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

We have previously isolated a partial cDNA clone encoding -40% of the rat Man II open reading frame (26) using a probe isolated by mixed oligonucleotide-primed amplification of cDNA (PCR-1) . Since Northern blots demonstrated a message size of -7.5 kb (adjusted downward from the previously published -8 kb; 26) we decided to perform subsequent rounds of screening using a library primed with both oligo(dT) and random hexamers (41) . Approximately 1 x 106 independent recombinants from the unamplified 3T3 Moremen We have also screened a human HepG2 cDNA library with the rat PCR amplification product (PCR-1) as a probe to isolate human cDNA clones for sequencing and for use as hybridization probes . These probes have been used to examine the expression of Man II in HEMPAS disease (12) , a heterogeneous disease characterized in some individuals by a deficiency in Man II . The sequence has also been used to design human Man II specific primers for chromosome mapping by PCR (see below) . The HepG2 library was screened (4 X 105 recombinants) and three independent clones were isolated . The longest of the clones (ti1.9 kb) contained -55 ofthe human Man II open reading frame and aligned to position -5 to +1,928 on the 3T3 sequence in Fig . 2 (Fig . 1 ) .