人类β-1,4-半乳糖苷转移酶Ⅲ基因的分离与克隆

ISOLATION AND CLONING OF HUMAN β1, 4-GALACTOSYLTRANSFERASE III GENE

以人、牛、鼠、鸡、蜗牛的β-1,4-半乳糖苷转移酶催化区的编码核苷酸序列为探针,在NCBI GenBank EST数据库中进行同源搜寻,获得若干有高度同源的EST.在拼接序列的两端设计引物,以从人胎盘cDNA文库中PCR扩增获得的片段为探针,在人胎盘cDNA分子库中步移获得一个长1,907bp的cDNA片段,包含一个长1,179bp的开放阅读框(ORF,open reading frame),编码393个氨基酸残基。该基因与已知的人类β1,4-GalTI的氨基酸同源性为43.8%,在蛋白质催化区的同源性更高达60.9%。表达谱分析发现该基因在人体16种组织中均有不同程度的表达,转录本大小约为2.4kb.通过该cDNA人/啮齿类杂种细胞株DNA Southern杂交将该基因定位在1号染色体。

Using the conservative nucleotide sequences encoding the catalytic domain of the β-1, 4-GalT genes in human, bovine, mouse, chick and snail as probes to search the NCBI GenBank EST database, several ESTs with high homology were obtained. Primers were designed in the flanking sequence of EST contig. Using the PCR product amplified in human placenta cDNA library as probe to perform 'walking' hybridization with human placenta cDNA library, a cDNA fragment with the length of 1,907 bp was cloned. It contained an open reading frame (ORF) with the length of 1,179 bp, which encodes 393 amino acid residues. The deduced amino acid sequence of this gene shares 43. 8% identity to the human β-1,4-GalTI and 60. 9% in the catalytic domain especially. The expression mapping showed that it was expressed in human most tissues with a single 2.4kb transcript, but the relative expression level of the transcript are vary. While this gene was mapped on chromosome 1 using the cDNA hybridization with human/rodent hybrid cell line DNA Southern blot panel.