May L T, Sehgal P B, LaForge K S, Inouye M
Virology. 1983 Aug;129(1):116-26. doi: 10.1016/0042-6822(83)90400-2.
We have investigated the expression of human interferon (IFN)-related transcripts in Sendai virus-induced lymphoblastoid (Namalwa) cells and in poly(I) X poly(C)-induced fibroblast (FS-4) cells by electrophoresis of RNA through agarose-CH3HgOH gels followed by (a) blot hybridization of the RNA using IFN-alpha 1 and IFN-beta 1 cDNA probes, and (b) translation of the eluted RNA into biologically active IFN using the Xenopus laevis oocyte assay. In Namalwa cells induced with or without prior treatment with bromodeoxyuridine we can detect IFN-alpha 1 hybridizable, translationally active, cytoplasmic RNA of lengths 0.3-0.6, 2-3, 3.5-5, and 7-8 kb in addition to the expected 0.8-1.4-kb RNA and IFN-beta 1-hybridizable, translationally active, cytoplasmic RNA of lengths 0.9 kb and approximately 4 kb. However the translationally active 1.8-kb IFN-alpha L mRNA(s) in Namalwa cells (P. B. Sehgal, May, LaForge, and Inouye, Proc. Nat. Acad. Sci. USA 79, 6932-6936, 1982) appears to have less than 60-70% nucleotide sequence homology with IFN-alpha 1 in the coding region because it does not cross-hybridize IFN-alpha 1 cDNA even under very relaxed hybridization conditions. In induced FS-4 cells we can detect IFN-beta 1-hybridizable and translationally active cellular RNA of lengths 0.3-0.4, 0.65, 0.9, 1.8, 2-3, 3.5-5, and 7-8 kb. No hybridization is detected in RNA of length 1.3 kb ("IFN-beta 2 mRNA"). Direct comparisons between the translation and hybridization data using dot-blot hybridization procedures confirm that the apparently subcistronic 0.35-kb IFN-beta 1 hybridizable-RNA species detected in FS-4 cells is translationally active.
我们通过将RNA在琼脂糖 - 甲基汞凝胶中进行电泳,随后进行以下操作,研究了仙台病毒诱导的淋巴母细胞样(Namalwa)细胞和聚肌苷酸 - 聚胞苷酸(poly(I) X poly(C))诱导的成纤维细胞(FS - 4)细胞中人类干扰素(IFN)相关转录本的表达:(a)使用IFN - α1和IFN - β1 cDNA探针与RNA进行印迹杂交;(b)使用非洲爪蟾卵母细胞检测法将洗脱的RNA翻译成具有生物活性的IFN。在经或未经溴脱氧尿苷预处理诱导的Namalwa细胞中,除了预期的0.8 - 1.4 kb RNA外,我们还能检测到长度为0.3 - 0.6、2 - 3、3.5 - 5和7 - 8 kb的可与IFN - α1杂交、具有翻译活性的细胞质RNA,以及长度为0.9 kb和大约4 kb的可与IFN - β1杂交、具有翻译活性的细胞质RNA。然而,Namalwa细胞中具有翻译活性的1.8 kb IFN - αL mRNA(P. B. Sehgal、May、LaForge和Inouye,《美国国家科学院院刊》79,6932 - 6936,1982)在编码区与IFN - α1的核苷酸序列同源性似乎不到60 - 70%,因为即使在非常宽松的杂交条件下,它也不能与IFN - α1 cDNA交叉杂交。在诱导的FS - 4细胞中,我们能检测到长度为0.3 - 0.4、0.65、0.9、1.8、2 - 3、3.5 - 5和7 - 8 kb的可与IFN - β1杂交且具有翻译活性的细胞RNA。在长度为1.3 kb的RNA(“IFN - β2 mRNA”)中未检测到杂交信号。使用斑点印迹杂交程序对翻译和杂交数据进行直接比较,证实了在FS - 4细胞中检测到的明显亚顺反子的0.35 kb可与IFN - β1杂交的RNA物种具有翻译活性。