| CRLP(Crn-like Protein) is a novel gene of complete coding sequence that is isolated from the human placenta cDNA library by a high throughput cDNA transfection screening assay established by our laboratory. The full length cDNA of CRLP is composed of about 2.7Kb nucleotides, which encodes a protein of 855 amino acids and its expected molecular weight is 100 KDa. The Genebank ID number of CRLP is AF258567.Bioinformatics analysis of the nucleotide sequence of CRLP has been done by referring to the database of the Genebank by utilizing appropriate softwares. The result of BLASTn analysis reveals that CRLP has 19 exons and 18 introns, and locates on human chromosome 19p13.3. The data of RH positioning analysis further confirm the above findings. The amino acid sequence analysis indicates that its homologs exist in rattus norvegicus, drosophila, elegans and yeast. The CRLP protein has 15 TPR motifs. The TPR is composed of 34 amino acids, among which 8 residues are highly conservative; while its secondary structure is principally identical.Previous studies have shown that TPR motif can mediate interaction among different proteins and also relates to cell cycle, transcription, protein transportation and other important functions implicating that CRLP may play important biological roles in cell activity.The result of multiple tissue Northern blot shows that the expression of CRLP gene is present in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancrea. The transcription of CRLP has also been tested in the cell lines of hepatocellular carcinoma , transformed hepatocyte , breast cancer, ovarian cancer, osteosarcoma,cervical cancer and hepatoblastoma by RT-PCR. Since CRLP gene is expressed in all these tested tissues andcell lines and structurally high conserved in evolution, it is implied that CRLP is probably a housekeeping gene.Preliminary research has been conducted on the promoter of CRLP. The segment of about 1Kb upstream to the translation initiation site of CRLP has been inserted into pGL3 vector encoding the reporter gene --- Firefly Luciferase without promoter. The data of promoter activity revealed by the dual luciferase reporter gene system indicate that the activity of this segment is 1/33 of SV40 promoter as control.A plasmid which can express the CRLP-EGFP fusion protein is constructed. After transfected with CRLP-EGFP in BEL7402 hepatocarcinoma cell, the protein is located in cell nucleus, implicating its possible function inside the nucleus.In order to study the physiological function of CRLP, its coding sequence is inserted into an expression plasmid pcDNA3.1. The cell growth curve and foci formation assay confirm that the overexpression of CRLP inhibits the cell proliferation. Since TPR motif is involved in transcription repression complex, it is suggested that CRLP might inhibit the cell growth by facilitating the formation of this complex. It has been reported that CRLP might interact with XPA in the DNA repair complex. The relationship between CRLP and DNA repair process has been further studied in cells after DNA damage with UV exposure, the apoptotic cells increase substantially when endogeneous CRLP is inhibited by its antisense sequcence. After cells are treated with chemiotherapy drugs, adriamycin or cisplatin, the cell growth curves show that the expression of CRLP is in favor of the increase of cell drug-resistanceï¼› while the downregulation of CRLP by its antisense sequence can reduce the cell drug-resistance. It is implicated that the participation of CRLP into the DNA repair complex possibly mediated by XPA is essential for assurance of DNA repair activity.The existence of numerous TPR motifs in CRLP indicates its multiple function in cell activity. Its 15 TPR motifs can be subdivided into 3 TPR tandem clusters(D1,D2,D3).For determining the biological significance of TPR motif in different region and TPR number as well as its spatial relationship, 5 truncated mutants(mut1,mut2,mut3,mut4,mut5) with deleted TPR at N-terminus,middle or...
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