Localization of the human glucosidase I gene to chromosome 2p12-p13 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids

• Published in: Genomics (San Diego, Calif.) Vol. 34; no. 3; pp. 442 - 444
• Main Authors: KALZ-FÜLLER, B, HEIDRICH-KAUL, C, NÖTHEN, M, BAUSE, E, SCHWANITZ, G
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier 15.06.1996
• Subjects: alpha-Glucosidases - genetics; Animals; Base Sequence; Biological and medical sciences; Chromosome Mapping; Chromosomes, Human, Pair 2; Classical genetics, quantitative genetics, hybrids; Cricetinae; DNA Primers; DNA Probes; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; Human; Humans; Hybrid Cells; In Situ Hybridization, Fluorescence; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Genetic mapping; Human; Gene; Enzyme; Transferases; Glycosyltransferases; Chromosome A2
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.1996.0313

The processing of N-linked glycoproteins is initiated by the action of glucosidase I, which cleaves specifically to the distal alpha -1,2 linked glucose residue in the Glc sub(3)-Man sub(9)-GlcNAc sub(2) oligosaccharide precursor after its en bloc transfer from dolichyl diphosphate to the nascent polypeptide chain. The resulting Glc sub(2)-Man sub(9)-GlcNAc sub(2) intermediate is then further modified by glucosidase II and several ER- and Golgi-resident mannosidases and glycosyltransferases, finally yielding a complex array of glycan structures. Glucosidase I has been purified from calf liver, pig liver, and yeast. The amino acid sequence of tryptic peptides of the pig liver enzyme provided the basis for the synthesis of degenerated oligonucleotides that were used to amplify a specific cDNA probe by PCR with porcine first-strand cDNA. Screening of a human hippocampus cDNA library with this probe resulted in the isolation of several glucosidase I-specific clones, which allowed the reconstruction of a full-length cDNA of 2881 bp. The oligonucleotide sequence showed no homology to other processing enzymes cloned so far. On transfection of COS 1 cells with the glucosidase I cDNA, a 95-kDa protein that displayed the same enzymatic properties as the enzyme isolated from pig liver was overexpressed. The expressed enzyme was found to carry one oligosaccharide chain of the high mannose type. Proteolytic and immunofluorescence studies showed that the expressed glucosidase I is a type II transmembrane protein located in the endoplasmic reticulum. Screening of a genomic human placenta library yielded two glucosidase I-specific clones that covered the entire coding sequence of the protein. Two genomic subclones, each carrying a 3.0-kb insert spanning the furthermost 5' and 3' regions, respectively, were generated in the pUC BM 20 vector. These clones were biotinylated with biotin-11-dUTP (Sigma) as probes for in situ hybridization using the Gibco BRL system for nick-translation. Fluorescence in situ hybridization was performed as described previously with minor modifications. In brief, metaphase spreads were prepared from human peripheral blood lymphocyte cultures treated with 5-bromodeoxyuridine (BrdU) during the final 6.5 h prior to harvesting. Slides were pretreated with RNase and pepsin, postfixed for 10 min in 3% paraformaldehyde, and stored in 70% ethanol until denaturation and hybridization. Probe DNA was denatured for 5 min at 75 degree C. After preannealing with an unlabeled cot1 DNA fraction, 500 ng of the probe DNA was hybridized to each denaturated chromosome preparation for 48 h at 37 degree C. Hybridization signals were detected immunohistochemically using fluorescein isothiocyanate conjugated to avidin D. Chromosome preparations were counter-stained with propidium iodide and 4,6-diamidino-2-phenylindole (DAPI) for 20 min, mounted in antifading solution, and evaluated with an epifluorescence microscope. Hybridization consistently gave positive signals on the short arm of chromosome 2 in the region of 2p12-p13. The chromosomal localization was confirmed by polymerase chain reaction (PCR) amplification of chromosome 2-specific DNA sequences from human/rodent somatic cell hybrid DNAs. Mapping panel 2, Version 2, consists of DNA isolated from 24 human/mouse and human/hamster hybrids, each retaining one intact human chromosome. The PCR primers (sense, 5' GGGAGGGAGCAGATACTGG 3'; antisense, 5' CAGCCTCACCCAGATGCTC 3') amplified a 409-bp-long exonic fragment extending from bp 1579-1987 of the cDNA. PCR was carried out in a 25- mu l total volume, using 80 ng genomic DNA, 10 pmol of each primer, 200 nM dNTPs, 1 U Taq DNA polymerase, 50 mM KCl, 1.5 mM MgCl sub(2), 0.01% gelatin, and 10 mM Tris-HCl (pH 8.3). After an initial 5-min denaturation at 95 degree C, 35 cycles were carried out consisting of 20 s at 94 degree C, 20 s at 55 degree to 57 degree C, and 20 s at 72 degree C, followed by a final extension step of 5 min at 72 degree C. Human genomic DNA and hybrid cell lines containing human chromosome 2 showed a positive signal for the human glucosidase I gene. Neither mouse nor hamster control DNAs gave an amplification signal, confirming the localization of the glucosidase I gene on chromosome 2.