| Large-scale sequencing of cDNA library has been proved to be a powerful tool for evaluation of the gene expression profile and identification of novel genes. For better understanding the biological functions and their related mechanism of human dendritic cells, a system for large scale sequencing of human dendritic cell cDNA library was established, and a novel full-length cDNA, calcium/calmodulin-dependent protein kinase II inhibitor protein (CaM-KIIN), was isolated from human dendritic cells by large-scale random sequencing of cDNA library.1. Cloning and mRNA expression analysis of human CaM-KIINHNA5B4 cDNA isolated from human dendritic cell cDNA library was composed of 591 base pair (bp) and contained a complete open reading frame (ORF) of 240bp potentially encoding a 79-aa protein. The protein has a molecular mass of 8657.69 Dalton and an isoelectric point of 5.16. The protein exhibited significant homology to the known Rattus norvegicus CaMK II inhibitor protein beta and alpha (98% identity and 98%positives, 65% identity and 78% positives, respectively). Based on its sequence similarity to the known CaMK II inhibitor proteins and its inhibition capacity of cell growth, the protein was dignated as human calcium/calmodulin-dependent protein kinase II inhibitor protein (CaM-KIIN). As illustrated by the hydrophobicity plot, the protein showed obvious hydrophilicity, and no signal peptide was predicted.The mRNA expression pattern of CaM-KIIN was analyzed by RT-PCR and Northern blot analysis. RT-PCR results showed differences in the intensity of message expression in different cells. In freshly isolated cells, relatively strong signal was seen in KLH-stimulated PBMC-derived DC, with lower levels of expression in unstimulated mature and immature DC. The expression was not detectable in monocytes, T cells and B cells. Different levels of CaM-KIIN expression were seen on cell lines including hemopoietic cell lines and some tumor cell lines, such as MCF-7, A172, HeLa, SMMC7721, PC3, A549, THP-1, U937, Jurkat, Raji and Daudi. No mRNA expression was detected in LoVo,CaoV-3, HL60, NB-4, and Reh.Northern blot analysis of CaM-KIIN showed a major band of 5.2kb. mRNA was detected in greatest quantities in kidney, liver, HeLa cell S3, lymphoblastic leukemia MOLT-4 and Burkitt's lymphoma Raji cells. Moderate expression observed in heart, skeletal muscle, placenta, and chronic myelogenous leukemia K-562 cells. Very low levels of expression were found in small intestine, colorectal adenocarcinoma SW480 and lung carcinoma A549. No expression in colon (no mucosa), thymus, spleen, lung, peripheral blood leukocyte, promyelocytic leukemia HL60 and melanoma G361. In brain, there is no 5.2kb but a strong 1.65 kb message expressed, suggesting an isoform of CaM-KIIN mRNA was detected. The same 1.65 kb transcript was also seen in HeLa and Raji cells. The high strigency washes used for this Northern blot tend to suggest that this is not due to a related gene, but rather to the presence of two transcripts.2. Fuctional study of human CaM-KIINTo further investigate whether human CaM-KIIN might regulate cell functions, we constructed pSPROT2.0 expression vector and transfected into LoVo, NIH3T3, and HeLa cells. Then we examined the difference of the morphology by phase contrast microscopy and the proliferation by MTT incorpration method. The results do suggest that there were significant difference between LoVo cells transfected with CaM-KIIN mRNA and control LoVo cells.We use a 40x phase-contrast objective to observe the effect of human CaM-KIIN on the growth of LoVo and HeLa cells. During the course of experiment, a remarkable change in the morphology of LoVo cells occurred. After 24 hrs of transfection, LoVo cells were less spread out than control and mock cells transfected with the pcDNA 3.1 vector, and contained more granules. After 48 hrs of transfection, cells were smaller and contracted. Necrotic cores appeared in the cells and increased continuously. Many cells are unattached and dead. Most of the cells died a...
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