Yokoyama K, Hou D X, Gao H, Tang X, Kitabayashi I, Nishikura K, Gachelin G
Gene Bank, Tsukuba Life Science Center, RIKEN (The Institute of Physical and Chemical Research), Ibaraki, Japan.
Cell Struct Funct. 1992 Dec;17(6):433-42. doi: 10.1247/csf.17.433.
Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E. coli (Ecogpt) into mouse erythroleukemia (MEL) cells by protoplast fusion. Specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of alpha-globin mRNA (Oligo-A); Other plasmids with antisense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3' untranslated region were ineffective. This antisense RNA efficiently reduced the production of alpha-globin to 9-18% of the endogenous level after induction with hexylmethylene-bis-acetoamide (HMBA). Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells. Thus, we propose a hypothesis that the early decline in levels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells.
通过原生质体融合，将以小鼠α-珠蛋白mRNA不同区域的核苷酸序列为基础、按反义或正义构型合成的、与68个碱基的寡聚脱氧核苷酸相对应的质粒DNA，与来自大肠杆菌的黄嘌呤-鸟嘌呤磷酸核糖转移酶基因（Ecogpt）一起导入小鼠红白血病（MEL）细胞。仅在用与α-珠蛋白mRNA的帽位点以及翻译起始位点相对应的反义68聚体的质粒转化的细胞中观察到α-珠蛋白合成的特异性抑制（寡聚物A）；其他与外显子/内含子连接区、各个外显子或3'非翻译区相对应的反义68聚体的质粒无效。在用己基亚甲基-双-乙酰乙酰胺（HMBA）诱导后，这种反义RNA有效地将α-珠蛋白的产量降低至内源性水平的9-18%。此外，大多数反义转化体在MEL细胞分化的早期阶段未显示c-myc基因表达有任何下降。因此，我们提出一个假说，即在MEL细胞分化过程中，c-myc mRNA水平的早期下降可能与珠蛋白合成程序无关且相互分离。
