【摘要】 目的:人和大鼠WIPI-3基因的电子克隆和序列分析。方法:综合运用基因电子拼接和比较基因组分析等生物信息学手段进行新基因的电子克隆和注释。通过拼接CR593190、NM₀19613和BC000974 cDNA序列获得人WIPI-3 cDNA全长序列,通过拼接EST序列CB727439、CB737031、BF557312、BG663387和预测的CDS获得大鼠WIPI3基因的cDNA序列。结果:成功克隆了人和大鼠自噬相关新基因WIPI-3的cDNA,上述两个新基因序列均得到了人类基因组序列和EST序列的双重支持,另外经RT-PCR验证电子克隆的基因序列也是正确的,而且序列均已被GenBank收录,其编号分别为:AM182326和NM₀01039587。其中,人WIPI-3基因修正了目前基因库中注释的WIPI-3序列,而大鼠基因则是国际上首次克隆,并被确定为参考序列。结论:基因电子拼接结合种属同源基因比较分析,可大大提高电子克隆的精确性,这种方法可有效用于新基因的克隆和注释。更多还原

【Abstract】 WD-repeat proteins are key components of many essential biological functions including cell cycle control,apoptosis,signal transduction pathways,RNA metabolism,chromatin assembly,vesicular trafficking and tumor phenotypes.The recently identified WIPI1,WIPI2,WIPI3 and WIPI4 genes constitute the WIPI subfamily of WD40 repeat proteins.Dysfunctional WIPI-dependent autophagy has been linked to tumor progression pathway in multiple cancer types.Because human WIPI3 cDNA（NM_<sub>019613） was an aberrant cDNA with frame shifts due to 106 base erroneous insertions and two base substitutions compared with the ESTs and human genome draft sequences,we identified and characterized human WIPI3 gene by a bioinformatics approach.Human WIPI3 gene,consisting of 10 exons,was located within human chromosome 17 genomic contig NT_<sub>010663.14 and mapped to human chromosome17q25.3 in the reverse orientation.Nucleotide sequence of human WIPI3 cDNA was determined in silico by assembling CR593190 cDNA,BC007838 cDNA and BC000974 cDNA.Two WIPI3 transcript variants with or without exon 1 and 2 were transcribed due to alternative splicing.In addition,17 single nucleotide polymorphisms were found within the human WIPI3 genomic sequence by dbSNP databases mining.Nucleotide sequence of rat WIPI3 cDNA was determined by assembling CB708542,CB727439,CB737031,BF557312,BG663387 ESTs and predicted CDS within rat genomic contig NT_<sub>030059.12 by TBLASTN with mouse WIPI3 protein as a query.Rat WIPI3 gene was located at chromosome 10q32.3.The WIPI3 proteins is extremely conserved and the length are quite comparable in all species,Human WIPI3 protein isoform1 showed 99% total-amino-acid identity with rat and mouse WIPI3, 97%.total-amino-acid identity with xenopus WIPI3,and 95% total-amino-acid identity with danio WIPI3.Human,rat,mouse,xenopus and danio WIPI3 were WD40 repeat homology proteins with 7-blade beta-propeller structure.dbEST expression profile showed that human WIPI3 gene up-regulated in several malignancies including stomach,large intestine,breast,ovary,kidney and pancreatic cancers.Normal tissues of testis and liver also showed over-expression.Part of these results were further verified by Gene Expression Omnibus（GEO） experimental databases.This is the first report on comprehensive characterization of human and rat WIPI3 gene.更多还原