Steps in the in silico and physical cloning and expression of GANC. (a) Computer-based identification of candidate clones and construction and analysis of a computer contig. Sequence for human acid α-glucosidase and the catalytic subunit of glucosidase II was used as “bait” in a TBLASTX and BLASTN search of the human EST database for “orphan” ESTs with homology to the two glucosidases. This procedure identified GB R95789 (clone 1) that contained a sequence tagged site (STS) WI-17962 mapping to chromosome 15q15 in radiation hybrid panels 3 and 4, which is consistent with the earlier mapping of GANC to chromosome 15 in somatic cell hybrids (5). The search simultaneously identified GB AA101464/3 (clone 2), a putatively “reversed” EST that contained sequence for the consensus catalytic site for family 31 glycosyl hydrolases (15) and an STS G31167 that also mapped to chromosome 15q15 in the radiation hybrid maps 3 and 4. Clone 2 additionally identified GB AA323316 (clone 3) that overlapped a conserved sequence in clone 2. Complete sequencing of all clones in both directions and comparison for homology against the sequence database revealed two areas that did not show homology. (b) Deletion of these two regions and “pasting together” the computer construct resulted in an ORF of an appropriate size. After deletion of the two areas of apparent nonhomology (confirmed by absence of these areas from sequence of cDNA obtained by RT-PCR of mRNA), the computer-edited cDNA gave an appropriate size ORF from the initiating methionine to a stop codon (2.745 kb and 914 codons) and homology throughout the ORF (see Table 2). (c) To facilitate deletion of the two areas of nonhomology from a cDNA containing the coding region, the initial construct tested for expression of neutral glucosidase was a hybrid. This hybrid contained a 5′ fragment from clone 2, (beginning 8 bp 5′ of the ATG with an added EcoRI site), ligated at an endogenous SacI site (2,371 bp) to a 3′ fragment derived by RT-PCR of mRNA from cell line GM02756 and extending 15 bp 3" of the stop codon with an added Xba site. (The first methionine in the construct was at bp 753–755 of the original full-length cDNA). We previously had determined that GM02756 expressed two different electrophoretically distinguishable alleles for GANC and that GM02756, therefore, did not contain a null allele lacking expression of enzyme activity. (d) Two allelic cDNA clones, differing in sequence at several sites, were obtained by RT-PCR of mRNA from cell line GM02756. The differing cDNA clones were found to express electrophoretically distinguishable allozymes (see Fig. 2 and Table 1).

Transient expression in Cos cells of cloned α-glucosidase C alleles. Cos cells were transiently transfected with the various cDNA clones in an expression vector. Cell lysates were prepared from nontransfected cells and from cells transfected with the two clones containing differing alleles derived from mRNA of the GM02756 cell line that is heteroallelic for type 2 and type 3 GANC alleles (see Fig. 1d and Table 1). Areas of α-glucosidase activity were visualized under long-wave UV light as fluorescent bands. GANAB is the gene name for human α-chain (catalytic unit) of glucosidase II on chromosome 11; GANC is the official gene name for human neutral α-glucosidase C on chromosome 15. Lane 1: cloned human GANC cDNA expressed in COS cells, showing activity comigrating with the product of human GANC type 3 allele. Lane 2: cloned human GANC cDNA expressed in COS cells, showing activity comigrating with the human type 2 allele. Lane 3: human cell line heterozygous for type 2 and type 3 GANC alleles and the source of mRNA for cloning the allelic cDNAs. Lane 4: human cell line null for GANC (fourfold greater amount of cell lysate applied).