TY - JOUR
T1 - Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned T-helper lymphocyte
AU - Sabath, Daniel E.
AU - Broome, H. Elizabeth
AU - Prystowsky, Michael B.
N1 - Funding Information:
D.E.S. was supportedb y NIH trainingg rant5 -T32-GM-07170,H .E.B. by NIH training grant T32-CA01940-13, the researchb y NIH grant GM-36962. The BIONET National ComputerR esourceis supportedb y NIH grant P41RR01685W. e thank Dr. Mitchell J. Weiss for assist-ante in the constructiono f the cDNA library,a nd Brenda McCaffertyf or performingt he GAPDH assays.
PY - 1990/7/16
Y1 - 1990/7/16
N2 - 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.
AB - 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.
KW - G and S phases
KW - Recombinant DNA
KW - T-cell proliferation
KW - cDNA
KW - cell cycle
KW - differential hybridization
KW - gene expression
KW - glycolysis
KW - growth factors
KW - signal transduction
UR - http://www.scopus.com/inward/record.url?scp=0025112855&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025112855&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(90)90087-8
DO - 10.1016/0378-1119(90)90087-8
M3 - Article
C2 - 2145197
AN - SCOPUS:0025112855
SN - 0378-1119
VL - 91
SP - 185
EP - 191
JO - Gene
JF - Gene
IS - 2
ER -