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

An unamplified BALB/c 3T3 cDNA library primed with a mixture of oligo(dT) and random hexamers and packaged into a XZAP II cloning vector (Stratagene) was obtained from D. J . G. Rees (Massachusetts Institute of Technology) (41) . An amplified version of the same library was used in the second round of screening. A similarly prepared amplified HepG2 cDNA library was obtained from E . Marcantonio (Columbia University, New York, N .Y.) . The packaged libraries were plated on XLl-Blue host cells and screened by plaque hybridization using standard procedures. The probe that was used for library screening and blot hybridizations (see below) was a 1,170 by Man II PCR amplification fragment (PCR-1) generated from a rat liver cDNA preparation by amplification with Man II-specific degenerate and inosine-substituted oligonucleotide primers designed using protein sequence data from the purified Man II polypeptide (26) .