**5. Perspectives**

Living organisms have evolved so as to acquire various anti-stress systems in response not only to exogenous stresses, but also to the intrinsic stresses faced by multicelluar organisms for physiological homeostasis. DNA repair systems in all organisms, immune systems in vertebrates, and the placental systems in mammals are some of the most fruitfully acquired systems. Several lines of evidence have shown that the expression of various integrated retrotransposons is induced by environmental stimuli, such as ultraviolet light, heat shock, or macromolecule synthesis inhibitors (Liu et al., 1995; Morales et al., 2003; Häsler et. al., 2007). Although the induction mechanism of expression has not been fully elucidated, these stresses may enhance promoter activity (Morales et al., 2003) or release the suppressive state of expression, resulting in the creation of new genetic materials. If the retrotransposon induction process is combined with tandem gene duplication, it is a much more efficient way to create a novel gene. Under such stressful conditions, novel genetic materials may play a role in adaptation to new environments.

Among the three Bcnt-related proteins in ruminants, the two paralogs p97Bcnt/Cfdp2 and p97Bcnt2 were generated by partial segmental duplication of the ancestral *Bcnt/Cfdp1* gene, followed by the insertion of an order-specific retrotransposon, resulting in the recruitment of the AP-endonuclease domain of the retrotransposon. Based on the 3-D remodeled structures of these recruited RTE domains and comparison of their protein sequences, they probably retain AP-endonuclease activity. Mammalian AP-endonuclease plays a role not only in direct DNA repair, but also in stimulating pathways for anti-stress activities. Although the latter activity depends mostly on the N-terminal 6-kDa region (Tell et al., 2009; Busso et al., 2010) which is dissimilar to those of p97Bcnt/Cfdp2 and p97Bcnt2, they contain intrinsically disordered regions other than the core RTE domains in both the N-terminal and C-terminal regions, including outside of the core RTE domains. These regions may serve as scaffolds for cellular protein-protein interactions and create novel functions as chimeric genes. The two paralogs distribute in both the cytosolic and nuclear fractions. The properties of intrinsically disordered proteins other than the AP-endonuclease domain and wide cellular distribution are similar to those of the APE1/Ref-1 molecule. Based on these considerations, we conclude that p97Bcnt/Cfdp2 and p97Bcnt2 have recruited the AP-endonuclease domain of a retrotransposon, which originally played an essential role in the integration of the retrotransposon into the genome, and that the two paralogs may have utilized APendonuclease activity to suppress cellular stress for survival. The cellular stress that might have induced the retrotransposition of Bov-B LINEs would increase the probability that the newly created genes would become fixed in a population. In addition, because these novel genes have chimeric origins, the original regulatory network of the ancestral *Bcnt/Cfdp1* gene may also have been modified to some extent. Therefore, we hypothesize that the two novel genes have become additional components of pre-existing regulatory networks for anti-stress activities. Although this hypothesis cannot explain why molecules containing the APendocnuclease domain, such as p97Bcnt/Cfdp2 and p97Bcnt2, are so rare despite the advantage of being able to regulate cellular activity, it will be intriguing to examine the functions of the three Bcnt-related proteins based on this working hypothesis.

### **6. Conclusion**

The *Bcnt/Cfdp1* gene comprises a unique gene family with three members in ruminants. The two paralogs, *p97Bcnt/cfdp2* and *p97Bcnt-2*, were created in ancient ruminants by a partial duplication of the ancestral *Bcnt/Cfdp1* gene followed by the insertion of an order-specific retrotransposon, Bov-B LINE. This type of combined process provides great potential to generate a novel gene because a novel function can be acquired under the guarantee of the original gene function. The ancestral Bcnt/Cfdp1 protein contains a highly conserved Cterminus of 82-amino acids (Bcnt-C) that is not present in either p97Bcnt/Cfdp2 or p97Bcnt2. Bcnt-C is found in all eukaryotes where it is known as the BCNT superfamily. A chicken Bcnt/Cfdp1 is a centromere protein while the yeast ortholog is a component of the chromatin-remodeling complex, suggesting that the ancestral Bcnt/Cfdp1 protein plays a role in the regulation of gene expression. The two paralogs, p97Bcnt/Cfdp2 and p97Bcnt2, recruited an AP-endonuclease domain of the retrotransposon during their generation process as a ~325 amino acid region (RTE domain) in the middle of the molecule. The three Bcnt-related proteins distribute in both the cytosolic and nuclear fractions, and include intrinsically disordered regions other than the core of RTE domains of the two paralogs. The 3-D structures of the core RTE domains can be remodeled as canonical AP-endonucleases with identical catalytic amino acid residues. Although as yet there is no direct evidence for it, the two paralogs probably retain AP-endonuclease activity. Because AP-endonuclease/ Redox effector factor 1 is one of the major regulators of cellular responses to various stresses, we propose that the recruited AP-endonuclease domains, which may have emerged in response to cellular stresses, may be utilized by the paralogs in cellular regulation. Therefore, the three Bcnt-related family members provide a good opportunity to examine dynamic changes in signaling networks that accompany novel genes.
