* 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
Correspondence: E-mail: siwast{at}libra.ls.m-kagaku.co.jp.
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Abstract |
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Key Words: gene duplication retrotransposable element-1 bovine pseudogene relaxed evolution
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Introduction |
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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.
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Materials and Methods |
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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 bcntp97. All primers were designed based on the common nucleotide sequences of cDNAs among bcntp97, 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 (66177th aa), h-type bovine Bcnt (66175th aa), p97Bcnt (65173th aa), and mouse Bcnt (66174th 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: 1531 nt for human BCNT, 1525 nt for h-type bovine bcnt, and 1519 nt for both bcntp97and 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).
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Results |
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Isolation of the h-Type bcnt and Its Processed Genes in Addition to bcntp97
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: bcntp97, 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 bcntp97and h-Type bcnt Genes in Bovine Cells and Brain
We next examined whether h-type bcnt is expressed in addition to bcntp97. 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 bcntp97 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).
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Discussion |
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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, dN/dS ratio between bcntp97 and h-type bcnt does not strongly support the idea that positive selection operates on the bcntp97 gene (fig. 7B), although the criterion by dN/dS > 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-1derived 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.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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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 2528, 2002.
Thomas Eickbush, Associate Editor
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