Moremen K W
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1989 Jul;86(14):5276-80. doi: 10.1073/pnas.86.14.5276.
A clone encoding Golgi mannosidase II (MII; GlcNAc-transferase I-dependent alpha 1,3(alpha 1,6) mannosidase), an enzyme involved in asparagine-linked oligosaccharide processing, was isolated from a rat liver lambda gt11 cDNA library by a method that employs a modification of the polymerase chain reaction. Specific oligonucleotide primers were designed from two regions of protein sequence and were combined in an amplification reaction with a single-stranded cDNA preparation derived from rat liver poly(A)+ RNA. Based upon mapping of the protein sequences 42 kDa apart on the MII polypeptide, the procedure was expected to generate an approximately 1150-base-pair amplification product representing a segment of the MII gene between the two primer regions. The size of the amplified product (1170 base pairs) was in close agreement with this predicted fragment size. The authenticity of the amplified fragment was confirmed by the agreement of the DNA sequence with additional protein sequence data. A 1474-base-pair clone was isolated from a cDNA library by plaque hybridization using the amplification fragment as a radiolabeled probe. The nucleotide sequence of this clone predicts a single continuous open reading frame and, based upon a polypeptide molecular mass of 117 kDa for the enzyme subunit, is consistent with the clone representing approximately 50% of the coding sequence of MII. Both the clone and the amplification product hybridized to a rat liver mRNA of approximately 8 kilobases, a message size approximately 4.7 kilobases larger than the size of the predicted open reading frame. This extensive non-coding information on the MII message is a feature common to two other Golgi processing enzymes, both of which contain most of the non-coding information on the 3' end of their messages. The function of these disproportionately large untranslated regions is not clear.
通过一种采用聚合酶链反应改进方法，从大鼠肝脏λgt11 cDNA文库中分离出一个编码高尔基体甘露糖苷酶II（MII；N-乙酰葡糖胺转移酶I依赖性α1,3（α1,6）甘露糖苷酶）的克隆，该酶参与天冬酰胺连接的寡糖加工。根据蛋白质序列的两个区域设计了特异性寡核苷酸引物，并将其与源自大鼠肝脏聚腺苷酸加尾RNA的单链cDNA制剂一起用于扩增反应。基于MII多肽上相距42 kDa的蛋白质序列定位，预计该程序将产生一个约1150个碱基对的扩增产物，代表两个引物区域之间的MII基因片段。扩增产物的大小（1170个碱基对）与预测的片段大小非常一致。扩增片段的真实性通过DNA序列与其他蛋白质序列数据的一致性得到证实。使用扩增片段作为放射性标记探针，通过噬菌斑杂交从cDNA文库中分离出一个1474个碱基对的克隆。该克隆的核苷酸序列预测有一个单一的连续开放阅读框，基于该酶亚基117 kDa的多肽分子量，该克隆与代表MII编码序列约50%的克隆一致。该克隆和扩增产物均与约8千碱基的大鼠肝脏mRNA杂交，该信使RNA大小比预测的开放阅读框大小约大4.7千碱基。MII信使RNA上这种广泛的非编码信息是另外两种高尔基体加工酶共有的特征，这两种酶的信使RNA的3'端都包含大部分非编码信息。这些不成比例的大非翻译区的功能尚不清楚。
