Gene Diagnosis Of Oculocutaneous Albinism/characterization Of Cell Division Cycle Associated 8(CDCA8) Promoter And Transcription Factores

Part 1 Gene diagnosis of oculocutaneous albinismObjective To identify the mutations of tyrosinase gene (TYR) and P gene in patients with oculocutaneous albinism.Methods PCR and DHPLC were applied to detect the mutations in all exons of TYR gene and P gene except for exon 1 of the P gene which is noncoding. DNA sequencing and PCR-RFLP were applied to affirm the mutation detected by DHPLC.Results A novel heterozygous mutation (C1349T) of exon 13 in the P gene was detected in patient 1. Patient 2 has the heterozygous mutation (C1349T) of exon 13 and a heterozygous mutation (G2323C) of exon 23 in the P gene. Patient 3 has the heterozygous mutation(G2323C) as well.The PCR-restriction enzyme digestion showed that OliI enzyme digestion location disappeared in patient 1 and patient 2, which verified the results of sequencing analysis. Moreover, there is no changes in OliI enzyme digestion location of the DNA samples obtained from 100 unrelated, phenotypically normal Chinese.Conclusion The heterozygous mutation (C1349T) is a novel mutation which has not been reported. Part 2 characterization of cell division cycle associated 8(CDCA8) promoter and transcription factoresCDCA8(cell divison cycle associated 8) is thought to play an important role in cell development and cell proliferation. It encodes a protein, termed Borealin/Dasra B, which is component of chromosomal passenger complex. Functional studies revealed that Borealin was 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 analysis in different tissues and cell lines demonstrated that CDCA8 was 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 over 5 months. Analysis of the CDCA8 promoter activities in different kind of cells revealed that CDCA8 promoter is significantly activated in undifferentiated ES cells and cancer cell lines but repressed in differentiated ES cells and normal primary cells.Objective To investigate transcriptional regulation of CDCA8 gene and reasons of its differential expression, we had basic studies in CDCA8 promoter and transcriptional factors.Methods Constructs the CDCA8 promoter-reporter vectors for the use of transgene mouse. The vectors were identified by detecting the expression of reporter genes after transient transfection into eucaryotic cells and microinjection into mouse fertilized ovum. Meanwhile, we forecasted the potential transcriptional factors binding to CDCA8 promoter(-1123bp/-53bp) by computer analysis and found the real transcriptional factors in hES, NTERA-2 and hEF by pull-down assay and protein/DNA array. Then we compared the transcriptional factors in these three cells in order to find out the possible reasons or regulation mechanisms for differential expression of CDCA8 gene.Results We detected the expression of reporter genes in hES, Hela, MCF-7 and mouse fertilized ovum which indicated that the promoter-reporter vectors were useful for transgene mouse. Meanwhile, we found out the real transcriptional factors binding to CDCA8 promoter(-1123bp/-53bp) in hES, NTERA-2 and hEE NF-Y and CREB are critical transcriptional factors and can certainly bind to CDCA8 promoter according to the results of protein/DNA array, EMSA and site-specific mutagenesis.Conclusion We successfully constructed the CDCA8 promoter-reporter vectors: pGL₃-LacZ-Plkb and EGFP-C3-Plkb. Results of protein/DNA array provides us many candidate factors for later reseach and reduce the research scope in the certain degree.