of Zoology, Academia Sinica, Taipei, Taiwan 115, ROC. sclee{at}sinica.edu.tw . Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Accepted for publication September 4, 2001.