Identification of okadaic-acid-induced genes by mRNA differential display in glioma cells.
To identify novel genes associated with apoptosis in glioma cells, we treated T98G glioma cells with okadaic acid (OA). Differential display using 15 random primers was performed on RNA extracted from these cells. Upregulated bands were excised from polyacrylamide gels and cloned. Northern blots were used to confirm RNA expression in T98G cells. 18 RNA fragments corresponding to the untranslated region of genes were identified and sequenced. Three unknown gene fragments were used to screen a fetal brain cDNA library resulting in three complete cDNA sequences. The three sequences corresponded to a human gene homologous to the yeast translation initiation factor Sui-1, a cAMP-regulated phosphoprotein, ARPP-16/19, and a novel gene designated O48. Transcription of Sui-1 increased in response to all stress factors tested, whereas ARPP only responded to OA. 2-kb and 4-kb O48 RNA species were identified. OA and stress factors increased 2-kb expression while K252a (protein kinase inhibitor) increased 4-kb expression. Differential display is effective for identifying genes associated with apoptosis. Novel genes may be identified by further analysis of the gene fragments identified in this study. The function of O48 is unknown.
Chin
L S
LS
Department of Neurosurgery and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA. lchin@smail.umaryland.edu
Singh
S K
SK
Wang
Q
Q
Murray
S F
SF
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000196:embryology / Q000378:metabolism; D001932:Brain Neoplasms / Q000235:genetics / Q000473:pathology*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D039642:Eukaryotic Initiation Factors; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D015723:Gene Library; D005910:Glioma / Q000235:genetics / Q000473:pathology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins; D019319:Okadaic Acid / Q000494:pharmacology*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D013382:Subtraction Technique; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D018076:DNA, Complementary; C410108:EIF1 protein, human; D039642:Eukaryotic Initiation Factors; C404083:LUC7L3 protein, human; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C064322:cyclic AMP-regulated phosphoprotein 16; C064321:cyclic AMP-regulated phosphoprotein 19; D019319:Okadaic Acid