Expression of the polyubiquitin gene early in the buprenorphine hydrochloride-induced apoptosis of NG108-15 cells.
To clone genes expressed early in the buprenorphine hydrochloride (Bph)-induced apoptosis of NG108-15 nerve cells, we adopted a previously reported rapid and simple differential display (DD) cloning procedure. Complementary DNA was generated from differentially expressed mRNAs by reverse transcription (RT) using a fully degenerate 6-mer oligonucleotide as the primer. PCR amplification was then conducted using a combination of three arbitrary but defined 10-bp nucleotide primers. The differentially generated DNA fragments were detected by agarose gel electrophoresis, and 9 were excised from the gel and subcloned into a sequencing vector. Three DNAs that were specifically expressed upon Bph-induced apoptosis of NG108-15 cells were sequenced. Their specific expression was then confirmed by reverse-transcription PCR. One was identified as the mouse polyubiquitin gene c, and the others remain unidentified. Northern and western blots indicated the transcription and translation of polyubiquitin early in Bph-induced apoptosis. The polyubiquitination of apoptotic cellular proteins was also confirmed.
Kugawa
Fumihiko
F
Department of Biological Pharmaceutical Sciences, College of Pharmacy, Nihon University, 7-7-1 Narashino-dai, Funabashi, Chiba 274-8555, Japan. jfkugawa@pha.nihon-u.ac.jp
Aoki
Masatada
M
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001483:Base Sequence; D002047:Buprenorphine / Q000494:pharmacology*; D003001:Cloning, Molecular; D015870:Gene Expression / Q000502:physiology*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025822:Ubiquitin C / Q000235:genetics* / Q000378:metabolism
D025822:Ubiquitin C; D002047:Buprenorphine