Björklund S, Skog S, Tribukait B, Thelander L
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Biochemistry. 1990 Jun 12;29(23):5452-8. doi: 10.1021/bi00475a007.
Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.
哺乳动物细胞中的核糖核苷酸还原酶由两个不同的亚基，即R1和R2蛋白组成，每个亚基单独存在时均无活性。R1蛋白在增殖细胞中过量存在，其水平在细胞周期中保持恒定。对酶活性起限制作用的R2蛋白的表达与S期严格相关。在本文中，我们使用反义RNA探针进行溶液杂交分析，以测量在离心淘洗的细胞以及通过异亮氨酸或血清饥饿同步化的细胞的细胞周期中R1和R2 mRNA的水平。在G0/G1期细胞中，这两种转录本的水平都非常低或无法检测到，随着细胞进入S期，其水平显著增加，然后在细胞进入G2 + M期时下降。R1和R2转录本平行增加，略早于S期细胞的增加，并达到相同水平。因此，先前获得的R1蛋白水平相对缺乏细胞周期依赖性变化，可能仅仅是R1蛋白半衰期长的结果。耐羟基脲、R2过量产生的小鼠TA3细胞与亲代细胞表现出相同的R1和R2转录本调控，但R2 mRNA水平高出40倍。
