The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus Laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes start at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed both in the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 only in the cytoplasm, indicating their different functions. Genetic mutations in human gene microcephalin1 (MCPH1) cause primary microcephaly, and during evolution of human being, the fast changes in this gene play important roles in the enlargement of brain size and the increase of cognitive ability. But the functions of MCPH gene in other species are still not clear. We cloned the Xenopus laevis MCPH A and B genes. Paralog B encodes a truncated form of MCPH and it is not known whether it is functional or not. The domain structure of MCPHA and the BRCT domains are well conserved suggesting its conserved role in development. However, in situ hybridization study showed that Xenopus MCPH genes are expressed in neural placode region at neurula stage and later in the branchial arch regions, but not clear in the brain. In the mouse, MCPH is reported to be strongly expressed in the brain. This clear difference might suggest different roles for MCPH during evolution. To screen for interacting factors of these proteins, we constructed a Xenopus yeast two-hybrid cDNA library. When synthesizing the double-strand cDNA, we used modified random primers and specifically designed adaptors. A complete SalI site formed at the 3’-end of the cDNA when the primer, and the adaptor ligated properly. The cDNAs were then introduced into the linearized vector directionally. We checked the ratio of empty colony, the average size of inserts, and the presence of several known gene in the library, the results showed that the construction of the cDNA library was successful.
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