Boege F
Biosci Rep. 1982 Jun;2(6):379-89. doi: 10.1007/BF01119300.
The presence of terminal nucleotidyl transferase activities catalyzing the addition of AMP, CMP, GMP, and UMP residues to the 3' ends of oligonucleotide primers was detected in healthy tomato plants. These enzyme activities copurify with RNA-dependent RNA polymerase during the initial stages of purification. Their separation from RNA-dependent RNA polymerase is finally achieved by DEAE chromatography: terminal transferase activities are retained on DEAE while RNA-dependent RNA polymerase does not bind in the presence of 20 mM MgCl2. Elution by a linear gradient of 0 to 400 mM NH4Cl releases all four terminal transferase activities from the DEAE column at a concentration of 270 mM NH4Cl, thus suggesting that they may belong to one enzyme molecule; this question, however, needs further clarification. The enzyme activities are completely dependent on the presence of an RNA primer and are strongly influenced by its base composition as well as its chain length. Characterization of the respective reaction products by electrophoresis on 15% polyacrylamide sequencing gels reveals striking differences as to the number of nucleotides added to a given primer. In the case of UMP transfer to U8 or A8 and in the case of GMP transfer to A8 only 1 to 6 nucleoside monophosphates are added to the 3' terminus of the oligonucleotide primer, whereas in the case of AMP transfer to A8 or U8, the CMP transfer to A8, and the GMP transfer to U8, longer chains of minimally 30 nucleotides are added to the respective primer. After gradient elution from DEAE the transferase preparation displays no nucleolytic activity when incubated in the presence of 3H-labelled ribosomal RNA or [3H]poly(A) X poly(U). Only in the case of [3H]poly(A) and [3H]poly(U) or [3H]poly(C) 10 to 15% of the radioactivity is transferred to acid-soluble counts.
在健康番茄植株中检测到了末端核苷酸转移酶活性，该酶可催化将AMP、CMP、GMP和UMP残基添加到寡核苷酸引物的3'末端。在纯化初期，这些酶活性与依赖RNA的RNA聚合酶一起被共纯化。最终通过DEAE色谱法将它们与依赖RNA的RNA聚合酶分离：在20 mM MgCl2存在的情况下，末端转移酶活性保留在DEAE上，而依赖RNA的RNA聚合酶不结合。用0至400 mM NH4Cl的线性梯度洗脱，在270 mM NH4Cl浓度下从DEAE柱上释放出所有四种末端转移酶活性，因此表明它们可能属于一个酶分子；然而，这个问题需要进一步阐明。酶活性完全依赖于RNA引物的存在，并受到其碱基组成及其链长的强烈影响。通过在15%聚丙烯酰胺测序凝胶上进行电泳对各自的反应产物进行表征，结果显示在添加到给定引物的核苷酸数量方面存在显著差异。在UMP转移到U8或A8以及GMP转移到A8的情况下，仅向寡核苷酸引物的3'末端添加1至6个核苷单磷酸，而在AMP转移到A8或U8、CMP转移到A8以及GMP转移到U8的情况下，向各自引物添加的是至少30个核苷酸的更长链。从DEAE进行梯度洗脱后，转移酶制剂在3H标记的核糖体RNA或[3H]聚（A）×聚（U）存在下孵育时不显示核酸酶活性。仅在[3H]聚（A）和[3H]聚（U）或[3H]聚（C）的情况下，10%至15%的放射性转移到酸溶性计数中。
