Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte.
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.
Sabath
D E
DE
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104.
Broome
H E
HE
Prystowsky
M B
MB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D016193:G1 Phase; D015971:Gene Expression Regulation, Enzymologic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D007376:Interleukin-2 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D006377:T-Lymphocytes, Helper-Inducer / Q000201:enzymology*; D014158:Transcription, Genetic
D007376:Interleukin-2; D012333:RNA, Messenger; D003609:Dactinomycin; D003513:Cycloheximide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases