Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA.
Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA.
复制标题
DOI：
10.1016/s0021-9258(19)40170-1
复制
发表时间：
1990-01
期刊：
影响因子：
--
通讯作者：
F. Yamamoto;J. Marken;T. Tsuji;T. White;H. Clausen;S. Hakomori
中科院分区：
文献类型：
--
作者：
F. Yamamoto;J. Marken;T. Tsuji;T. White;H. Clausen;S. Hakomori
根据部分氨基酸序列，克隆了组织血型A基因（A transferase）特异性初级基因产物UDP-GalNAc：Fuc α 1-2Gal α 1-3GalNAc transferase的cDNA，并进行了序列测定。从高表达A抗原的人胃癌细胞系MKN 45中提取Poly（A）+ RNA，用于构建λ gt 10 cDNA文库。将简并的合成寡脱氧核苷酸用于聚合酶链反应，以检测cDNA中目标序列的存在（存在试验），并在用放射性标记的聚合酶链反应扩增片段筛选文库后鉴定正确的克隆（鉴定试验）。核苷酸序列分析揭示了1062个碱基对的编码区，编码41 kDa的蛋白质。疏水性图分析显示存在三个结构域：N-末端短延伸、跨膜疏水区和长C-末端结构域（迄今为止克隆的所有糖基转移酶的共同特征）。Southern杂交分析表明，该DNA不代表一个多基因家族。未发现限制性片段长度多态性与ABO血型相关。在来自表达A、B、AB或H抗原的细胞系的mRNA的北方杂交中检测到条带。这些结果表明，ABO基因序列基本上非常相似（差异极小），O基因不能编码A或B转移酶可能是由于结构差异，而不是A或B转移酶表达失败。
Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.