Cloning And Characteristics Of Two Novel Gene XVAP019,FAM92A1

Objective: Cloning and characteristics of the Xenopus larevis novel gene xVAP019 and its human homologous gene FAM92A1.Methods. Xenopus larevis were here used as model animal. The Xenopus larevis novel gene xVAP019 was cloned by 3' end RT-PCR from a 5'express sequence tag (5'EST) (GenBank accession numbers CV973659), which was screened from Xenopus larevis oocyte cDNA pool. Its homologs and conserved domain were gained by bioinformatics analysis. Semi-quantitative RT-PCR and whole-mount in situ hybridization were used to detect the spatial and temporal expression of xVAP019. We cloned the completed read frame (ORF) of xVAP019 and constructed plasmid pGEMT-xVAP019, and which was used to sythesis xVAP019 mRNA and Morpholino in vitro. By microinjection xVAPO19 mRNA and Morpholino in vivo, we studied the impact of xVAP019 overexpression and downregulation on Xenopus larevis embryonic development. Othermore, the human homologue gene FAM92A1 ofxVAP019 was cloned here, and two novel transcript variants of FAM92A1 were found. Express plasmid pcDNA3.1-FAM92Als were constructed and transfected into SKOV3 cells, then cell cycle and apoptosis analysis were done by flow cytometry, MTT analysis, Hoechst 33258 staining. Synchronize cells in different cycle phase, then examined FAM92A1 expression at the mRNA level in different cell cycle phase by semi-quantitative RT-PCR. The construct containing FAM92A1-271 fused with EGFP was introduced into Hela cells by lipofectamine2000 mediated transfect, and add DAPI staining. The results revealed by directed fluorescence microscopy view. The p53, Rb, p27,caspase3,caspase 8,caspase 9 gene expression was analysed by semi-quantitative RT-PCR when FAM92Als over-expression in SKOV3 cells.Results: (1) We have identified a novel Xenopus gene (xVAP019) encoding a DUF1208 domain containing protein (2) By bioinformatics analysis, we known that xVAP019 belongs to conserved domain DUF1208 family and contained several Protein kinase C (PKC), Casein kinase 2 (CK2) and a coiled coil domain.(3) Using whole-mount in situ hybridization and RT-PCR, we found abundant xVAP019 maternal transcripts at animal hemisphere during the cleavage stages and blastula stages. During gastrulation xVAP019 is differentially expressed with high levels in the animal helf and highest in marginal zone, then is expressed widely at neurula stages with strongest signal in the prospective CNS regions and the epidermal ectoderm. Subsequently xVAP019 was expressed predominantly in the head, the eyes, the otic vesicle, branchial arches, spinal cord, notochord, somites and tailbud. It is absent or very weak in the endoderm. (4)Injected a morpholino oligo complementary to xVAP019 mRNA or injected a caped xVAP019 mRNA caused most of embryos died during gastrulation and neurulation. Overexpression of xVAP019 mRNA also led to eye defect, shorten interocular distance, small body and abnormal pigment formation in parts of the survival embryos. Similar effect was induced by injection of the xVAP019 human homologous gene FAM92Al.(5)We have also identified two novel transcripts variants (FAM92A1-251, FAM92A1-289) of FAM92A1 and constructed their eukaryocyte express plasmid pcDNA3.1 -FAM92A 1-271, pcDNA3.1 -FAM92A 1-251 and pcDNA3.1-FAM92A1-289. (6) We demonstrated the presence of FAM92Als in different cells, and found FAM92Al-271 and FAM92Al-289 were highly expressed in both cancerous and normal cells, while FAM92Al-251 was consistently expressed at a low level. (7) Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis observed as reducing MTT values.(8) Using semi-quantitative RT-PCR, we found that FAM92A1 s express main in S-phase. (9) When FAM92A1 s over-expression, p53,Rb, caspase3,caspase8 have a increased mRNA level,while no caspase9 mRNA was detected. (10)The subcellular location experiment showed that FAM92A1 localizes in nuclear.Conclusion: The novel gene xVAP019 is high conserved in vertibrates and express in ectoderm and axis mesoderm. Our results suggest that xVAP019 is essential for the normal ectoderm and axis mesoderm differentiation and embryos survival. This investigation is the first in vivo study examining the role of this novel gene and reveals an important role of xVAP019 in embryonic development. FAM92A1 gene has different transcript variants. FAM92A1 protein is located in nuclear and may take part in the regulation of cell proliferation and apoptosis.