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

Human Man II-specific primers were synthesized to assay for the presence of the human Man II gene in a human/hamster somatic cell hybrid panel . DNA isolated from a panel of 25 human/hamster hybrids (50 pg/ml, BIOS Corp .) was screened by PCR using primers with the following sequence : sense primer-GCTCGGATGCTACTAGA (3/17 mismatch with murine Man 11) ; antisense primer-TCTTAACTTTAAACTTGGA (7/19 mismatch with murine Man II) bracketing a 178 by of the human Man II gene which was not interrupted by introns (see Fig . 1 ) . Amplification reactions (25 pl) containing 50 mM KCI, 10 mM Tris HCl (pH 8.3), 1.5 mM MgC12, 200 pM each dNTP, 50 ng genomic DNA, 0.5 AM each primer, and 0.6 U Taq polymerase. A temperature cycle of 92°C (1 min), 55°C (1 min), and 72°C (2 min) was repeated for 35 cycles followed by an extension of 4 min at 72°C. Amplification products were resolved on a 2 % agarose gel containing ethidium bromide and scored for the presence of the 178-bp human Man II amplification fragment . A faint 178-pb band as well as several characteristic nonspecific bands were found in hamster controls and in human/hamster hybrids lacking chromosome 5, but the human amplification product could be unambiguously detected above this background by a greater yield, the presence of unique HindUI and EcoRl sites, and the DNA sequence of the human Man II cDNA (data not shown) .