Dissertation
Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Molecular Cloning and Characterization of Cell Division Cycle Associated8(CDCA8/HESPRG3) Promoter

Author MiaoCongXiu
Tutor LuGuang
School Central South University
Course Genetics
Keywords CDCA8gene promoter human embryonic stem cells transcriptional regulation
CLC Q78
Type PhD thesis
Year 2006
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CDCA8(cell division cycle associated8)/HESPRG3(Homo sapiens pluripotent embryonic stem cell-related gene No3) is thought to play an important role in cell development and cell proliferation. It encodes a protein, termed Borealin/Dasra B, which is components of chromosomal passenger complex. Chromosomal passenger complex shows a dynamic localization pattern during mitosis, appearing on the chromosome arms and inner centromeres from prophase through metaphase and then transferring to the spindle midzone and midbody from anaphase through cytokinesis. Chromosomal passenger complex plays crucial roles during mitosis and cell division. Its abnormalities cause inappropriate chromosomal segregation and cell division which may cause auneuploidy leading to cancer. Chromosomal passenger proteins includes Aurora B, Survivin, INCENP,and Borealin/Dasra B. Functional studies revealed that Borealin is required for targeting of the passenger holocomplx to centromeres, correction of kinetochore attachment errors and stability of the bipolar spindle in human cells. Express pattern anlysis in different tissues and cell lines demonstrated that CDCA8/HESPRG3is expressed highly in human undifferentiated ES cells compared with the differentiated ES cells and lack detectable transcription in human multiple tissues of both the adult and fetus aborted at pregnancy over5months. To investigate transcriptional regulation of CDCA8/HESPRG3, we identified transcription start sites, isolated and characterized the promoter, analyzed cis-acting elements and the effect of the promoter in vivo. We hope that the results will elucidate molecular mechanisms underlying the the mechanism of CDCA8/HESPRG3.Our results are as follows:the transcription start sites of CDCA8/HESPRG3are located at200bp,194bp and175bp upstreame of translation start site, one of which is alternative splicing. A longest2319bp sequence of NM018101is identified in GenBank and represents the full-length transcript of CDCA8/HESPRG3. The basal promoter of CDCA8/HESPRG3is located at-321/-196bp region, that drives the transcription of reporter gene in both human embryonic stem cells(hES) and multiple tumor cells and the highest activity region is located in a1071bp fragment of in length; Transient expression assays in different cells reveals that transcription of CDCA8/HESPRG3is significantly activated in undifferentiated ES cells and cancer cell lines but repressed in differentiated ES cells and normal primary cells. Computer analysis shows that the basal promoter of human CDCA8/HESPRG3contains neither canonical TATA box and nor initiator core element but contains CCAAT box, that is recognized by NF-Y family transcription factors. EMSA(electrophoretic gel mobility shift assay) confirms that the predicted NF-Y binding sites and CREB binding site do have the capability to bind related nuclear proteins. The luciferase activity assay of wild type and mutants shows that NF-Y and CREB are indispensable in the transcription of CDCA8/HESPRG3. The effect analysis of promoter in vivo reveals that the activation of the CDCA8/HESPRG3promoter is cancer-specific and can effectively induce report gene expression in breast tumor in vivo.In summary, we have identified CDCA8/HESPRG3promoter and a cis-acting element. Results establish the basis for further studies on the mechanism regulating the expression of the CDCA8/HESPRG3gene.

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