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Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana)
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 26 (2003) 18–25 www.elsevier.com/locate/ympev
Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana) Jaap J. Beintema,* Heleen J. Breukelman, Jean-Yves F. Dubois, and Hayo W. Warmels Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Received 13 December 2001; received in revised form 5 June 2002
Abstract Phylogenetic analyses based on primary structures of mammalian ribonucleases, indicated that three homologous enzymes (pancreatic, seminal and brain ribonucleases) present in the bovine species are the results of gene duplication events, which occurred in the ancestor of the ruminants after divergence from other artiodactyls. In this paper sequences are presented of genes encoding pancreatic and brain-type ribonuclease genes of pronghorn (Antilocapra americana). The seminal-type ribonuclease gene could not be detected in this species, neither by PCR amplification nor by Southern blot analyses, indicating that it may be deleted completely in this species. Previously we demonstrated of a study of amino acid sequences of pancreatic ribonucleases of a large number of ruminants the monophyly of bovids and cervids, and that pronghorn groups with giraffe. Here we present phylogenetic analyses of nucleotide sequences of ribonucleases and other molecules from ruminant species and compare these with published data. Chevrotain (Tragulus) always groups with the other ruminants as separate taxon from the pecora or true ruminants. Within the pecora the relationships between Bovidae, Cervidae, Giraffidae, and pronghorn (Antilocapra) cannot be decided with certainty, although in the majority of analyses Antilocapra diverges first, separately or joined with giraffe. Broad taxon sampling and investigation of specific sequence features may be as important for reliable conclusions in phylogeny as the lengths of analyzed sequences. Ó 2002 Published by Elsevier Science (USA).
1. Introduction In classical phylogenetic studies diagnostic morphological features, which are of value for deriving evolutionary relationships, are skillfully selected. In contrast modern molecular evolutionary studies handle large numbers of character states and use statistical methods for the same purpose. Protein chemists interested in the molecular evolution of a protein with known three-dimensional structure may be more comparable with classical morphologists than with their fellow molecular biologists when they select specific structural features, which tell something about the evolutionary history of a protein. Therefore it should be regretted that findings from evolutionary studies on amino acid sequence studies obtained in the years 1965–1985 are often ne-
* Corresponding author. Fax: +31-50-3634165. E-mail address: [email protected] (J.J. Beintema).
glected in current reviews on molecular evolution (e.g., Matthee et al., 2001). Our studies on the molecular evolution of the enzyme ribonuclease in mammals started by determining amino acid sequences, with special emphasis on enzymes isolated from the pancreas of artiodactyls and rodents (Beintema et al., 1988). A novel result of our studies was the grouping of pronghorn (Antilocapra americana) with giraffe (Giraffa camelopardalis) in trees derived by maximum-parsimony (Beintema and Lenstra, 1982; Beintema et al., 1979, 1986, 1988). At that time the pronghorn was still grouped as single representative of the family Antilocapridae with the family Bovidae in the superfamily Bovoidea (Morris, 1965). Diagnostive structural features, shared by ribonucleases of pronghorn and giraffe are three conservative replacements of hydrophobic amino acids in the interior of the molecule, which are rarely replaced in other mammalian ribonucleases (Beintema et al., 1988). Recently Van Dijk et al. (2001) described the
1055-7903/02/$ - see front matter Ó 2002 Published by Elsevier Science (USA). PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 2 9 5 - 6
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use of protein sequence signatures for phylogenetic purposes. The grouping of pronghorn with giraffe was supported by sequences of fibrino-peptides from these species (giraffe: unpublished sequence, communicated by Dr. R.F. Doolittle; Goodman et al., 1982). Since then nucleotide sequences became available of pronghorn and other true ruminants or pecora (bovids, deer, and giraffe) and also of the primitive ruminant species chevrotain (Tragulus), which allowed comparison of these more recent results with the earlier obtained ones from amino acid sequences. In studies demonstrating the monophyly of bovids from mitochondrial ribosomal DNA sequences (Allard et al., 1992; Gatesy et al., 1992) other pecorans were used as outgroup, in which pronghorn was an outside clade of the other pecorans, or pronghorn grouped with giraffe, respectively. However, in the database a stretch of only 457 nucleotides is shared by the two latter species (giraffe: AF151090; pronghorn: M55540), which includes the sequences of tRNA-Phe and the 50 region of 12S ribosomal RNA. More reliable are phylogenetic studies on ruminant phylogeny from larger data sets. Sequences of j-casein
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genes (Cronin et al., 1996), b-caseins (Gatesy et al., 1996), and of other available nuclear and mitochondrial nucleotide sequences were analyzed separately and in different tandem alignments by Gatesy et al. (1999). An extremely comprehensive analysis of ruminant and other artiodactyl sequences was published recently by Matthee et al. (2001), who sequenced eight nuclear genes of more than 20 artiodactyls (about 7000 nucleotide positions in exons and introns) and derived trees from these data with very sophisticated methods. Matthee et al. (2001) also review in their paper much other biological evidence about the phylogeny of ruminants. They also analyzed mitochondrial cytochrome b sequences from artiodactyls, but did no find a robust phylogeny. However, Randi et al. (1998) obtained useful data from sequences of this gene with a larger taxon sampling, especially of cervids. A study of the entire single-copy genome by DNA hybridization of several ruminants with camels and pig as outgroups (J.A.W. Kirsch, B. Brunner, K. Martin, C.G. Sibley, personal communication) showed strong evidence for a first divergence of pronghorn, and weaker support for grouping bovids and cervids, separate from giraffe.
Fig. 1. Proposed phylogenetic relationships of pronghorn and other pecorans and ruminants, derived from molecular data. * Bovidae are not always found to be monophyletic (- - - -).
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Proposed topologies of the evolution of ruminants, including an analysis of molecular and morphological data by Liu et al. (2001), are summarized in Fig. 1. There is essentially an absolute support from molecular data for the monophyly of the classical ruminant taxa: ruminants, pecorans (ruminants without chevrotain, Tragulus), cervids and bovids. But the relationships within the pecorans (bovids, cervids, giraffids, and pronghorn) is unstable, although from the comprehensive evidence presented by Matthee et al. (2001) it may be concluded that bovids group with cervids, and that the giraffids and pronghorn diverge first, either separately or as a monophyletic clade. Ribonuclease is a digestive enzyme, secreted at a high level by bovine pancreas. Two paralogous enzymes have been isolated from other bovine tissues, namely seminal (Suzuki et al., 1987) and brain ribonucleases (Watanabe et al., 1988), which are products of two gene duplications in an ancestral ruminant (Breukelman et al., 2001). Bovine seminal RNase is synthesized at high levels in seminal vesicles (DÕAlessio et al., 1972). Bovine brain RNase is expressed in the brain (Sasso et al., 1991) and in lactating mammary glands (Zhao et al., 2001). In previous papers we published nucleotide sequences of ribonucleases from several true ruminants or pecora (Breukelman et al., 1998; Confalone et al., 1995), chevrotain (Breukelman et al., 2001) and other artiodactyls (Kleineidam et al., 1999a). Here we present sequences of pronghorn ribonucleases, not only of the pancreatictype gene, but also of the paralogous brain-type gene, and present evidence that the gene encoding the seminaltype gene may be deleted in this species. Evolutionary trees were derived of ruminant ribonucleases and of recently deposited sequences of ruminant prion proteins (Raymond et al., 2000; Wopfner et al., 1999), and compared with those presented in Fig. 1.
2. Materials and methods Genomic DNA of pronghorn (A. americana) was isolated (Bothwell et al., 1990) from ethanol-preserved tissue, kindly provided by Dr. F. Catzeflis (Montpellier, France). Amplification of ribonuclease 1 sequences was performed by the hot-start procedure (Ampliwax PCR Gem 100, Perkin–Elmer) with different oligonucleotide pairs (Eurosequence BV, Groningen) in different reactions. As forward primers oligonucleotide B/SP (50 -GGGGATCC GGGTCCAGCCTTCCCTGGG-30 ), with a BamHI site (in italics) added by a linker (Breukelman et al., 1998), or oligonucleotide 5SP (50 -ATGGCTCTGCAGTCC/ TCT-30 ), encoding the N-terminus of the signal peptide (amino acids )26 to )21) of the three paralogous enzymes of ox with an internal PstI site (in italics), were used. As reverse primers, oligonucleotide E/B (50 -GGGA-
ATTCCTTGAG TTATTGCCCTCAAGTCAGGG-30 ), containing a linker with a EcoRI site (in italics) (Breukelman et al., 1998) and oligonucleotide AS (50 -GT/ CTCGGCCT/CAGGT G/AGAGA-30 ) (Confalone et al., 1995), were used. Oligonucleotide PrS (50 -CTGGCTTG CATTTCCCC-3), which is complementary to the region of nt 111–127 of various seminal-type ribonuclease genes, was used as a reverse primer for an effort to amplify part of the seminal-type ribonuclease sequence of pronghorn. PCR amplifications were performed in a total volume of 100 ll containing 500 ng high mol. wt. genomic DNA, 100 pmol of forward primer, 100 pmol of reverse primer, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris–Cl (pH 9.0), 1.5 mM MgCl2 , 0.01% gelatin, 0.1% Triton X-100, and 0.5 U Super Taq polymerase (HT Biotechnology). A denaturation step of 5 min was followed by 30 cycles of 1 min at 94 °C, 1 min 47–53 °C, and 1 min at 72 °C. The last segment was extended by 5 s per cycle. The specific products were purified by electrophoresis, extracted by centrifugation and cloned into pCR 2.1 (Invitrogen). DNA was sequenced by the dideoxynucleotide method (Sanger et al., 1977) using the sequencing kit (Pharmacia). The new sequences reported were the result of three independent PCRs, and were deposited in the EMBL GenBank under accession numbers AJ271301 (pancreatic ribonuclease) and AJ271302 (brain-type ribonuclease). Nucleotides 1–372 of the mature pancreatic and braintype ribonuclease sequences of ox (Carsana et al., 1988; Sasso et al., 1991), sheep, giraffe (Confalone et al., 1995), hog deer, roe deer (Breukelman et al., 1998), pronghorn, and chevrotain (Breukelman et al., 2001) were aligned, and treated by parsimony and likelihood analyses, using the programs PAUP Version 4.0b4a (Swofford, 1998). The PROTPARS program of PAUP, which takes into account codon differences between amino acids, was used for parsimony analysis of translated protein sequences. Confidence levels were estimated using bootstrap percentages (BP: Felsenstein, 1985) with the parsimony approach (heuristic search, 1000 replicates) and with the maximum likelihood approach (100 replicates). Aligned sequences of several other molecules from ruminants and other artiodactyls obtained from the nucleotide database were analyzed similarly.
3. Results and discussion 3.1. Pronghorn ribonucleases A ribonuclease-specific amplification product of 471 base pairs (bp) was obtained with primers 5SP and AS. The sequences of the clones obtained with this amplification product were determined, all encoding a sequence that showed the highest identity with the bovine pancreatic ribonuclease gene. Fig. 2A shows the nucleotide
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Fig. 2. Nucleotide sequences encoding ribonucleases in pronghorn. (A) Pancreatic ribonucleases of ox (Ox-A) and pronghorn (Pr-A). (B) Brain-type ribonucleases of ox (Ox-B) and pronghorn (Pr-B). The primary structures of bovine pancreatic and brain ribonucleases and the predicted ones of pronghorn are indicated in the one letter code. I, identical nucleotide; stop, stop codon; -, deletion of one nucleotide.
sequence encoding a part of the signal peptide and the complete mature protein compared with the same part of the bovine ribonuclease gene (Carsana et al., 1988). A high percentage of identity (93%) can be observed between these two genes. The amino acid sequence of bovine pancreatic ribonuclease and the predicted amino acid sequence of pronghorn pancreatic ribonuclease, which is identical to the previously determined one by Beintema et al. (1979), are also presented in Fig. 2A. The ratio of non-synonymous to synonymous substitutions in pronghorn pancreatic RNase compared with bovine ribonuclease A is 1:2. This ratio is in agreement with the ratio expected for a gene having a function.
A sequence that showed the highest identity (93%) with the bovine brain ribonuclease gene was obtained with clones containing a ribonuclease-specific amplification product of 490 bp with primers 5SP and E/B. This nucleotide sequence compared with the same part of the bovine brain ribonuclease gene (Sasso et al., 1991) is shown in Fig. 2B, together with the amino acid sequences of the two encoded proteins. In previous studies of other pecoran species the used primer pair also yielded ribonuclease-specific amplification products of about 580 bp (Breukelman et al., 1998). Now this product was not found and therefore the sequence of pronghorn brain-type ribonuclease presented in Fig. 2B
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Fig. 2. (continued)
does not include the C-terminal region of the molecule and the stop codon. None of the primer combinations 5SP-AS, 5SP-E/B, B/ SP-AS, or B/SP-E/B yielded a seminal-type ribonuclease sequence in pronghorn. Therefore oligonucleotide PrS, complementary to the region of nt 111–127 of several ruminant seminal-type ribonucleases and encoding amino acids 37–43 of this protein, was designed. With this primer and primer 5SP or primer B/SP it was still impossible to detect a seminal-type ribonuclease sequence.
Most ruminant seminal-type ribonucleases show characteristics of pseudogenes with deletions and insertions in the coding region of the gene resulting in frameshifts (Breukelman et al., 1998; Kleineidam et al., 1999b). Our failure to find a seminal-type ribonuclease sequence in the pronghorn genome might be explained by deletion of the complete gene and not only part of it. Therefore we looked again at previously obtained Southern blots of genomic DNA of pronghorn and other ruminants with ribonuclease specific probes, pre-
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sented in Figs. 1–3 in Breukelman et al. (1993) and available in unpublished material. These figures show three cross-reacting bands in investigated ruminants: ox, water buffalo, sheep, goat, black-tailed deer, and giraffe, when a probe is used encoding amino acids 4–124 and a few nucleotides downstream of the stop codon of pancreatic bovine ribonuclease, while probes with a sequence of the 30 untranslated region of this protein react less with brain-type ribonuclease genes. However, in Fig. 1D in Breukelman et al. (1993), which shows Southern blots of pronghorn, not three but two bands are visible. This suggests indeed that the seminal-type gene in this species is deleted completely. But concluding from Southern blots that a gene is absent may be erroneous as we experienced in investigating the number of genes encoding camel ribonucleases by Southern blotting (Breukelman et al., 1993) and PCR amplification (Kleineidam et al., 1999a).
Fig. 3. Maximum-parsimony (A) and maximum-likelihood tree (B) of tandemly aligned nucleotide sequences of pancreatic-type and braintype ribonuclease sequences of chevrotain (Tragulus javanicus; outgroup), pronghorn (A. americana), giraffe (G. camelopardalis), ox (Bos taurus), sheep (Ovis aries), hog deer (Axis porcinus), and roe deer (Capreolus capreolus). Bootstrap percentages are indicated at the nodes.
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3.2. Phylogenetic analyses Phylogenetic trees of nucleotide sequences of pancreatic-type and brain-type ribonucleases from pronghorn and other ruminants were derived with the maximum-parsimony method, with the chevrotain sequence as outgroup. Four most parsimonious tree were obtained (results not shown). But as previously observed (Breukelman et al., 1998, 2001), there was no congruency between the pancreatic-type and brain-type ribonuclease subtrees. Tandemly aligned nucleotide sequences of the two ribonuclease types were investigated with the maximumparsimony and maximum-likelihood methods. Only one tree was found with the maximum-parsimony method, which is presented in Fig. 3A. The two bovid sequences are not monophyletic in this tree, and the bootstrap percentages are rather low. Pronghorn diverges first, while giraffe groups with the deer sequences. A maximum-parsimony tree of translated amino acid sequences derived with the PROTPARS method also yielded one tree with the same topology. The maximum-likelihood tree has another topology (Fig. 3B), with giraffe and pronghorn grouping together and these two again with a monophyletic clade of the two bovids. But the bootstrap percentages are low. It is interesting that the topology of this latter tree is identical to the one presented in Fig. 1A, derived from amino acid sequences of pancreatic ribonucleases. We also derived trees both from nucleotide sequences and amino acid sequences (PROTPARS method) of other molecules present in the database, and obtained similar results as presented in the original publications by Randi et al. (1998), Cronin et al. (1996), and Gatesy et al. (1996, 1999). A phylogenetic analysis of prion protein sequences of artiodactyls and other mammals was published (Wopfner et al., 1999) before a pronghorn sequence was available. We made an analysis including the latter species. As no sequence of the prion protein from a chevrotain species (Tragulus) is yet available in the database, we used the camel and pig sequences as outgroups for our analyses. The deposited sequence of the giraffe prion protein is shorter at both termini than the other ones. Therefore we used an alignment with a 50 sequence corresponding with the CKKRP amino acid sequence (which includes the C-terminal residue of the signal peptide and the N-terminus op the mature sequence) and a 30 sequence corresponding with the ILLISFL amino acid sequence, which lacks six amino acid residues before the stop codon in other deposited sequences. An interesting feature of prion proteins is a repeated octapeptide sequence GGGWGQPH. This repeat occurs four times in most investigated artiodactyl prion proteins, and five times in those from the majority of investigated alleles of ox and several closely related species, and in those from goitered gazelle, lama, giraffe (Wopfner et al., 1999) and prong-
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Fig. 4. Maximum-likelihood analysis of nucleotide sequences (706 positions) of prion proteins of pig (Sus scrova, LO7623), camel (Camelus dromedarius, CDPRPCELF), giraffe (G. camelopardalis, AF113942), pronghorn (A. americana, AF156187), American elk (Cervus elaphus nelsoni, AF156183), ox (Bos taurus; D10614), and sheep (Ovis aries, AF180389). Bootstrap percentages of the maximumparsimony (with identical topology of the tree; upper values) and maximum-likelihood analyses (lower values) are indicated at the nodes.
However, the phylogeny of the four pecoran taxa: Bovidae, Cervidae, Giraffidae, and Antilocapra, remains unresolved. In most analyses pronghorn (Antilocapra) diverges first, either separately, or together with giraffe (Gatesy et al., 1999; Matthee et al., 2001). The latter topology is supported by other molecular features, like the presence of three rarely observed amino acid acid replacements in pancreatic ribonucleases, and the presence of an additional octapeptide repeat in prion proteins. Thus not only sequence data, but also other molecular features are valuable for deriving phylogenetic relationships (Van Dijk et al., 2001), as also Matthee et al. (2001) conclude from the analysis of indels in the sequences determined by them. Another point is that the four pecoran taxa diverged in a rather short time span about 30 million years ago (Breukelman et al., 2001). May it be that the three successive divergences occurred in populations of an ancestral species, which still had the capacity for interbreeding, and that therefore separate genes may show different phylogenies?
Acknowledgments horn. So the number of repeats is not a strong phylogenetic feature. C-terminal of these repeats is a heptapeptide sequence GGGGWGQ in ruminants and pig, and GGGWGQ in camel. Nucleotide sequences of prion proteins were analyzed by the maximum-parsimony and the maximum-likelihood methods. The former method yielded only one tree with an identical topology as obtained with the latter method. The maximum-likelihood tree is presented in Fig. 4, with bootstrap percentages obtained with both methods. In these trees giraffe diverges first, followed by pronghorn, while deer and bovids group together.
4. Conclusion In all analyses which include chevrotain (Tragulus) and other ruminant and non-ruminant artiodacyl sequences, this taxon groups with the ruminants as first diverging clade (Fig. 1, Breukelman et al., 2001). The deer (Cervidae) are always found to be monophyletic, but the Bovidae are sometimes found to be diphyletic (Figs. 1C and 3A). This may improve with a broader taxon sampling of bovid taxa. A phylogenetic analysis of amino acid sequences of pancreatic ribonucleases from ten bovids yielded a monophyletic taxon, with a topology corresponding to other biological data (Beintema and Lenstra, 1982; Beintema et al., 1986). The broader taxon sampling in our previous protein studies may be superior to the increase in number of analyzed nucleotide positions in the current work.
This research was supported by the European Community Project ERB-FMRX-CT98-221. We thank Dr. W.T. Stam, Department of Marine Biology for his advice using the PROTPARS program.
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Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana) Jaap J. Beintema,* Heleen J. Breukelman, Jean-Yves F. Dubois, and Hayo W. Warmels Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Received 13 December 2001; received in revised form 5 June 2002
Abstract Phylogenetic analyses based on primary structures of mammalian ribonucleases, indicated that three homologous enzymes (pancreatic, seminal and brain ribonucleases) present in the bovine species are the results of gene duplication events, which occurred in the ancestor of the ruminants after divergence from other artiodactyls. In this paper sequences are presented of genes encoding pancreatic and brain-type ribonuclease genes of pronghorn (Antilocapra americana). The seminal-type ribonuclease gene could not be detected in this species, neither by PCR amplification nor by Southern blot analyses, indicating that it may be deleted completely in this species. Previously we demonstrated of a study of amino acid sequences of pancreatic ribonucleases of a large number of ruminants the monophyly of bovids and cervids, and that pronghorn groups with giraffe. Here we present phylogenetic analyses of nucleotide sequences of ribonucleases and other molecules from ruminant species and compare these with published data. Chevrotain (Tragulus) always groups with the other ruminants as separate taxon from the pecora or true ruminants. Within the pecora the relationships between Bovidae, Cervidae, Giraffidae, and pronghorn (Antilocapra) cannot be decided with certainty, although in the majority of analyses Antilocapra diverges first, separately or joined with giraffe. Broad taxon sampling and investigation of specific sequence features may be as important for reliable conclusions in phylogeny as the lengths of analyzed sequences. Ó 2002 Published by Elsevier Science (USA).
1. Introduction In classical phylogenetic studies diagnostic morphological features, which are of value for deriving evolutionary relationships, are skillfully selected. In contrast modern molecular evolutionary studies handle large numbers of character states and use statistical methods for the same purpose. Protein chemists interested in the molecular evolution of a protein with known three-dimensional structure may be more comparable with classical morphologists than with their fellow molecular biologists when they select specific structural features, which tell something about the evolutionary history of a protein. Therefore it should be regretted that findings from evolutionary studies on amino acid sequence studies obtained in the years 1965–1985 are often ne-
* Corresponding author. Fax: +31-50-3634165. E-mail address: [email protected] (J.J. Beintema).
glected in current reviews on molecular evolution (e.g., Matthee et al., 2001). Our studies on the molecular evolution of the enzyme ribonuclease in mammals started by determining amino acid sequences, with special emphasis on enzymes isolated from the pancreas of artiodactyls and rodents (Beintema et al., 1988). A novel result of our studies was the grouping of pronghorn (Antilocapra americana) with giraffe (Giraffa camelopardalis) in trees derived by maximum-parsimony (Beintema and Lenstra, 1982; Beintema et al., 1979, 1986, 1988). At that time the pronghorn was still grouped as single representative of the family Antilocapridae with the family Bovidae in the superfamily Bovoidea (Morris, 1965). Diagnostive structural features, shared by ribonucleases of pronghorn and giraffe are three conservative replacements of hydrophobic amino acids in the interior of the molecule, which are rarely replaced in other mammalian ribonucleases (Beintema et al., 1988). Recently Van Dijk et al. (2001) described the
1055-7903/02/$ - see front matter Ó 2002 Published by Elsevier Science (USA). PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 2 9 5 - 6
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use of protein sequence signatures for phylogenetic purposes. The grouping of pronghorn with giraffe was supported by sequences of fibrino-peptides from these species (giraffe: unpublished sequence, communicated by Dr. R.F. Doolittle; Goodman et al., 1982). Since then nucleotide sequences became available of pronghorn and other true ruminants or pecora (bovids, deer, and giraffe) and also of the primitive ruminant species chevrotain (Tragulus), which allowed comparison of these more recent results with the earlier obtained ones from amino acid sequences. In studies demonstrating the monophyly of bovids from mitochondrial ribosomal DNA sequences (Allard et al., 1992; Gatesy et al., 1992) other pecorans were used as outgroup, in which pronghorn was an outside clade of the other pecorans, or pronghorn grouped with giraffe, respectively. However, in the database a stretch of only 457 nucleotides is shared by the two latter species (giraffe: AF151090; pronghorn: M55540), which includes the sequences of tRNA-Phe and the 50 region of 12S ribosomal RNA. More reliable are phylogenetic studies on ruminant phylogeny from larger data sets. Sequences of j-casein
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genes (Cronin et al., 1996), b-caseins (Gatesy et al., 1996), and of other available nuclear and mitochondrial nucleotide sequences were analyzed separately and in different tandem alignments by Gatesy et al. (1999). An extremely comprehensive analysis of ruminant and other artiodactyl sequences was published recently by Matthee et al. (2001), who sequenced eight nuclear genes of more than 20 artiodactyls (about 7000 nucleotide positions in exons and introns) and derived trees from these data with very sophisticated methods. Matthee et al. (2001) also review in their paper much other biological evidence about the phylogeny of ruminants. They also analyzed mitochondrial cytochrome b sequences from artiodactyls, but did no find a robust phylogeny. However, Randi et al. (1998) obtained useful data from sequences of this gene with a larger taxon sampling, especially of cervids. A study of the entire single-copy genome by DNA hybridization of several ruminants with camels and pig as outgroups (J.A.W. Kirsch, B. Brunner, K. Martin, C.G. Sibley, personal communication) showed strong evidence for a first divergence of pronghorn, and weaker support for grouping bovids and cervids, separate from giraffe.
Fig. 1. Proposed phylogenetic relationships of pronghorn and other pecorans and ruminants, derived from molecular data. * Bovidae are not always found to be monophyletic (- - - -).
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Proposed topologies of the evolution of ruminants, including an analysis of molecular and morphological data by Liu et al. (2001), are summarized in Fig. 1. There is essentially an absolute support from molecular data for the monophyly of the classical ruminant taxa: ruminants, pecorans (ruminants without chevrotain, Tragulus), cervids and bovids. But the relationships within the pecorans (bovids, cervids, giraffids, and pronghorn) is unstable, although from the comprehensive evidence presented by Matthee et al. (2001) it may be concluded that bovids group with cervids, and that the giraffids and pronghorn diverge first, either separately or as a monophyletic clade. Ribonuclease is a digestive enzyme, secreted at a high level by bovine pancreas. Two paralogous enzymes have been isolated from other bovine tissues, namely seminal (Suzuki et al., 1987) and brain ribonucleases (Watanabe et al., 1988), which are products of two gene duplications in an ancestral ruminant (Breukelman et al., 2001). Bovine seminal RNase is synthesized at high levels in seminal vesicles (DÕAlessio et al., 1972). Bovine brain RNase is expressed in the brain (Sasso et al., 1991) and in lactating mammary glands (Zhao et al., 2001). In previous papers we published nucleotide sequences of ribonucleases from several true ruminants or pecora (Breukelman et al., 1998; Confalone et al., 1995), chevrotain (Breukelman et al., 2001) and other artiodactyls (Kleineidam et al., 1999a). Here we present sequences of pronghorn ribonucleases, not only of the pancreatictype gene, but also of the paralogous brain-type gene, and present evidence that the gene encoding the seminaltype gene may be deleted in this species. Evolutionary trees were derived of ruminant ribonucleases and of recently deposited sequences of ruminant prion proteins (Raymond et al., 2000; Wopfner et al., 1999), and compared with those presented in Fig. 1.
2. Materials and methods Genomic DNA of pronghorn (A. americana) was isolated (Bothwell et al., 1990) from ethanol-preserved tissue, kindly provided by Dr. F. Catzeflis (Montpellier, France). Amplification of ribonuclease 1 sequences was performed by the hot-start procedure (Ampliwax PCR Gem 100, Perkin–Elmer) with different oligonucleotide pairs (Eurosequence BV, Groningen) in different reactions. As forward primers oligonucleotide B/SP (50 -GGGGATCC GGGTCCAGCCTTCCCTGGG-30 ), with a BamHI site (in italics) added by a linker (Breukelman et al., 1998), or oligonucleotide 5SP (50 -ATGGCTCTGCAGTCC/ TCT-30 ), encoding the N-terminus of the signal peptide (amino acids )26 to )21) of the three paralogous enzymes of ox with an internal PstI site (in italics), were used. As reverse primers, oligonucleotide E/B (50 -GGGA-
ATTCCTTGAG TTATTGCCCTCAAGTCAGGG-30 ), containing a linker with a EcoRI site (in italics) (Breukelman et al., 1998) and oligonucleotide AS (50 -GT/ CTCGGCCT/CAGGT G/AGAGA-30 ) (Confalone et al., 1995), were used. Oligonucleotide PrS (50 -CTGGCTTG CATTTCCCC-3), which is complementary to the region of nt 111–127 of various seminal-type ribonuclease genes, was used as a reverse primer for an effort to amplify part of the seminal-type ribonuclease sequence of pronghorn. PCR amplifications were performed in a total volume of 100 ll containing 500 ng high mol. wt. genomic DNA, 100 pmol of forward primer, 100 pmol of reverse primer, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris–Cl (pH 9.0), 1.5 mM MgCl2 , 0.01% gelatin, 0.1% Triton X-100, and 0.5 U Super Taq polymerase (HT Biotechnology). A denaturation step of 5 min was followed by 30 cycles of 1 min at 94 °C, 1 min 47–53 °C, and 1 min at 72 °C. The last segment was extended by 5 s per cycle. The specific products were purified by electrophoresis, extracted by centrifugation and cloned into pCR 2.1 (Invitrogen). DNA was sequenced by the dideoxynucleotide method (Sanger et al., 1977) using the sequencing kit (Pharmacia). The new sequences reported were the result of three independent PCRs, and were deposited in the EMBL GenBank under accession numbers AJ271301 (pancreatic ribonuclease) and AJ271302 (brain-type ribonuclease). Nucleotides 1–372 of the mature pancreatic and braintype ribonuclease sequences of ox (Carsana et al., 1988; Sasso et al., 1991), sheep, giraffe (Confalone et al., 1995), hog deer, roe deer (Breukelman et al., 1998), pronghorn, and chevrotain (Breukelman et al., 2001) were aligned, and treated by parsimony and likelihood analyses, using the programs PAUP Version 4.0b4a (Swofford, 1998). The PROTPARS program of PAUP, which takes into account codon differences between amino acids, was used for parsimony analysis of translated protein sequences. Confidence levels were estimated using bootstrap percentages (BP: Felsenstein, 1985) with the parsimony approach (heuristic search, 1000 replicates) and with the maximum likelihood approach (100 replicates). Aligned sequences of several other molecules from ruminants and other artiodactyls obtained from the nucleotide database were analyzed similarly.
3. Results and discussion 3.1. Pronghorn ribonucleases A ribonuclease-specific amplification product of 471 base pairs (bp) was obtained with primers 5SP and AS. The sequences of the clones obtained with this amplification product were determined, all encoding a sequence that showed the highest identity with the bovine pancreatic ribonuclease gene. Fig. 2A shows the nucleotide
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Fig. 2. Nucleotide sequences encoding ribonucleases in pronghorn. (A) Pancreatic ribonucleases of ox (Ox-A) and pronghorn (Pr-A). (B) Brain-type ribonucleases of ox (Ox-B) and pronghorn (Pr-B). The primary structures of bovine pancreatic and brain ribonucleases and the predicted ones of pronghorn are indicated in the one letter code. I, identical nucleotide; stop, stop codon; -, deletion of one nucleotide.
sequence encoding a part of the signal peptide and the complete mature protein compared with the same part of the bovine ribonuclease gene (Carsana et al., 1988). A high percentage of identity (93%) can be observed between these two genes. The amino acid sequence of bovine pancreatic ribonuclease and the predicted amino acid sequence of pronghorn pancreatic ribonuclease, which is identical to the previously determined one by Beintema et al. (1979), are also presented in Fig. 2A. The ratio of non-synonymous to synonymous substitutions in pronghorn pancreatic RNase compared with bovine ribonuclease A is 1:2. This ratio is in agreement with the ratio expected for a gene having a function.
A sequence that showed the highest identity (93%) with the bovine brain ribonuclease gene was obtained with clones containing a ribonuclease-specific amplification product of 490 bp with primers 5SP and E/B. This nucleotide sequence compared with the same part of the bovine brain ribonuclease gene (Sasso et al., 1991) is shown in Fig. 2B, together with the amino acid sequences of the two encoded proteins. In previous studies of other pecoran species the used primer pair also yielded ribonuclease-specific amplification products of about 580 bp (Breukelman et al., 1998). Now this product was not found and therefore the sequence of pronghorn brain-type ribonuclease presented in Fig. 2B
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Fig. 2. (continued)
does not include the C-terminal region of the molecule and the stop codon. None of the primer combinations 5SP-AS, 5SP-E/B, B/ SP-AS, or B/SP-E/B yielded a seminal-type ribonuclease sequence in pronghorn. Therefore oligonucleotide PrS, complementary to the region of nt 111–127 of several ruminant seminal-type ribonucleases and encoding amino acids 37–43 of this protein, was designed. With this primer and primer 5SP or primer B/SP it was still impossible to detect a seminal-type ribonuclease sequence.
Most ruminant seminal-type ribonucleases show characteristics of pseudogenes with deletions and insertions in the coding region of the gene resulting in frameshifts (Breukelman et al., 1998; Kleineidam et al., 1999b). Our failure to find a seminal-type ribonuclease sequence in the pronghorn genome might be explained by deletion of the complete gene and not only part of it. Therefore we looked again at previously obtained Southern blots of genomic DNA of pronghorn and other ruminants with ribonuclease specific probes, pre-
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sented in Figs. 1–3 in Breukelman et al. (1993) and available in unpublished material. These figures show three cross-reacting bands in investigated ruminants: ox, water buffalo, sheep, goat, black-tailed deer, and giraffe, when a probe is used encoding amino acids 4–124 and a few nucleotides downstream of the stop codon of pancreatic bovine ribonuclease, while probes with a sequence of the 30 untranslated region of this protein react less with brain-type ribonuclease genes. However, in Fig. 1D in Breukelman et al. (1993), which shows Southern blots of pronghorn, not three but two bands are visible. This suggests indeed that the seminal-type gene in this species is deleted completely. But concluding from Southern blots that a gene is absent may be erroneous as we experienced in investigating the number of genes encoding camel ribonucleases by Southern blotting (Breukelman et al., 1993) and PCR amplification (Kleineidam et al., 1999a).
Fig. 3. Maximum-parsimony (A) and maximum-likelihood tree (B) of tandemly aligned nucleotide sequences of pancreatic-type and braintype ribonuclease sequences of chevrotain (Tragulus javanicus; outgroup), pronghorn (A. americana), giraffe (G. camelopardalis), ox (Bos taurus), sheep (Ovis aries), hog deer (Axis porcinus), and roe deer (Capreolus capreolus). Bootstrap percentages are indicated at the nodes.
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3.2. Phylogenetic analyses Phylogenetic trees of nucleotide sequences of pancreatic-type and brain-type ribonucleases from pronghorn and other ruminants were derived with the maximum-parsimony method, with the chevrotain sequence as outgroup. Four most parsimonious tree were obtained (results not shown). But as previously observed (Breukelman et al., 1998, 2001), there was no congruency between the pancreatic-type and brain-type ribonuclease subtrees. Tandemly aligned nucleotide sequences of the two ribonuclease types were investigated with the maximumparsimony and maximum-likelihood methods. Only one tree was found with the maximum-parsimony method, which is presented in Fig. 3A. The two bovid sequences are not monophyletic in this tree, and the bootstrap percentages are rather low. Pronghorn diverges first, while giraffe groups with the deer sequences. A maximum-parsimony tree of translated amino acid sequences derived with the PROTPARS method also yielded one tree with the same topology. The maximum-likelihood tree has another topology (Fig. 3B), with giraffe and pronghorn grouping together and these two again with a monophyletic clade of the two bovids. But the bootstrap percentages are low. It is interesting that the topology of this latter tree is identical to the one presented in Fig. 1A, derived from amino acid sequences of pancreatic ribonucleases. We also derived trees both from nucleotide sequences and amino acid sequences (PROTPARS method) of other molecules present in the database, and obtained similar results as presented in the original publications by Randi et al. (1998), Cronin et al. (1996), and Gatesy et al. (1996, 1999). A phylogenetic analysis of prion protein sequences of artiodactyls and other mammals was published (Wopfner et al., 1999) before a pronghorn sequence was available. We made an analysis including the latter species. As no sequence of the prion protein from a chevrotain species (Tragulus) is yet available in the database, we used the camel and pig sequences as outgroups for our analyses. The deposited sequence of the giraffe prion protein is shorter at both termini than the other ones. Therefore we used an alignment with a 50 sequence corresponding with the CKKRP amino acid sequence (which includes the C-terminal residue of the signal peptide and the N-terminus op the mature sequence) and a 30 sequence corresponding with the ILLISFL amino acid sequence, which lacks six amino acid residues before the stop codon in other deposited sequences. An interesting feature of prion proteins is a repeated octapeptide sequence GGGWGQPH. This repeat occurs four times in most investigated artiodactyl prion proteins, and five times in those from the majority of investigated alleles of ox and several closely related species, and in those from goitered gazelle, lama, giraffe (Wopfner et al., 1999) and prong-
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Fig. 4. Maximum-likelihood analysis of nucleotide sequences (706 positions) of prion proteins of pig (Sus scrova, LO7623), camel (Camelus dromedarius, CDPRPCELF), giraffe (G. camelopardalis, AF113942), pronghorn (A. americana, AF156187), American elk (Cervus elaphus nelsoni, AF156183), ox (Bos taurus; D10614), and sheep (Ovis aries, AF180389). Bootstrap percentages of the maximumparsimony (with identical topology of the tree; upper values) and maximum-likelihood analyses (lower values) are indicated at the nodes.
However, the phylogeny of the four pecoran taxa: Bovidae, Cervidae, Giraffidae, and Antilocapra, remains unresolved. In most analyses pronghorn (Antilocapra) diverges first, either separately, or together with giraffe (Gatesy et al., 1999; Matthee et al., 2001). The latter topology is supported by other molecular features, like the presence of three rarely observed amino acid acid replacements in pancreatic ribonucleases, and the presence of an additional octapeptide repeat in prion proteins. Thus not only sequence data, but also other molecular features are valuable for deriving phylogenetic relationships (Van Dijk et al., 2001), as also Matthee et al. (2001) conclude from the analysis of indels in the sequences determined by them. Another point is that the four pecoran taxa diverged in a rather short time span about 30 million years ago (Breukelman et al., 2001). May it be that the three successive divergences occurred in populations of an ancestral species, which still had the capacity for interbreeding, and that therefore separate genes may show different phylogenies?
Acknowledgments horn. So the number of repeats is not a strong phylogenetic feature. C-terminal of these repeats is a heptapeptide sequence GGGGWGQ in ruminants and pig, and GGGWGQ in camel. Nucleotide sequences of prion proteins were analyzed by the maximum-parsimony and the maximum-likelihood methods. The former method yielded only one tree with an identical topology as obtained with the latter method. The maximum-likelihood tree is presented in Fig. 4, with bootstrap percentages obtained with both methods. In these trees giraffe diverges first, followed by pronghorn, while deer and bovids group together.
4. Conclusion In all analyses which include chevrotain (Tragulus) and other ruminant and non-ruminant artiodacyl sequences, this taxon groups with the ruminants as first diverging clade (Fig. 1, Breukelman et al., 2001). The deer (Cervidae) are always found to be monophyletic, but the Bovidae are sometimes found to be diphyletic (Figs. 1C and 3A). This may improve with a broader taxon sampling of bovid taxa. A phylogenetic analysis of amino acid sequences of pancreatic ribonucleases from ten bovids yielded a monophyletic taxon, with a topology corresponding to other biological data (Beintema and Lenstra, 1982; Beintema et al., 1986). The broader taxon sampling in our previous protein studies may be superior to the increase in number of analyzed nucleotide positions in the current work.
This research was supported by the European Community Project ERB-FMRX-CT98-221. We thank Dr. W.T. Stam, Department of Marine Biology for his advice using the PROTPARS program.
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