POSTER NO: 208

Screening and cloning the related genes sensitive/resistant to inhalation anesthetic (sevoflurane) in Drosophila

¹Hong Liu, ²Xiaomeng Ren, ¹Lanying Chen, ²Jin Liu
¹Division of Biochemistry, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beilishi Road 167#, Beijing 100037, ²Division of Anesthesia-Purfusion, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beilishi Road 167#, Beijing 100037

The inhalation anesthetics have been introduced into clinical practice for 150 years. They have become more widely used and important therapeutic agents. However, despite over a century of research, the molecular mechanisms of action for inhaled anesthetics are not clear. In this study, we try to find some new genes related to inhaled anesthetic or to verify some promising molecular targets that have been considered. To identify differential expressed genes in difference strains of Drosophila, the differential display reverse transcription-PCR (DDRT-PCR) method was applied to compare mRNA levels from sevoflurane-sensitive strain, sevoflurane-resistant strain and wild type strain. Each of the three strains was divided into two groups, experimental group induced with sevoflurane and control group induced without sevoflurane. The total RNA was extracted from each strain (1-2 days old). Then reverse transcription and DD-PCR were performed. Later the PCR products were separated on denaturing polyacrylamide gel using silver staining or fluorescent differential display (FDD). After recovering and reamplifying the differential display cDNAs, we used the both methods of reverse Northern blotting and Northern blotting to identify the positive cDNA fragments. Subsequently, the positive cDNAs were sequenced and searched for the homologous sequences by BLAST algorithm at NCBI and TIGR sites. Finally, 63 differential expressed cDNA fragments were isolated by DDRT-PCR. 5 cDNAs were identified by Northern blotting among 7 candidate positive fragments screened with reverse Northern blotting. Compared with wild type strain, the expression level of fragment 8# increased in resistant strain, while decreased in sensitive strain. On the contrary, the expression level of fragment 41# decreased in resistant strain. In the sevoflurane-induced group the expression level of fragment 17IH increased comparing with control group, whereas the expression level of 45# and 38# decreased. The five positive cDNA fragments were analyzed using BLAST with public database (NCBI BLAST site and Institute for Genome Research, TIGR). Fragment 8#, 41# and 38# are located on Chr.2, which were identical with the result of the previous genetic researches in our lab. The others are located on Chr.3. Among these fragments, only 38# is homologous to calmodulin mRNA of Drosophila, the others are all unknown genes on their function. Our results should provide a useful clue on study of the molecular mechanisms of the inhalation anesthetics.