Institute of Zoology, Academia Sinica, Taipei, Taiwan
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Abstract |
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Introduction |
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Jeffery et al. (2000)
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 1980
; Kimura 1983
; Gillespie 1991
; Ohta 1995
; Yang and Nielsen 2000
; Yang, Nielsen, and Hasegawa 2000a
). This method has been applied to many areas of research, such as the evolution of duplicate genes (Zhang, Rosenberg, and Nei 1998
; Lynch and Conery 2000
), the diversifying selection of abalone sperm lysin (Lee, Ota, and Vacquier 1995
; Yang, Swanson, and Vacquier 2000c
), and the evolution of reproductive genes (Wycoff, Wang, and Wu 2000
). 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. 2000b
).
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, 1994
; Hughes and Hughes 1993
; Zhang 2000
).
Fish have two PGI loci in contrast to only one in terrestrial vertebrates as detected by isozyme electrophoresis (Dando 1980
; Fisher et al. 1980
). It has been suggested that two rounds of gene duplication can account for the multilocus isozymes in fish (Holland et al. 1996
), 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)
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.
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Materials and Methods |
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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 1990
; DNASTAR 1994
). 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)
. Amino acid sites within vertebrate PGIs under diversifying selection were detected using the method of Yang et al. (2000b)
. Rates of conservative and radical nonsynonymous nucleotide substitutions were calculated using the method of Zhang (2000)
. Phylogenetic trees for protein sequences were constructed using the Neighbor-Joining (NJ) method (Saitou and Nei 1987
) or protein sequence parsimony method (Propars) implemented in the PHYLIP package (Felsenstein 1993
). Majority-rule consensus trees were obtained from 100 bootstrap replicates with hagfish PGI as the outgroup.
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Results |
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The proposed active sites of human PGI for substrate binding include Lys211, Gln354, Glu358, Gln512, Lys519, and His389 (Read et al. 2001
) 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)
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., 2000
) 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 2000
). The sequence is located at position 185189 in our alignment (fig. 1
). An inverted repeat (DINS) at position 506510 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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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)
.
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Discussion |
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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)
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)
. 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. 2000
). Kisters-Woike, Vangierdegom, and Müller-Hill (2000)
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)
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. 2000
), 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. 1999
; Chou et al. 2000
). 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)
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.
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Acknowledgements |
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Footnotes |
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Keywords: gene duplication
nonsynonymous substitution
purifying selection
radical amino acid change
synonymous substitution
Address for correspondence and reprints: Sin-Che Lee, Institute of Zoology, Academia Sinica, Taipei, Taiwan 115, ROC. sclee{at}sinica.edu.tw
.
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