Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

Clone MII-8 containing the entire Man II open reading frame inserted into the EcoRl site of pBluescript II (see Fig . 1 ) was linearized by digestion with BamHI and transcribed in vitro using the T7 polymerase promoter of pBluescript II and the in vitro transcription kit from Stratagene. RNA synthesis was carried out for 2 h at 37°C in a 40 pl reaction volume containing 40 mM Tris HCl (pH Z5), 50 mM NaCl, 8 mM M9Clz, 2 mM spermidine, 0.3 mM M~G(5)ppp(5)G, 30 mM l7rT, 80 U RNasin, 0.5 mM each ATP, UTP, and CTP, 0.17 mM GTP, 10 U of T7 polymerase, and 5,ug of the linearized clone MII-8 template. After the addition of GTP to 0.5 mM the reaction was continued for another 16 h at 37°C . The reaction mixture was extracted twice with phenol/chloroform/isoamyl alcohol followed by ethanol precipitation . Control reactions contained plasmid without insert or plasmid with a noncoding insert.