Cloning, Expression Of ALEX2 CDNA And Purification Of The Protein And Its Bioinformatics Analysis

ALEX2 (Arm proteins lost in epithelial cancers, X chromosome. 2) is the member of the ALEX family of proteins which includes ALEX1, ALEX2, and ALEX3. Members of the ALEX family of proteins were found to co-localized at the same region in Xq21.33-q22.2 and they all cotain arm repeat domains. Li the N-terminal parts of the proteins they all have putative transmembrane domain. ALEX1 and ALEX2 transcripts are found in almost all human tissues except peripheral blood leukocytes. The expressions of ALEX proteins'mRNA are either significantly reduced or completely lost in human carcinomas but not in other tumor types. This suggests that the ALEX proteins play a role in tumor suppression, possibly by being involved in the regulation of normal cell growth.The arm repeats were first noticed in the segment polarity gene armadillo in Drosophila, which is required both for cell polarity and for morphogenesis during Drosophila embryogenesis. Arm repeat proteins have a conserved three-dimensional structure: each arm repeat is an amino acid motif of about 42 residues. A single arm repeat consists of three a -helics. Tandem arm repeat fold together and interact extensively with one another to form a right-handed superhelix of helices, which creats a surface for protein-proteininteractions. A number of proteins cotaining homologous motifs have been cloned and sequenced. Their common feature is an arm repeat which has been identified in 6-13 imperfect tandemly repeated units in all members of this family. Members of this family have been implicated in a variety of processes such as tumorigenesis, development and maintenance of tissue integrity.In our research, we explored the background of ALEX2 through gene cloning, bioinformatic analysis, and in situ hybridization study. Four experiments were included as follows:1. By bionformatic analysis, we found ALEX2' cDNA sequence had an open reading frame(ORF) encoding putative 632 amino acid protein, and ALEX2 had at least six exons with the ORF locating in one exon. Sequence analysis of the ALEX2 polypeptide revealed an armadillo repeat domain, and a transmembrane domain in its N-terminal parts. Phylogenetic analysis showed that ALEX2 was more closely related to ALEX1.2. Using hybridization in situ, we analyzed the expression of ALEX2 mRNA in tissues of prostate and breast ALEX2 mRNA was found mainly located in cytoplasm and distributed granularly and sporadically: all prostate hyperplasia specimens were stained positively. The positive staining was found only in one case of prostatic adenocarcinoma samples. The expression of ALEX2 mRNA was observed in parts of normal breast specimens, and no expression was found in breast carcinoma. The expresson pattern of ALEX2 mRNA was similar with its family proteins, which suggests that ALEX2 may play an important role in the process of tumorigenesis and development.3. The total RNA was extracted from human placenta brain tissue. Then the 440bp fragment of cDNA encoding ALEX2 was amplified by RT-PCR method using the isolated total RNA as the template. The PCR product was cloned into PGEM-T easy vector and the DNA sequence was found...