Lipson K E, Baserga R
Department of Pathology, Temple University School of Medicine, Philadelphia, PA 19140.
Proc Natl Acad Sci U S A. 1989 Dec;86(24):9774-7. doi: 10.1073/pnas.86.24.9774.
We have used the technique of reverse transcription coupled to the polymerase chain reaction to detect mRNA precursors [heterogeneous nuclear RNA (hnRNA)] transcribed from the thymidine kinase (TK) gene of human diploid fibroblasts. With this method, the amplification products of both hnRNA (containing the introns) and mature mRNA can be detected on Southern blots with appropriate hybridization probes. With the experimental conditions used, the sensitivity of the technique is such that TK mRNA can be detected in as few as 20 S-phase cells. TK hnRNA is maximally expressed early in the S phase of the cell cycle after quiescent human fibroblasts are stimulated to proliferate. At this point, the ratio of TK hnRNA to TK mRNA is 1:155. A small amount of TK hnRNA can be detected in populations of cells that appear to be quiescent. However, the presence of the precursor in these populations correlates with the number of cells still cycling. No TK hnRNA can be detected in truly quiescent human diploid fibroblasts, suggesting that in these cells, the TK gene is not transcribed in G0.
我们运用逆转录与聚合酶链反应相结合的技术，来检测人二倍体成纤维细胞胸苷激酶（TK）基因转录产生的mRNA前体[核不均一RNA（hnRNA）]。通过这种方法，使用合适的杂交探针，在Southern印迹上就能检测到hnRNA（含内含子）和成熟mRNA的扩增产物。在所采用的实验条件下，该技术的灵敏度极高，以至于在少至20个处于S期的细胞中就能检测到TK mRNA。静止的人成纤维细胞受到刺激开始增殖后，TK hnRNA在细胞周期的S期早期表达量最高。此时，TK hnRNA与TK mRNA的比例为1:155。在看似静止的细胞群体中能检测到少量的TK hnRNA。然而，这些细胞群体中前体的存在与仍在进行循环的细胞数量相关。在真正静止的人二倍体成纤维细胞中检测不到TK hnRNA，这表明在这些细胞中，TK基因在G0期不转录。
