Expression of human terminal deoxynucleotidyl transferase in Escherichia coli

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.     

基于模板切换的mRNA5'末端全长扩增方法

本文介绍一种称为CapFinder的技术,可用于克隆基因mRNA序列的5'末端非翻译区全长。该技术是利用某些反转录酶在反转录达到mRNA的5'末端帽结构时表现出很高的加尾活性(主要添加dC)这一特点,在反转录体系中加入一种带GGG的寡核苷酸序列,当反转录反应到达mRNA模板的5'末端帽结构时,切换到以该寡核酸为模板继续进行反转录反应,即可合成完整的cDNA一链,且在其3'末端还带有一段额外的寡核苷酸序列。用GGG寡核苷酸序列为上游引物和基因特异性的下游引物进行PCR即可扩增得到mRNA5'末端非翻译区的全长。利用该技术克隆了棉铃虫幼虫中肠Bt毒素受体E-钙粘素基因的5'末端非翻译区序列。