Sabath D E, Broome H E, Prystowsky M B
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104.
Gene. 1990 Jul 16;91(2):185-91. doi: 10.1016/0378-1119(90)90087-8.
A cDNA library was constructed using mRNA from interleukin 2 (IL2)-stimulated cloned murine T lymphocytes to isolate cDNA clones of mRNAs that were induced by IL2 and present at maximal levels in late G1/early S phase of the cell cycle. When the library was screened by differential hybridization, over half of the clones isolated were found to cross-hybridize, indicating that there was a predominant IL2-induced mRNA in these cells. This cDNA was identified as encoding murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). The in vitro translation product of this cDNA was a 36-kDa protein using both hybridization-selected RNA and in vitro transcribed RNA. We estimate that GAPDH mRNA comprises approx. 0.7% of total mRNA in the cloned T cells in late G1. GAPDH mRNA is induced two- to fivefold over resting levels upon IL2 stimulation, due in part to an increased rate of transcription. GAPDH enzymatic activity is induced approx. sevenfold over resting levels. The induction of GAPDH mRNA is inhibited only slightly by CHX under conditions in which cell proliferation is inhibited. In addition, the induction of GAPDH is directly due to the effect of IL2, and not in conjunction with any serum components, since IL2 will induce GAPDH mRNA under serum-free conditions. Finally, when genomic DNA is probed with a full-length GAPDH cDNA, a complex pattern of bands is observed, whereas if a 5' end probe is used, a much simpler pattern is obtained, indicating that many of the GAPDH pseudogenes in the murine genome lack 5' sequence information.
利用来自白细胞介素2（IL2）刺激的克隆鼠T淋巴细胞的mRNA构建了一个cDNA文库，以分离那些由IL2诱导且在细胞周期的G1晚期/ S期早期达到最高水平的mRNA的cDNA克隆。当通过差异杂交筛选该文库时，发现超过一半的分离克隆发生交叉杂交，表明这些细胞中存在一种主要的IL2诱导的mRNA。该cDNA被鉴定为编码鼠甘油醛-3-磷酸脱氢酶（GAPDH；EC 1.2.1.12）。使用杂交选择的RNA和体外转录的RNA，该cDNA的体外翻译产物是一种36 kDa的蛋白质。我们估计GAPDH mRNA在G1晚期的克隆T细胞中约占总mRNA的0.7%。IL2刺激后，GAPDH mRNA诱导至静息水平的两到五倍，部分原因是转录速率增加。GAPDH酶活性诱导至静息水平的约七倍。在细胞增殖受到抑制的条件下，CHX仅轻微抑制GAPDH mRNA的诱导。此外，GAPDH的诱导直接归因于IL2的作用，而不是与任何血清成分共同作用，因为IL2在无血清条件下也能诱导GAPDH mRNA。最后，当用全长GAPDH cDNA探测基因组DNA时，观察到复杂的条带模式，而如果使用5'端探针，则会获得简单得多的模式，这表明鼠基因组中的许多GAPDH假基因缺乏5'序列信息。
