Molecular Biology and Evolution

MBE Advance Access originally published online on June 27, 2003

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Mol. Biol. Evol. 20(9):1556-1563. 2003
DOI: 10.1093/molbev/msg168
© 2003 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

A Transposable Element-Mediated Gene Divergence that Directly Produces a Novel Type Bovine Bcnt Protein Including the Endonuclease Domain of RTE-1

Shintaro Iwashita^*,,, Naoki Osada^,1, Tomohito Itoh, Mariko Sezaki^*, Kenshiro Oshima^||, Etsuko Hashimoto^||, Yuko Kitagawa-Arita^||, Ichiro Takahashi, Tohru Masui^¶, Katsuyuki Hashimoto and Wojciech Makalowski^#

^* Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan
Yokohama National University, Yokohama, Japan
National Institute of Infectious Diseases, Tokyo, Japan
Shirakawa Institute of Animal Genetics, Fukushima, Japan
^|| Hitachi Instruments Service Co., Ltd., Tokyo, Japan
^¶ National Institute of Health Sciences, Tokyo, Japan
^# Pennsylvania State University

E-mail: siwast{at}libra.ls.m-kagaku.co.jp.

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
Ruminant Bcnt protein with a molecular mass of 97 kDa (designated p97Bcnt) includes a region derived from the endonuclease domain of a retrotransposable element RTE-1. Human and mouse Bcnt proteins lack the corresponding region but have a highly conserved 82-amino acid region at the C-terminus that is not present in p97Bcnt. By screening a bovine BAC library, we found two more bcnt–related genes: human-type bcnt (h-type bcnt) and its processed pseudogene. Whereas the pseudogene is localized on chromosome 26, both bcnt^p97 and the h-type bcnt genes are found on bovine chromosome 18, a synteny region of human chromosome 16 on which human BCNT is localized. Complete nucleotide sequencing of the BAC clone reveals that the bcnt^p97 and h-type bcnt genes are located just 6 kb apart in a tandem manner. The two h-type bcnt and bcnt^p97genes are active at both the transcriptional level and the protein level. H-type bovine Bcnt is more like human BCNT than p97Bcnt, when compared at their N-terminal regions. However, phylogenetic analysis using the N-terminal region of the bcnt gene family revealed that the duplication of bovine genes occurred within the bovine lineage with significantly accelerated substitution in bcnt^p97. This acceleration was not ascribed definitely to positive selection. After duplication, one of the bovine bcnt genes recruited the endonuclease domain of an intronic RTE-1 repeat accompanied by the accelerated substitution at the 5'-ORF, resulting in creation of a novel type of Bcnt protein in bovine.

Key Words: gene duplication • retrotransposable element-1 • bovine • pseudogene • relaxed evolution

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
Gene diversity is accompanied by gene duplication or polyploidization (Sidow 1996) resulting in nonfunctionalization (pseudogene) or neofunctionalization for preservation. The concept of subfunctionalization as an alternative way of preserving duplicated genes has been proposed to resolve the distinctively different rates between the remaining duplicated genes and the mutation rate for preservation by beneficial change (Force et al. 1999). This concept is complementation of degenerate elements in the duplicated gene.

Transposable elements (TEs), such as a short or long interspersed DNA sequence elements (SINE or LINE), may generate many subfunctional alleles and thus are key factors in gene divergence (Makalowski 2000; Long 2001). Although a recent survey of vertebrate cDNAs suggests many TE-derived cassettes within open reading frames (ORF), no exhaustive functional study has been done to confirm the biological significance of such cassettes (Sorek, Ast, and Graur 2002; Lorenc and Makalowski 2003). Bcnt, named after Bucentar, with a molecular mass of 97 kDa (now designated p97Bcnt), was discovered as the first protein that includes a domain derived from the Ruminentia-specific retroposon (Szemraj et al. 1995; Nobukuni et al. 1997; Takahashi et al. 1998). The recruited cassette coincides with the endonuclease domain of an RTE-1 element, a class of LINEs that is widely distributed from C. elegans to mammals (Youngman, van Luenen, and Plasterk 1996; Malik and Eickbush 1998; fig. 1). Human and mouse Bcnt proteins (also called CFDP1) lack the unique region but have a highly conserved 82-amino acid (aa) region at the C-terminus that is not present in p97Bcnt (Takahashi et al. 1998; Diekwisch et al. 1999). In addition, whereas p97Bcnt contains two intramolecular repeat (IR) units at the C-terminus, human and mouse Bcnt proteins contain only one IR unit in the middle of the molecule. The large difference in the structural organization between ruminant p97Bcnt and other mammalian Bcnt proteins raises the question of whether another copy of the bcnt gene that preserves the ancestral gene structure exists in the bovine genome. Therefore, we searched for an ancestral bcnt gene that would include sequences corresponding to the highly conserved C-terminal region by screening a bovine BAC library.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Schematic representation of the structural relationships among p97Bcnt, human BCNT, and ORF structures and the enzymatic domains of retrotransposable L1 and RTE-1 elements. The numbers above the rectangles of p97Bcnt and human BCNT indicate aa residues. The gray box in the middle of p97Bcnt shows the region derived from the apurinic-apyrimidic endonuclease (APE) domain of RTE-1. The N-terminal shaded boxes show the acidic regions, and the horizontal striped boxes represent intramolecular repeat (IR) units consisting of 40 aa. The black box at the C-terminus of human BCNT indicates a conserved 82-aa region. The APE and reverse transcriptase (RT) domains are indicated by gray and meshed regions, respectively. The L1 element has another restriction enzyme–like endonuclease domain (REL-ENDO) in the C-terminus. The assignment of these domains (APE, RT, and REL-ENDO) is according to Malik and Eickbush (1998)

 
In this article, we show two more bcnt-related genes in the bovine genome in addition to bcnt^p97. One is an active h-type bcnt gene that encodes a 297-aa protein with the conserved 82-aa C-terminus, and the other is its processed pseudogene. Furthermore, by presenting the genomic nucleotide sequence of bcnt^p97and h-type bcnt, their chromosomal localization, and the expression of their mRNAs and proteins, we provide evidence that the integration of an RTE-1 element results in a novel type protein with an endonuclease domain of RTE-1.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
Isolation of Chicken bcnt cDNA
An E1 chick embryo cDNA library (RZPD number 573, Berlin) was screened with the full-length mouse bcnt cDNA (Takahashi et al. 1998), which was excised from pT7T3D by Not I/Xho I digestion.

Isolation of Bovine BAC Clones
A bovine BAC library (RZPD number 750) was screened by two probes, the full-length mouse bcnt cDNA as described above and a 200-bp polymerase chain reaction (PCR) fragment corresponding to part of the C-terminal region of the h-type bovine bcnt cDNA (see Results).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
MDBK cells, a bovine cell line from adult kidney, were cultured in F-12 medium supplemented with 10% fetal calf serum as described previously (Iwashita et al. 1999). Total MDBK cell RNA was isolated with an RNeasy kit (Qiagen). RT-PCR was carried out with a kit (Life Technol.) using the above RNA and a set of primers (sense 5'-cagcatggaggaattcgactcyraaga-3' and anti-sense 5'-tcaaggtttcattttgctcagcctgagatc-3') for h-type bovine bcnt and a set of primers (sense 5'-cgctgcggactggggggagttgcggagtca-3' and anti-sense 5'-tttatttcacaaactgtatcagctttcagc-3') for bcnt^p97. All primers were designed based on the common nucleotide sequences of cDNAs among bcnt^p97, human BCNT and mouse bcnt.

Northern Blotting Analysis
Poly⁺A RNA was prepared from the total RNA of MDBK cells by oligo (dT)-Latex (Roche Japan), and 2.5 µg was transferred to a membrane (Bidyne plus) after separation in a 1% agarose gel with formaldehyde according to the manufacturer's protocol. To prepare a probe, an RT-PCR fragment of 904 bp corresponding to the ORF of h-type bovine bcnt was cloned once into pGEM-T (Promega). The fragment was excised by Eco RI/ Spe I digestion and subjected to random primer labeling (Amersham) with [-³²P] dCTP (specific activity of the probe: 2.4 x 10⁹dpm/µg). Hybridization was carried out in buffer (Stratagene) for 2 hr at 60°C. The filter was washed with 2x SSC containing 0.1% SDS and then with 0.1x SSC containing 0.1% SDS at 60°C. The image was processed using a Fuji Image analyzer (FLA-2000).

Genomic Analysis of BAC Clones
The BAC DNA was purified twice by CsCl₂ centrifugation and subjected to fragmentation by shearing force (HydroShear, Genemachine). The two sheared DNA populations, approximately 2 kb or 5 kb in length, were ligated to the pUC18 vector at the Sma I site after blunt-ending of the fragments and dephosphorylation of the vector with bacterial alkaline phosphatase. The ligated products were electroporated into E. coli DH5. The DNA inserts of pUC18 were amplified by PCR using a set of M13 forward and reverse primers. The PCR fragments were used for sequencing analysis as template DNA after treatment of the reaction mixtures with exonuclease I and shrimp alkaline phosphatase. The total sequence was obtained from a combination of 2,016 end sequences (giving 9.8x coverage) from the shotgun library using an M13 universal primer and Big dye terminator in an ABI3700 sequencer (Applied Biosystems). Sequence assembly was accomplished using PHRAP software (Ewing et al. 1998). The two gaps in the above assembly were closed by direct sequencing of BAC DNA using the primers designed based on the neighboring sequences.

Preparation of Antiserum Against h-Type Bcnt
Antibody against the cystidinyl peptide (EELAIHNRGKEGYIERKA, 18 aa located at the C-terminus of human BCNT) was prepared in a guinea pig (Hartley, 5-week-old) by injecting the peptide coupled to keyhole limpet hemocyanin through the cysteine residue three times (Takara, Ootsu). Brain extracts of S-100 from bovine and Wistar rats were prepared as described previously (Kobayashi et al. 1993; Nobukuni et al. 1997).

Immunoblotting
Western blotting was carried out using CDP-star (Tropix, Bedford) as a substrate. Alkaline phosphatase-linked goat anti-mouse IgG (Tropix, Bedford) and goat anti-guinea pig IgG (Jackson Immuno Research lab.) were used to detect p97Bbcnt and h-type Bcnt, respectively.

Fluorescence In Situ Hybridization (FISH) Analysis
BAC DNAs were labeled by nick translation with Spectrum Green-dUTP or Spectrum Orange-dUTP (Vysis) according to the manufacturer's protocol. Metaphase chromosomes from bovine lymphocytes were prepared by bromodeoxyuridine-thymidine double block and stored at -80°C until use. The chromosomes were denatured on a glass slide by incubation in 70% formamide in 2x SSC solution at 75°C for 5 min, quenched in ice-cold 70% ethanol, and dehydrated through ice-cold 100% ethanol. The labeled DNA probes were denatured with salmon testes DNA and bovine Cot5 DNA at 75°C for 10 min in LSI/WCP hybridization buffer (Vysis) and put on the denatured slide, covered with parafilm, and incubated overnight at 37°C. The slide was washed 3 times for 10 min in 50% formamide in 2x SSC, and for 10 min in 2x SSC and 0.1% NP-40 in 2x SSC for 5 min at 37°C, after rinsing in 2x SSC at room temperature. The slide was counterstained with DAPI (Vectashield, Vector Laboratories) and covered with a glass coverslip. FISH images were observed under an Axioplan2 fluorescence microscope (Zeiss) equipped with a cooled CCD camera (Hamamatsu Photo). Digitized images were captured separately and merged using the ISIS digital image analysis system (Zeiss).

Phylogenetic Analysis
The chicken bcnt cDNA with an incomplete 5' region was set as an outgroup for phylogenetic tree construction. We used a part of each N-terminal region corresponding to human BCNT (66–177th aa), h-type bovine Bcnt (66–175th aa), p97Bcnt (65–173th aa), and mouse Bcnt (66–174th aa). These sequences were aligned using ClustalX with default parameters (Thompson et al. 1997) and the obtained alignment was used to calculate the phylogenetic tree. The Neighbor-Joining method (Saitou and Nei 1987) was used as implemented in MEGA suite (Kumar, Tamura, and Nei 1994). Kimura's 2-parameter model was used for evolutionary distance correction (Kimura 1980), and 1,000-replica bootstrap was used for tree topology significance. For testing positive selection, the nucleotide sequences of ORFs corresponding to first four exons were used: 1–531 nt for human BCNT, 1–525 nt for h-type bovine bcnt, and 1–519 nt for both bcnt^p97and mouse bcnt. These sequences were aligned using ClustalX with default parameters. The obtained alignment was used to construct a maximum likelihood phylogenetic tree by the PAML program package (Yang 1997). For testing positive selection, we used the log-likelihood test by a program implemented in PAML (Yang 1998).

	Results

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
Isolation of the Chicken bcnt Gene
Because human and mouse Bcnt proteins have almost the same C-terminal regions (98% identity over 82 aa), a region not present in bovine p97Bcnt, we estimated the gene organization of the ancestor bcnt by elucidating a chicken homolog. By screening a chicken embryo cDNA library with the full-length mouse bcnt cDNA, we isolated seven candidates, and five of them encoded the IR and C-terminal 82-aa region, while the others were unrelated clones. The C-terminal region is highly conserved, and the IR region is less conserved (fig. 2A and B). These data suggest that the ancestor bcnt should include the highly conserved C-terminal region.

Isolation of the h-Type bcnt and Its Processed Genes in Addition to bcnt^p97
The above results prompted us to search for a prototype bcnt encoding the C-terminal 82-aa region in the bovine genome. Four independent clones were isolated and classified into three groups by direct sequencing: bcnt^p97, human-type bcnt containing the highly conserved 3' ORF, and its processed bcnt gene. The last gene is a fragment very similar to human BCNT cDNA without any intron (in BAC clone 1 and 2), as shown below.

Expression of Both the bcnt^p97and h-Type bcnt Genes in Bovine Cells and Brain
We next examined whether h-type bcnt is expressed in addition to bcnt^p97. We carried out RT-PCR using total RNA from MDBK cells, a bovine kidney epithelial cell line, and detected a band of approximately 900 bp in addition to a 2.9-kb fragment that reflects the nearly full-length bcnt^p97 cDNA (Nobukuni et al. 1997; fig. 3A). Nucleotide sequence analysis of the 904-bp fragment revealed the exact ORF of h-type bcnt. These two expected transcripts were also detected by Northern blotting (fig. 3B). Furthermore, the h-type Bcnt protein was detected in bovine brain extract by immunoblotting with an antibody against the peptide corresponding to part of the C-terminal region (fig. 3C). Both rat and h-type bovine Bcnt proteins show doublet bands with molecular masses of about 43 kDa. The doublet bands might be caused by protein phosphorylation as similar, as in the case of p97Bcnt (Iwashita et al. 1999).

View larger version (38K): [in this window] [in a new window]   FIG. 3. Expression of both p97Bcnt and h-type Bcnt. (A) RT-PCR was carried out using a set of primers for bcntp97 (1 and 2) or a set of primers for h-type bovine bcnt (3 and 4) in the presence (1 and 3) or absence (2 and 4) of cDNAs prepared from total RNA of MDBK cells. The products were separated in 1.2% agarose gel and stained with ethidium bromide. (B) For Northern blotting, Poly+A RNA of MDBK cells was transferred onto a membrane after separation in 1% agarose gel with formaldehyde. The membrane was hybridized with a 32P-labeled 904-bp PCR fragment corresponding to the ORF of h-type bcnt. The filter was washed and its image was processed with an Image analyzer. Size markers (kb) are shown on the left (M). (C) For Western blotting, 40 µg of rat or bovine extract was separated in 12% SDS polyacrylamide gels and subjected to immunoblotting. Two left lanes: with anti-p97Bcnt monoclonal antibodies; four middle and right lanes: with anti-C-terminal region peptide antibody in the absence (two middle lanes) or presence of antigen peptide at a final concentration of 100 µM (two right lanes) to show the specificity. The arrows indicate h-type Bcnts showing doublets, probably a result of the phosphorylation (Iwashita et al. 1999)

 
Characterization of Three bcnt-Related Genes
The bcnt processed gene has exactly the same sequence as part of the h-type bcnt cDNA, except for one base missing, which causes a frameshift and premature stop codon just at the beginning of the IR region, even if the gene is transcribed and translated (data not shown). When the nucleotides in the 5' regions of the ORF corresponding to the N-terminal region are compared, bovine h-type bcnt shows 88% identity to human BCNT and 84% identity to bcnt^p97. The N-terminal region of bovine h-type Bcnt is 88% identical to that of human BCNT and only 72% identical to that of p97Bcnt at the amino acid level (fig. 4). It is noteworthy that the C-terminal 82-aa of h-type Bcnt matches almost completely those of human BCNT and mouse Bcnt, whereas the IR region is variable (fig. 2). As a whole molecule, the h-type bovine Bcnt is 91% identical to human BCNT. These results show that bovine h-type bcnt is more similar to human BCNT than to bcnt^p97.

View larger version (46K): [in this window] [in a new window]   FIG. 4. Similarities among bovine h-type Bcnt, p97Bcnt, human BCNT, and mouse Bcnt. The aa sequences of the N-terminal regions of bovine h-type Bcnt, p97Bcnt, human BCNT, and mouse Bcnt were aligned. The different aa residues in each alignment lane are indicated by shaded boxes, and identical aa residues among the four N-terminal regions are indicated by asterisks (*) at the top of each aligned lane

 

View larger version (41K): [in this window] [in a new window]   FIG. 2. Multiple alignments of the IR and C-terminal regions of the Bcnt family proteins. Bovine h-type Bcnt was identified in this paper. Multiple alignment of p97Bcnt, bovine h-type Bcnt, human BCNT, mouse Bcnt, and chicken Bcnt (AB080800) proteins was achieved using the Clustal W program (Thompson 1997). (A) IR regions: P97Bcnt has two IR units; its N-terminal IR is designated IR-1 and its C-terminal IR is designated IR-2. (B) The C-terminal region. Identical aa residues among the six IR regions or four C-terminal regions are indicated by asterisks (*) at the top of each aligned lane

 
Chromosomal Localization
By Southern blotting of isolated BAC clones with oligonucleotides designed on each exon, we noticed that a BAC clone with a 92-kb insert (clone 4) included both bcnt^p97and human-type bcnt. To clarify the chromosomal relationship of these three bcnt-related genes, bcnt^p97, h-type bcnt, and the processed gene, FISH analysis was carried out using these BAC DNAs. As shown in figure 5, BAC clone 4, which was expected to carry the whole bcnt^p97 and most of h-type bcnt, and BAC clone 3, which was expected to carry the 3' part of h-type bcnt, were mapped on chromosome 18, and the processed gene (in BAC clone 1 and 2) was mapped on chromosome 26. The fact that bcnt^p97 is mapped on chromosome 18 is consistent with previous data obtained by screening a bovine/hamster hybrid panel (Iwashita et al. 2001). We also independently examined the chromosomal localization of h-type bcnt by screening the above bovine/hamster hybrid panel, and the results were consistent with those obtained from the above FISH analysis (data not shown). These data indicate that the bcnt^p97and h-type bcnt genes are localized on chromosome 18, a synteny of human chromosome 16 on which human BCNT maps (Takahashi et al. 1998), whereas the h-type bcnt processed gene is located separately on chromosome 26.

View larger version (61K): [in this window] [in a new window]   FIG. 5. Chromosomal localization of bcntp97, h-type bcnt, and its processed gene. Metaphase chromosomes of bovine lymphocytes were incubated with BAC clone DNAs double-labeled with Spectrum Green-dUTP or Spectrum Orange-dUTP. (A) Green-dye–labeled BAC 1 and orange-dye–labeled BAC 2. (B) Green-dye–labeled BAC 1 and orange-dye-labeled BAC 4. (C) Green-dye–labeled BAC 3 and orange-dye–labeled BAC 1. (D) Green-dye–labeled BAC 3 and orange-dye–labeled BAC 4

 
Gene Organization of bcnt^p97 and h-Type bcnt
We determined the complete nucleotide sequence of BAC clone 4 and revealed the exon-intron relationship as shown in figure 6 (top). However, the clone did not contain the whole h-type bcnt lacking the sequence downstream of the IR region (exon 5) where a large 95 kb intron exists in the case of human BCNT. We found that BAC clone 3 contained exons 6 and 7, resulting in covering the whole h-type bcnt. These results are completely consistent with the above FISH analysis. We also compared the nucleotide sequences with the corresponding region of human chromosome 16 by pairwise blast (fig. 6, bottom), and can summarize the results as follows:

1. The two genes, bcnt^p97and h-type bcnt, are localized just 6 kb apart in a tandem manner.
   
2. The gene organization of h-type bcnt is quite similar to that of human BCNT, and many homologous nucleotide fragments were found in the corresponding introns.
   
3. Six IRs or their related nucleotide fragments were found, including the two IRs in bcnt^p97 (termed IR4 and 5) and one IR 6 in h-type bcnt. The other three IRs (IR1, IR2, and IR3) are located upstream of the 5'UTR of bcnt^p97 (fig. 6). We examined the nucleotide sequences of the neighboring regions of each IR (total 2 kb range, one kb of the 5'-upstream region and one kb of the 3'- downstream region) and found more homologous fragments between IR5 and IR6, than between IR4 and IR6, (data not shown). These results suggest that IR 5 in bcnt^p97 is derived from the ancestor bcnt, whereas IR4 might have been produced by another local duplication.
   
4. Except for the region neighboring the 3'UTR of bcnt^p97 (red boxes in fig. 6, lower), whose corresponding region is not present in either human-type bcnt or human BCNT cDNAs, no significant homologous region corresponding to bcnt^p97 is found in the human genome.
   

View larger version (17K): [in this window] [in a new window]   FIG. 6. Schematic presentation of the gene organization of bcntp97and human-type bcnt, and comparison with its corresponding region in the human genome. The complete nucleotide sequence of BAC 4 with a 92-kb insert was determined. Each exon is indicated by a vertical bar and is numbered. The bcntp97 gene consists of 8 exons, and the human-type bcnt gene consists of 7 exons. Three similar IR units (IR') are located in the upstream 5' region of the bcntp97 gene. (Bottom) Two nucleotide sequences were compared by a pairwise blast program (Tatusova and Madden 1999) with default between the above bovine BAC nt sequence (92 kb) and its corresponding human 92-kb region in gi | 20561873. Red boxes represent the regions neighboring the 3'UTR of bcntp97, whose corresponding region is not present in either human-type bcnt cDNA or human BCNT cDNA

 
Phylogenetic Analysis
The DNA sequence analysis strongly suggests that the RTE-1–involved duplication of the bcnt gene occurred within the bovine lineage and was not an ancient duplication with one member having been lost in human/mouse genome. To examine the evolutionary relationship between p97Bcnt and h-type Bcnt, a phylogenetic tree was constructed (fig. 7A). The tree, based on the limited amino acid sequences of the N-terminal regions, setting a chicken Bcnt as an outgroup, supports the view that the divergence of the bovine bcnt genes occurred relatively recently. Furthermore, it also shows a significantly longer branch in p97Bcnt (Bos with L1) than in h-type Bcnt (Bos h-type), indicating an accelerated substitution rate in the p97Bcnt gene. To examine whether the substitution acceleration in p97Bcnt reflects a positive selection, we used nucleotide sequences of mouse bcnt, h-type bovine bcnt, and bcnt^p97 (Bos with L1) corresponding to 1–531 nt of human BCNT ORF and constructed a maximum-likelihood–based phylogenetic tree (fig. 7B). The maximum-likelihood test indicated that the d_N (non-synonymous substitution per site)/d_S (synonymous substitution per site) values of the bcnt^p97 lineage were significantly greater than those of other lineages. However, the model of positive selection in the bcnt^p97 lineage, where the value of d_N/d_S generally exceeds one, was not significantly demonstrated (fig. 7B).

View larger version (13K): [in this window] [in a new window]   FIG. 7. Phylogenetic analysis and likelihood ratio tests. (A) Phylogenetic tree of the bcnt-related gene family. Amino acid sequences of N-terminal regions of several Bcnt proteins, which correspond to human BCNT 66–177th aa, were aligned using ClustalX with default parameters and subjected to phylogenetic tree analysis by the Neighbor-Joining method. Kimura's 2-parameter model (Kimura 1980) was used for evolutionary distance corrections, and 1,000-replica bootstrap for tree topology significance. (B) Maximum-likelihood estimates. The nucleotide sequences corresponding to the first four exons of the bcnt genes (1–531 nt of human BCNT ORF) were used. These alignments were used to construct a maximum likelihood phylogenetic tree by the PAML program package. The numbers of dN/dS were presented below each branch. The maximum likelihood test for detection of positive selection was performed according to the method described by Yang (1998). The result indicated the acceleration of substitution in the bcntp97 branch (P < 0.001) but dN/dS > 1 in the bcntp97 branch (indicating a positive selection) was not significant (P > 0.05)

 

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
Human and mouse Bcnt proteins have a highly conserved C-terminal region (98% identity over 82 aa residues) which is absent from bovine p97Bcnt. Instead, bovine p97Bcnt includes an RTE-1–derived endonuclease domain followed by two copies of an IR module at the C-terminus (Iwashita et al. 2001; fig. 1). A chicken Bcnt gene structure suggests that the human/mouse gene is an ancestral genomic structure and that the bovine one must be a recent evolutionary addition (fig. 2). Indeed, screening of a bovine genomic library revealed another copy of the bovine Bcnt gene that, in its structure, resembles the ancestral gene—seven exons, including one that codes an IR module (fig. 6). This protein has a highly conserved C-terminus, exactly as in the human and mouse Bcnt proteins (fig. 2B). We also found its processed gene that shows only one bp deletion compared with bovine h-type bcnt cDNA, suggesting that its retroposition must have occurred very recently.

The bovine h-type Bcnt protein shows a molecular mass of about 43 kDa (fig. 3C). As in p97Bcnt (Nobukuni et al. 1997), the apparent molecular size of 43 kDa is significantly larger than the calculated mass of 33 kDa, which might be caused by the slower mobility on SDS-PAGE due to the acidic N-terminal region (Nobukuni et al. 1997; Iwashita et al. 1999). Therefore, although mouse Bcnt has been reported to be 27 kDa (Diekwisch et al. 1999), this might be wrong as a result of a high background. Functional studies showed that both forms of Bcnt are expressed in a number of bovine tissues and that they produce active proteins (Iwashita et al. unpublished data).

Both h-type and p97 Bcnt-coding genes are localized linearly on bovine chromosome 18, and that suggests tandem duplication as a mechanism of p97Bcnt gene creation (fig. 6). Our phylogenetic analysis shows the duplication of an ancestral bcnt gene within the bovine lineage, followed by relaxed evolution of one copy, as seen in the significantly longer branch of the bcntp97 gene (fig. 7A). Many cases have been cited to demonstrate that such an accelerated substitution rate associates with a new functional gene under a positive Darwinian selection after gene duplication (Long and Langley 1993; Ohta 1994; Nurminsky et al. 1998). However, d_N/d_S ratio between bcnt^p97 and h-type bcnt does not strongly support the idea that positive selection operates on the bcntp97 gene (fig. 7B), although the criterion by d_N/d_S > 1 is not absolutely required. Therefore we concluded that the new gene might be under relaxed evolution that enables fast diversification of duplicated genes, although the alternative possibility of adaptive evolution cannot be excluded at this point.

Regardless of relaxed evolution or adaptive evolution, it is likely that, after duplication, one of the duplicated copies lost its 3' exon but instead recruited an RTE-1–derived endonuclease domain. In addition, an IR coding exon got duplicated and another genomic piece was recruited to create an exon constituting the unique 3' part of the p97Bcnt mRNA.

It should be noted that the effect of RTE-1 insertion in the bovine bcnt gene creates a novel protein, whereas TEs are often selfish elements with deleterious effects to the genome (Long 2001). Compared with a long process of gene evolution by nucleotide substitution, a RTE-1 insertion can drastically change the coding region quickly, probably resulting in a new protein function. It is likely that p97Bcnt functions through the two IR domains, and that the ancestral type Bcnt works through the conserved C-terminal region. The described evolution of the Bcnt locus enabled us, for the first time, to infer in detail how the transposable elements contribute to protein divergence (Lorenc and Makalowski 2003). We are now investigating the differential distribution of p97Bcnt and h-type Bcnt in bovine brain tissues.

	Supplementary Material

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
The nucleotide sequences reported in this article appear in the DDBJ/EMBL/GenBank databases as AB080800 (chicken bcnt cDNA), AB081004 (human-type bovine bcnt cDNA), AB081003 (human-type bovine processed bcnt), and AB081095 (a bovine BAC clone including bcnt^p97 and human-type bcnt).

	Acknowledgements

TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 
We are grateful to Tomoko Ohta and Norihiro Okada for suggestions; to Shin-ichi Makino, Kenji Tsuge, and Shinya Kaneko for technical advice; to President Yoshitaka Nagai for ongoing support and encouragement; and to Margaret Dooley-Ohto for editing the manuscript. This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (no. 13575027).

	Footnotes

 
¹ Present address: Department of Ecology and Evolution, University of Chicago.

A portion of this paper was presented as a preliminary report at the 12th International Workshop: Beyond the Identification of Transcribed Sequences, held in Vienna, Virginia, October 25–28, 2002.

Thomas Eickbush, Associate Editor

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TOP Abstract Introduction Materials and Methods Results Discussion Supplementary Material Acknowledgements Literature Cited

 

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Accepted for publication May 13, 2003.

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