Mansur N R, Meyer-Siegler K, Wurzer J C, Sirover M A
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140.
Nucleic Acids Res. 1993 Feb 25;21(4):993-8. doi: 10.1093/nar/21.4.993.
The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant.
在正常人细胞中研究了甘油醛-3-磷酸脱氢酶（GAPDH）/尿嘧啶DNA糖基化酶（UDG）基因的细胞周期调控。使用包含1.3 kb GAPDH/UDG cDNA的质粒（pChug 20.1），通过Northern印迹分析监测稳态RNA水平。使用抗人胎盘GAPDH/UDG单克隆抗体免疫沉淀放射性标记的蛋白质，以确定37 kDa GAPDH/UDG蛋白的生物合成。稳态GAPDH/UDG mRNA水平的增加具有细胞周期特异性。观察到一种双相模式，导致GAPDH/UDG mRNA量增加19倍。37 kDa GAPDH/UDG蛋白的生物合成表现出类似的双相调控，增加了7倍。脉冲追踪实验显示新合成的37 kDa蛋白的半衰期非常短，不到1小时，这与先前记录的许多癌基因的半衰期相当。当DNA合成达到最大值时，GAPDH/UDG mRNA水平在24小时时显著降低。这些结果将GAPDH/UDG基因定义为细胞周期调控基因，其表达具有与DNA合成相关的特征性时间序列。不稳定的37 kDa单体的细胞周期合成表明这种多维蛋白可能具有调节功能。此外，当细胞的增殖状态不保持恒定时，细胞周期中GAPDH/UDG基因的调节可能会妨碍其用作报告基因。
