Fluorescent differential display analysis of gene expression in apoptotic neuroblastoma cells.
Identification of differentially expressed genes will provide leads in the elucidation of the molecular mechanisms underlying neuronal cell death associated with neurodegenerative disorders. Using a high-throughput fluorescent differential display (FDD) system based on an automated DNA sequencer, we analyzed global patterns of gene expression during the apoptosis of neuroblastoma SH-SY5Y cells induced by a neurotoxin, colchicine. Initial screening of approximately 24000 cDNA bands displayed with 320 primer combinations has revealed 263 fragments showing differential expression patterns, suggesting that approximately 1% of transcripts are modulated in their expression level. Of these differentially displayed bands, we cloned 18 fragments composed of 17 distinct species and confirmed differential expression of each species by reverse transcription-PCR or Northern blot hybridization, thereby proving the reliability of the approach. These include eight derived from seven known genes, five homologous to expressed sequence tags (ESTs), and five totally lacking any homology to those deposited in the database. Among these, a novel transcript SAI1 induced prominently was characterized further and revealed to encode a putative RNA-binding protein NAPOR (neuroblastoma apoptosis-related RNA-binding protein), containing three copies of evolutionarily conserved RNA recognition motif. Since several RNA-binding proteins have been known to play crucial roles in other apoptosis systems, it is conceivable that NAPOR is also involved in the process of neuronal cell death.
Choi
D K
DK
Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan.
Ito
T
T
Mitsui
Y
Y
Sakaki
Y
Y
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D000067878:CELF Proteins; D003001:Cloning, Molecular; D003078:Colchicine / Q000494:pharmacology; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D016601:RNA-Binding Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D000067878:CELF Proteins; C117363:CELF2 protein, human; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D003078:Colchicine