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The Journal of Immunology, 1999, 162: 4094-4100.
Copyright © 1999 by The American Association of Immunologists

Molecular Cloning of a Glycosylphosphatidylinositol-Anchored Molecule CDw1081 ,2

Akira Yamada3,*,{dagger}, Keisuke Kubo{dagger}, Toshikazu Takeshita§, Nanae Harashima*, Koichiro Kawano*, Takashi Mine*, Kimitaka Sagawa{ddagger}, Kazuo Sugamura§ and Kyogo Itoh*,{dagger}

* Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy, and Departments of {dagger} Immunology and {ddagger} Transfusion Medicine, Kurume University School of Medicine, Kurume, Japan; and § Department of Microbiology and Immunology, Tohoku University School of Medicine, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23–24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
CDw108, also known as the John-Milton-Hagen (JMH)4 human blood group Ag, is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is preferentially expressed on activated lymphocytes, some leukemic cell lines, and E (1, 2, 3, 4, 5). CDw108 is not only expressed on peripheral blood cells but also on the thymus (6). Most GPI-anchored molecules are associated with the src-family tyrosine kinases, and some of these molecules play important roles in cell activation (7, 8, 9, 10, 11, 12). The physical association of the src-family tyrosine kinases with CDw108 has also been described previously (7). This accumulating evidence suggests that CDw108 may play an important role in lymphocyte development and activation. The presence of GPI-anchor proteins (i.e., CD55 (decay-accelerating factor) and CD59) on the HIV-1 virions and their contribution to HIV-1 infection has been reported (13, 14, 15, 16). Host lymphocyte-derived CDw108 is also incorporated into the HIV-1 envelope during the budding stage, and it is suspected that CDw108 is involved in HIV-1 infection (17). Nonetheless, the molecular characteristics and the biological function of CDw108 have yet to be fully studied. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Antibodies

Spleen cells from a BALB/c mouse that had been immunized with PHA-activated human PBMCs were fused with the murine myeloma cell line NS-1. The mAbs KS-2 (IgG2a) and H105 (IgM) (6) were initially screened as mAbs specific for T cell activation Ags. These two mAbs recognized the same molecule, later designated as CDw108 (5, 6). Immunoprecipitation analysis indicated that the molecular mass of the KS-2/H105 was ~80-kDa under both reducing and nonreducing conditions.

Cells

Human PBMCs were prepared by Ficoll-Conray density gradient centrifugation. PBMCs were cultured with 0.1% PHA-P (Difco, Detroit, MI) containing 10% FCS-RPMI 1640 medium at 37°C for 48 h and used as PHA-activated PBMCs. The leukemic cell lines HPB-ALL (T cell) and NALM-6 (B cell) as well as an esophageal cancer cell line, TE9 (18), were maintained in our laboratory and cultured in 10% FCS-RPMI 1640 medium.

Surface labeling of cells and immunoprecipitation analysis

Cells (1 x 107) were washed twice with PBS and labeled with 37 MBq of Na125I (IMS-30; Amersham, Arlington Heights, IL) by lactoperoxidase-catalyzed iodination as described previously (19). After iodination, cells were washed three times with PBS and subsequently lysed in 1 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, and 0.02% NaN3) at room temperature for 10 min and then on ice for 20 min. The cell lysate was centrifuged at 12,000 rpm for 20 min at 4°C and further precleared overnight at 4°C with formalin-fixed Staphylococcus aureus Cowan I (Mercian, Tokyo, Japan). The precleared lysate was incubated at 4°C overnight with 5 µl of ascites fluid of the mAb KS-2 or control normal mouse serum and subsequently precipitated with rabbit anti-mouse IgG-coupled protein A-AffiGel beads (Bio-Rad, Richmond, CA). Precipitates were washed twice with the lysis buffer, once with the lysis buffer containing 0.5 M NaCl, once with 0.5% deoxycholate, and once with the lysis buffer alone. Immunoprecipitates were solubilized with SDS-PAGE sample buffer and analyzed by 7.5% or 6% SDS-PAGE under reducing conditions. Gels were then fixed, dried, and subjected to autoradiography.

Pulse-chase labeling of cells

Cells (2 x 107) were preincubated at 37°C for 2 h in methionine- and cysteine-free RPMI 1640 (select amine kit, Life Technologies, Rockville, MD) supplemented with 1% dialyzed FCS. Subsequently, the cells were incubated with 52 MBq/ml of a [35S]methionine and [35S]cysteine mixture (Pro-mix L-[35S] in vitro cell labeling mix; Amersham) in the methionine/cysteine-free medium at 37°C for 5 min. After methionine and cysteine were added at a final concentration of 250 mM each, cells were incubated at 37°C for an additional 2–120 min to allow for the maturation of glycosylated CDw108 molecules; cells (2.5 x 106) were then lysed in 0.25 ml of the lysis buffer. The cell lysates were used for further analysis by immunoprecipitation. To inhibit N-linked glycosylation, 1 or 3 µg/ml tunicamycin (Wako Pure Chemical, Osaka, Japan) was added to each step of the labeling protocol, and cells were lysed at 0 or 30 min. When the cells were treated with 10 µg/ml of tunicamycin, the labeling efficiency of the cells was markedly decreased due to a toxicity of tunicamycin.

Glycosidase digestion

Immunoprecipitates from pulse-chase-labeled CDw108 transfectants were denatured with 1 µl of 10% SDS at 95°C for 5 min. After the denaturation, 50 µl of the lysis buffer supplemented with 0.5% 2-ME was added to the precipitates and incubated with 0.4 U of peptide-N-glycosidase F (PNGaseF) (Boehringer Mannheim, Tokyo, Japan) or 2 mU of O-glycosidase (OGase) (EC3.2.1.97; Boehringer Mannheim) at 37°C for 20 h unless stated otherwise. After digestion, SDS sample buffer was added and subjected to SDS-PAGE. Partial digestion with various concentrations of PNGaseF was performed at 37°C for 10 min to estimate the number of N-linked oligosaccharide chains.

Isolation and N-terminal amino acid sequencing of CDw108

The CDw108 molecule was immunoprecipitated with the mAb KS-2 from Triton X-100-solubilized HPB-ALL cells (equivalent to 109 cells) and subsequently separated by two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) as described previously (19). After the separation, the appropriate protein spot was electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and subjected to an automatic protein sequencer (477A; Perkin Elmer Applied Biosystems, Foster City, CA).

cDNA cloning and sequencing of CDw108

The partial cDNA fragment (60 base pairs (bp) in length) of the CDw108 gene encoding the N-terminal peptide was amplified from HPB-ALL cells by RT-PCR with degenerated primer pairs; next, the PCR products were cloned into a plasmid vector, pCRII, with a TA cloning system (Invitrogen, San Diego, CA). The nucleotide sequences of the cloned cDNA fragments were determined using an automatic DNA sequencer (ALFexpress DNA sequencer; Pharmacia, Uppsala, Sweden). One primer pair (K1: 5'-CA(C/T)CTGAG(A/G)AGCGGAC-3' and K8: 5'-ACG(G/A)TC(C/T)TGCCC(C/G)ACATG-3') successfully amplified the 60 bp of the CDw108 cDNA fragment whose deduced amino acid sequence matched 19 of 20 amino acids (aa) of the N-terminal amino acid sequence under the following conditions: denaturing at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 1 min for 25 cycles. A total of 25-mer of the synthetic oligonucleotide KS-2gt (5'-ATCTTCGCCGTCTGGAAAGGCCATG-3') matched to the amplified sequence was used as a probe and a primer for the following screening from the cDNA library. A cDNA library from HPB-ALL cells was screened with the Gene Trapper cDNA positive selection system (Life Technologies) using the KS-2gt probe/primer. The cDNA library was created using the SuperScript plasmid system for cDNA synthesis and plasmid cloning (Life Technologies). Briefly, the cDNA library was primed with oligo(dT)-NotI primer and ligated to SalI adapter. After NotI digestion, the cDNA library was size-selected to remove excess adapter and unidirectionally ligated to a plasmid expression vector, pCMVSPORT2.0 (Life Technologies), at the NotI/SalI site. An oligo(dT)-primed cDNA library from HPB-ALL cells ligated into the EcoRI site of {lambda}ZAPII (Stratagene, La Jolla, CA) and a placenta cDNA library (5'-stretch plus cDNA library; Clontech, Palo Alto, CA) were also used to screen the full-length CDw108 cDNA clone. Colony or plaque hybridization for the screening of the cDNA libraries was performed as follows: a total of 1 x 106 colonies or plaques were transferred to nylon filters (Hybond-N+; Amersham), hybridized with 32P-labeled probe overnight at 65°C in 7% SDS/1 mM EDTA/0.5 M Church’s phosphate buffer (0.25 M Na2HPO4, pH 7.2) (20), and washed in 1% SDS plus 40 mM of Church’s phosphate buffer at 65°C. The nucleotide sequences of both strands of the cloned cDNA were determined with multiple primers using the automatic DNA sequencer.

Transient transfection and flow cytometric analysis

An insert of a cDNA clone, ZH5, encoding the CDw108gene from the HPB-ALL/{lambda}ZAPII library was excised with pBluescript and further subcloned into a plasmid expression vector, pCR3.1 (Invitrogen) at the EcoRI site. Two clones, pCR3-ZH5.11 and pCR3-ZH5.23, with opposite directions were used for transfection. Plasmid pCR3-ICAM-1 containing the entire open reading frame (ORF) of human ICAM-1 (CD54) cDNA with sense direction was also used as a control. TE9 cells (4 x 105 cells) were precultured for 18 h in one well of a six-well plate with 10%FCS-RPMI 1640 medium. The cells were rinsed twice with PBS and transfected with 2 µg of pCR3-ZH5.11, pCR3-ZH5.23, or pCR3-ICAM-1 and with 10 µl of Lipofectamine (Life Technologies, Gaithersburg, MD) in 1 ml of Opti-MEM (Life Technologies) at 37°C. At 6 h after the addition of the DNA and Lipofectamine mixture, 2 ml of the 10% FCS-RPMI 1640 was added to the culture. The cell surface expression of the CDw108 or CD54 was examined at 2 days posttransfection. Cells (2 x 105) were stained with FITC-conjugated anti-CDw108 (KS-2) or anti-CD54 (YH370) mAb on ice for 30 min. After washing with PBS, cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment was performed as follows: cells were washed twice with PBS and incubated with 1 U/ml of PI-PLC (EC3.1.4.10; Boehringer Mannheim) in PBS at 37°C for 1 h.

Chromosome mapping of CDw108 gene

A fragment of the CDw108 gene spread out on two exons with one intron was amplified by PCR from a panel of radiation hybrid clones of whole human genome (Gene Bridge 4 Radiation Hybrid Panel; Research Genetics, Huntsville, AL) (21). PCR amplifications were performed with a sense primer, KS205S (position 344–362 of the cDNA; 5'-CACGGTGAATATCGGCTCC-3'), and an antisense primer, KS357A (494–475; 5'-AGTGCCATTCACCAGGTTCC-3'), under the following conditions: denaturing at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min for 35 cycles. The PCR products were subsequently dot-blotted on a nitrocellulose filter and hybridized with the 32P-end-labeled sequence-specific oligonucleotide probe KS254S (393–409; 5'-TGCGAGAACTACATCAC-3') in a hybridization buffer (3 M tetramethyl ammonium chloride, 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.1% SDS, and 5x Denhardt’s solution) at 54°C for 2 h. This probe specifically hybridized with the human, but not the hamster, CDw108 gene; even the critical sequence of the hamster CDw108 gene was not identified. After the incubation, the filter was washed with tetramethyl ammonium chloride solution (identical with the hybridization buffer but without 5x Denhardt’s solution) at 58°C and subjected to autoradiography. The results obtained were analyzed using the radiation hybrid map software of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research (http://www-genome.wi.mit.edu/cig-bin/contig/rhmapper.pl).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Nucleotide sequence and deduced amino acid sequence of CDw108cDNA

Human CDw108 cDNA clones were identified by the screening of a plasmid library generated from a leukemic T cell line, HPB-ALL, with N-terminal peptide-encoding oligonucleotide probe/primer. The obtained 1-kbp cDNA fragment was used as a probe for further screening of both the HPB-ALL phage library and a human placenta cDNA library to obtain longer cDNA clones, because the fragment was truncated. The cDNA sequence of the longest clone from the HPB-ALL library, ZH5, revealed an ORF of 1998 bp encoding a protein of 666 aa following the Kozak motif (22) (Fig. 1Go). The ORF contained 46 aa of the N-terminal signal peptide (M1-G46), 602 aa of the extracellular domain (H47-A648), and a GPI-anchor motif for an anchor addition in the C-terminal (23) (a cluster of three small residues (A648-S650) and a subsequent C-terminal 16 hydrophobic residue (L651-H666)). Thus, a possible cleavage/anchor addition site (the {omega} site) is A648. The predicted molecular mass of the ORF after cleavage with the GPI-anchoring enzyme is 68 kDa. There are five putative N-linked glycosylation sites. Furthermore, the RGD (Arg-Gly-Asp) sequence is found in the ORF at position 267–269, and this sequence was originally identified in fibronectin as a cell attachment sequence (24, 25) (Fig. 2Go). Previous studies suggest the existence of an intrapeptide disulfide bond(s), because the observed molecular mass of the CDw108 was 76 kDa and 80 kDa under nonreducing and reducing conditions, respectively (4, 5). A total of 19 cysteine residues were contained in the ORF, and some of the cysteines might be formed of disulfide bond(s). Homology analysis by basic local alignment search tool (BLAST) search indicated that the nucleotide sequence of the CDw108 gene was different from those of any known genes, including the genes encoding the other GPI-anchor proteins. A study using JMH alloimmune Abs suggested that CDw108 had variant types with different reactivities to the JMH alloantibodies (26). The molecular mass of the variant CDw108 is the same as that of the normal one, and it most likely represents polymorphic alleles at the JMH locus (27). Several cDNA clones encoding CDw108 were also obtained from the placenta cDNA library as well as from the HPB-ALL library (data not shown). There were no mutations or polymorphisms in these sequences. Identification of the variant-type CDw108 allele from individuals expressing variant JMH will be necessary.



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  		FIGURE 1. Nucleotide sequence and deduced amino acid sequence of CDw108 cDNA. The N-terminal 20-mer peptide at position H47 to D65 and the GPI-anchor motif at position A648 to H666 is underlined. Putative N-linked glycosylation sites and the RGD cell attachment sequence are also underlined.

 


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  		FIGURE 2. Molecular structure of CDw108. Numbers indicate amino acid residues. • indicate putative N-linked glycosylation sites.

 
Analysis of CDw108 transfectants

Cells of the human esophageal cancer cell line, TE9, were transiently transfected with a cDNA clone, pCR3-ZH5.11, which contained the entire ORF of the CDw108 gene. The cell surface expression of CDw108 on the transfectants was analyzed by FACScan with the anti-CDw108 mAb KS-2 (Fig. 3Go). A high level of CDw108 expression was observed on the CDw108 transfectants. Expression of CDw108 on the transfectants was also detected by the other anti-CDw108 mAbs (H105 and MEM-150, a standard anti-CDw108 mAb) (3, 4) (data not shown). In contrast, the transfectants with an antisense CDw108 clone, pCR3-ZH5.23, did not express CDw108 on the cell surface (Fig. 3Go). After treatment with PI-PLC, the expression of CDw108 on the HPB-ALL and the CDw108 transfectants markedly decreased (Fig. 3Go). However, in the control experiment, the expression of CD54 (ICAM-1), a type-I membrane protein, on ICAM-1 transfectants was not affected by the treatment (data not shown). Immunoprecipitation analysis of the transfectants also confirmed the expression of CDw108 on the transfectants (Fig. 4GoA). An 80-kDa band was precipitated from both the lysates of HPB-ALL and the CDw108 transfectants but not from the lysate of the parental TE9 cells. A 70-kDa band was also observed in the transfectants. High-resolution gel analysis with 6% SDS-PAGE indicated that the 80-kDa band was composed of two bands of 80 kDa and 78 kDa (Fig. 4GoB, surface label). The 70-kDa band was also composed of two bands (71 kDa and 68 kDa). Pulse-chase analysis indicated that the 78-kDa form was cotranslationally produced with the 80-kDa mature form appearing within a few minutes (Fig. 4GoB). In other words, the 78-kDa band was dominant even if both 78-kDa and 80-kDa bands were observed immediately after (0 min) short (5 min)-pulse labeling. The content of the 80-kDa band at 0 min varied in different experiments (Fig. 4Go, B and C). The density of both bands became equal at 6 min, and then the relative content of the 80-kDa band gradually increased until 120 min (Fig. 4GoB); the 78-kDa band was still observable at 4 h (data not shown).



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  		FIGURE 3. FACScan analysis of CDw108 transfectants. TE-9 cells were transiently transfected with CDw108 cDNA (TE-9/ZH5.11) or with a control antisense construct (TE-9/ZH5.23). PI-PLC-treated or untreated HPB-ALL cells and transfectants were stained with FITC-conjugated anti-CDw108 mAb and analyzed on a FACScan.

 


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  		FIGURE 4. Immunoprecipitation analysis of CDw108 protein. A, Cell lysates from the surface 125I-labeled CDw108 transfectants, parental TE-9 cells, and HPB-ALL cells were immunoprecipitated with anti-CDw108 (KS-2) or control normal mouse serum (NMS) and analyzed on a 7.5% SDS-PAGE under reducing conditions. This experiment was repeated twice; one representative data set is shown. B, Cell lysates from pulse-chase-labeled CDw108 transfectants were immunoprecipitated with anti-CDw108 and analyzed on a 6% SDS-PAGE under reducing conditions. Immunoprecipitate from the surface-labeled transfectants was also analyzed. Data shown are representative of three experiments. C, CDw108 transfectants were pulse-labeled and chased for 30 min or unchased (0 min) in the presence of tunicamycin (1 or 3 µg/ml). Controls were similarly pulse-chase-labeled without tunicamycin. The pulse-chase-labeled cells were subsequently lysed and immunoprecipitated with anti-CDw108. Data shown are representative of three experiments. The seventh lane from the left is a long exposed film of the sixth lane (sample treated with 3 µg/ml tunicamycin and chased for 30 min). D, Immunoprecipitates with anti-CDw108 from the lysate of pulse-labeled and 30 min-chased CDw108 transfectants were digested with PNGaseF or OGase at 37°C for 20 h. Controls were treated similarly without any glycosidases. Immunoprecipitate from the surface-labeled transfectants was also digested with PNGaseF and analyzed. Data shown are representative of two experiments. E, Immunoprecipitates with anti-CDw108 from the lysate of pulse-labeled and 30 min-chased CDw108 transfectants were digested with various concentrations of PNGaseF at 37°C for 10 min. Data shown are representative of two experiments.

 
The treatment of cells with tunicamycin, an inhibitor of N-linked glycosylation, suggested that the molecular mass of the unglycosylated precursor of CDw108 was 65 kDa (Fig. 4GoC). In this experiment, N-linked glycosylation was not completely inhibited even when the cells were treated with 3 µg/ml of tunicamycin and when five extra-faint bands, which might be differentially glycosylated, were observed (Fig. 4GoC). The digestion of immunoprecipitates from pulse-chase labeled CDw108 transfectants using PNGaseF, which cleaves all types of N-glycans, was further performed to confirm the molecular mass of the precursor (Fig. 4GoD, pulse label). Only a 65-kDa band was observed in a sample treated with PNGaseF. In contrast, treatment with an OGase, which cleaves O-glycans, did not affect the mobility and relative ratio of the 78-kDa and 80-kDa bands. These results suggested that the molecular mass of the precursor of CDw108 was 65 kDa and that all of the oligosaccharide chains of CDw108 were N-linked. Partial digestion with various concentrations of PNGaseF was further performed to estimate the number of N-linked oligosaccharide chains (Fig. 4GoE). Five differentially deglycosylated forms were observed in this experiment and differences between each band were ~2–3 kDa. These results together with tunicamycin treatment suggest that there are five oligosaccharide chains; this number matches the number of putative N-glycosylation sites. All of these results indicated that the cloned cDNA encoded the native form of CDw108.

The 71-kDa and 68-kDa bands observed in the surface-labeled samples were not seen in the pulse-labeled sample. The molecular masses of these two bands were close to those of incompletely glycosylated forms of CDw108. The 71-kDa and 68-kDa bands disappeared after treatment with PNGaseF, and a 60-kDa band, in addition to the 65-kDa unglycosylated form of CDw108, was observed (Fig. 4GoD, surface label). These results suggest that the 71-kDa and 68-kDa bands found in surface-labeled samples are some other associated molecules whose biosynthetic rates are low, rather than incompletely glycosylated forms of the CDw108. Further analysis may be needed to identify these molecules.

Tissue distribution of CDw108 mRNA expression

Northern blot analysis of the CDw108 mRNA in HPB-ALL cells indicated that the size of the CDw108 mRNA was ~3.5 kilobases (kb); the same size was also found in PHA-activated PBMCs but not in resting PBMCs or NALM-6 cells (Fig. 5GoA). Expression of the CDw108 mRNA in a panel of normal tissues was further analyzed by Northern blotting (Fig. 5GoB). High expression of the CDw108 mRNA was found in the placenta, testis, and spleen, and a weak signal was detected in the brain and thymus. CDw108 mRNA was not expressed in the prostate, uterus, small intestine, colon, heart, lung, liver, skeletal muscle, kidney, or pancreas. These results suggest that CDw108 may play an important role not only in the lymphoid organs but in other organs as well. The estimated size of CDw108 in the tissues was ~3.5 kb, which was the same size as the HPB-ALL and PHA-activated PBMCs. The discrepancy between the size of the mRNA and that of the cloned cDNA suggested the existence of an unidentified sequence of ~800 bp in the 5'- and/or 3'-untranslated regions.



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  		FIGURE 5. Northern blot analysis of CDw108 mRNA. A, mRNA expression in the NALM-6 cells, HPB-ALL cells, resting PBMCs, and PHA-activated PBMCs. B, mRNA expression in the various normal tissues.

 
Chromosome mapping of CDw108 gene

Chromosome mapping of the CDw108 gene was performed by the radiation hybrid mapping method (21). A genomic DNA panel of hybrids with 1000-kb resolution was made from hybrid cells of irradiated human HFL cells and hamster A23 cells and was used as a template for PCR (21). The PCR with the primer pair KS205S and KS357A amplified the 449-bp fragment from genomic DNA isolated from PBMCs (Fig. 6GoA). The PCR product contained 298-bp of an intron with GT-AG rule agreement (28). A fainter band of the same size of PCR product was also amplified from hamster A23 cell-derived genomic DNA under the same conditions. Therefore, the PCR products from the radiation hybrid panel were dot-blotted and subsequently hybridized with the sequence-specific oligonucleotide probe KS254S. This probe specifically hybridized with the product from the human HFL cell-derived genomic DNA (Fig. 6GoB, third column from the right of the bottom lane) but not with the hamster A23 genomic DNA (Fig. 6GoB, second column from the right of the bottom lane). Database analysis for the radiation hybrid mapping indicated that the CDw108 gene was located on the middle of chromosome 15, 5.23 centirays distal from the WI-6247 marker gene, compatible with the classical 15q23–24 (Fig. 6GoC). This region is close to the following metabolic disease-associated genes: polycystic ovary syndrome with hyperandrogenemia, Tay-Sachs disease, GM2-gangliosidosis, HexA pseudodeficiency, type-IIA glutaricacidurea, and type-I tyrosinemia (29). Therefore, it will be important to investigate the involvement of the CDw108 gene in the onset or pathogenesis of these diseases.



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  		FIGURE 6. Chromosome mapping of the CDw108 gene. A, Genomic DNA from PBMCs was amplified by PCR with KS205S and KS357A primers. The 449 bp of the amplified fragment contained 298 bp of intron. B, The same fragments were amplified from the Gene Bridge 4 Radiation Hybrid Panel (Research Genetics), dot-blotted on a nitrocellulose filter, and hybridized with the sequence-specific oligonucleotide probe KS254S. C, Chromosomal localization of the CDw108 gene.

 


   Footnotes
 
1 This study was supported in part by grants for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 The GenBank accession number of the human CDw108 is AF069493. Back

3 Address correspondence and reprint requests to Dr. Akira Yamada, Department of Immunology, Kurume University School of Medicine, Asahi-machi 67, Kurume, Fukuoka 830, Japan. E-mail address: Back

4 Abbreviations used in this paper: JMH, John-Milton-Hagen; GPI, glycosylphosphatidylinositol; ORF, open reading frame; PI-PLC, Phosphatidylinositol-specific phospholipase C; PNGaseF, peptide-N-glycosidase F; OGase, O-glycosidase. Back

Received for publication June 15, 1998. Accepted for publication January 7, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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