Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

Tetsuo Takahashi1, Risako Honda and Yoshihisa Nishikawa

Laboratory for Glycobiology and Glycotechnology, Department of Industrial Chemistry, School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka City, Kanagawa 259–1292, Japan

Received on August 2, 1999; accepted on August 20, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The assembly of the lipid-linked oligosaccharide, Glc3Man9GlcNAc2-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene (ALG1), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S.cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.

Key words: cDNA cloning/EST database/glycosylation/lipid-linked oligosaccharide/mannosyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In higher eukaryotes, glycoproteins are present mainly on the cell surface and as extracellular molecules and they function in intercellular, molecule-to-cell, and intermolecular interactions. The most common glycans of glycoproteins, Asn-linked oligosaccharides, serve as the recognition signals in these interactions and in the maintenance of the physico-chemical properties of glycoproteins. The biosynthesis of Asn-linked oligosaccharides consists of three stages: first, assembly of lipid-linked oligosaccharides in rough ER membrane by glycosyltransferases; second, transfer of fully assembled lipid-linked oligosaccharides (Glc3Man9GlcNAc2-P-P-Dol) to Asn residues at the -Asn-X-Ser/Thr- consensus sequence in nascent proteins by oligosaccharyltransferase; and third, processing of Asn-linked oligosaccharides in rough ER and Golgi by glycosyltransferases and glycosidases. The biosynthesis of lipid-linked oligosaccharides is highly conserved among eukaryotes and is catalyzed by fourteen glycosyltransferases in an ordered stepwise manner (Kukuruzinska et al., 1987Go; Herscovics and Orlean, 1993Go). However, in mammals, none of the glycosyltransferases involved in the synthesis of lipid-linked oligosaccharides has been well characterized except the hamster (Zhu and Lehrman, 1990Go) and mouse (Rajput et al., 1992Go) GlcNAc-1-P transferase.

We have studied the effect of alterations of the biosynthesis of lipid-linked oligosaccharides on cell growth, for example, the reversible G1 arrest of the human Burkitt lymphoma cell line, Raji, by tunicamycin (Nishikawa et al., 1980Go) and the isolation of a mouse FM3A mutant G258 cell line that has temperature-sensitive defects in both cell growth and the synthesis of lipid-linked oligosaccharides (Nishikawa, 1984Go). The biochemical lesion of the G258 mutant may have resulted from a defect in GDP-mannose dependent mannosylation of Man3GlcNAc2-P-P-Dol (mannosyltransferase IV) (Nishikawa, 1991Go). We have reported on a human gene that complements the defect of the synthesis of lipid-linked oligosaccharide of the G258 mutant (Kataoka et al., 1997Go, 1999). The latter study prompted us to attempt the isolation of other GDP-mannose dependent mannosyltransferase genes. The present study reports the identification of the human gene, designated Hmat-1, that must encode mannosyltransferase I (MT-I) which catalyzes the following reaction:

GlcNAcß1->4GlcNAc-P-P-Dol + GDP-Man ->

Manß1->4GlcNAcß1->4GlcNAc-P-P-Dol + GDP

MT-I catalyzes the first mannosylation step in the synthesis of lipid-linked oligosaccharides and is the only mannosyltransferase which forms the ß1->4 mannose linkage in the lipid-linked oligosaccharides (Couto et al., 1984Go). The MT-I gene has already been isolated from yeast (Albright and Robbins, 1990Go), nematode (Wilson et al., 1994Go), and slime mold (Lee et al., 1997Go). In this study, we have cloned and characterized the cDNA containing the Hmat-1 gene. This is the first report on the cloning of a mammalian mannosyltransferase gene involved in the early assembly of lipid-linked oligosaccharides.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human EST clones which partially encode a protein homologous to the yeast MT-I
Using the entire amino acid sequence deduced from the yeast Saccharomyces cerevisiae ALG1 gene (Albright and Robbins, 1990Go), we searched the human EST database for the human counterpart of the yeast ALG1 gene. Seven homologous EST clones were detected (Figure 1). Comparison among their nucleotide sequences revealed that they partially overlap with each other, suggesting that they are parts of one human gene (Figure 1).



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Fig. 1. Relative position of the detected human EST clones and the PCR primers. The GenBank accession numbers are shown in the left column. The position and size of the partial nucleotide sequences obtained from each EST clone are shown as horizontal lines. Based on the nucleotide sequences of these EST clones, we designed the gene-specific forward primer (A1-1F) and reverse primer (A1-2R) as shown by the arrows.

 
Partial cloning of the human counterpart cDNA of the yeast ALG1 gene
Based on coincident sequences among these seven cDNAs, gene-specific PCR primers were designed to screen a human cDNA library. AA083625, W38780 and R52790 (GenBank accession numbers) were used to design the A1-1F primer, and R52790, AA256650, W25302, AA493371 and AA024843 (GenBank accession numbers) were used to design the A1-2R primer (Table I, Figure 1). PCR with A1-1F and A1-2R primers using the {lambda}gt10 human fetal brain cDNA library as a template yielded a 430 bp fragment (Figure 2, lane B) which corresponded to the estimated distance between A1-1F and A1-2R (Figure 1).


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Table I. PCR primers used in this study
 


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Fig. 2. Agarose gel electrophoresis of human cDNAs obtained from a human fetal brain cDNA library. A, Sty I-digested {lambda} DNA (size marker DNA); B, PCR product with A1-1F and A1-2R primers; C, cDNA insert from HR1-3; D, cDNA insert from HR1-4; E, PCR product with A1-10F and A1-8RS primers.

 
Using the 430 bp PCR product as a probe, the cDNA library was rescreened by plaque hybridization. Two cDNA clones, designated HR1-3 (about 1.1 kb) and HR1-4 (about 1.4 kb), were obtained (Figure 2, lanes C and D) and subjected to nucleotide sequencing as described in Materials and methods. Both HR1-3 and HR1-4 contained the nucleotide sequence of the 430 bp PCR product. Restriction enzyme mapping and sequencing of these two cDNAs showed that they partially overlap with each other and share a common ORF (Figure 3). However, this ORF was incomplete; HR1-3 contained neither a putative initiation nor termination codon, whereas HR1-4 contained a termination codon (TAA, nucleotides 1408–1410, Figure 4) but not a polyadenylation signal sequence, suggesting that there is a relatively long 3' untranslated region in this gene (Figure 4).



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Fig. 3. Restriction map and relative position of the two cDNA clones HR1-3 and HR1-4 and EST clone AA675921. Arrows above show the gene-specific primers. The bold bar below shows the coding region of the Hmat-1 gene (Hmat-1 Coding Region).

 


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Fig. 4. Nucleotide sequence of the Hmat-1 gene. Amino acid sequence deduced from the Hmat-1 gene is illustrated below in bold.

 
Isolation and nucleotide sequencing of the Hmat-1 cDNA
With the nucleotide sequence of HR1-3 as a probe, we performed a homology search of the human EST database and detected the EST clone, AA675921 (GenBank accession number). Comparison of the nucleotide sequence of AA675921 with those of HR1-3 and HR1-4 showed that these three cDNAs partially overlap with one other (Figure 3). AA675921 contained the putative initiation codon (Figure 4, nucleotides 16–18) of the gene, judging from the similarity of both the amino-terminal sequence and size of the human protein to those of the S.cerevisiae and Caenorhabditis elegans MT-I proteins (Figure 5).



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Fig. 5. Comparison of the deduced amino acid sequence of the Hmat-1 protein (Hmat-1 ptn) with those of the yeast Saccharomyces cerevisiae (ALG1 ptn) and the nematode Caenorhabditis elegans (T26A5. 4 ptn). Upper asterisks (*) indicate the amino acid residues which are highly conserved between human, yeast and nematode.

 
We therefore designed A1-10F and A1-8RS primers from the sequences of AA675921 and HR1-4, respectively, for the cloning of the longer cDNA (Table I, Figure 3). PCR with these primers and human fetal brain cDNA as template allowed the amplification of about 1.8 kb cDNA (Figure 2, lane E), which corresponded with the estimated distance between A1-10F and A1-8RS (Figure 3). By subcloning and sequencing this 1.8 kb cDNA, we identified several bases which had not been characterized in AA675921 (data not shown) and obtained the complete ORF, which we designated the Hmat-1 gene (Figures 3, 4). The nucleotide sequence of the Hmat-1 gene has been registered in the GenBank database (accession number AB019038).

Amino acid sequence deduced from the Hmat-1 gene
Based on the nucleotide sequence of the 1.8 kb Hmat-1 cDNA, we deduced the amino acid sequence (Figure 4). The predicted protein consists of 464 amino acid residues equivalent to a 52.5 kDa molecular weight. It has no potential N-glycosylation site (-Asn-X-Ser/Thr-), although the S.cerevisiae MT-I protein has four such sites (Albright and Robbins, 1990Go). Moreover, it has a hydrophobic region at the amino terminus (Figure 5, amino acids 6–20) followed by short hydrophilic and hydrophobic regions similar to the S.cerevisiae MT-I protein.

The amino acid sequence of the protein that the Hmat-1 gene encodes was compared with those of both the S.cerevisiae MT-I (Albright and Robbins, 1990Go) and the C.elegans T26A5.4 gene product (Wilson et al., 1994Go) which was predicted to be MT-I (Figure 5). The Hmat-1 gene product showed 36% identity with both MT-I proteins.

The Hmat-1 gene can complement the yeast alg1-1 mutation
To ascertain whether the 1.8 kb Hmat-1 cDNA encodes the human MT-I, we subcloned it into the S.cerevisiae episomal expression vector, YEp352GAP, which contains URA3 gene and GAP promoter sequence (Yoko-o et al., 1993Go). Then, with the resultant recombinant plasmid, we transformed the S.cerevisiae PRY56 strain, which has the alg1-1 mutation (Huffaker and Robbins, 1982Go). The PRY56 strain did not grow on the plate of YPD medium at 36°C nor on the plates of SD medium lacking uracil [SD(Ura-) medium] at either 26°C or 36°C (Figure 6B–D). The mock-transformant grew on the SD(Ura-) plate at 26°C (Figure 6B), but did not grow on either the YPD or SD(Ura-) plates at 36°C (Figure 6C,D). Only the transformant with the Hmat-1 cDNA grew under the nonpermissive condition for the alg1-1 mutant (i.e., incubation at 36°C for 3 days, Figure 6C,D). To prove the presence of the introduced Hmat-1 cDNA, PCR with A1-10FX and A1-8RS primers was performed using as templates DNA from the PRY56 strain, the mock-transformant and the transformant with the Hmat-1 cDNA. The 1.8 kb PCR product was detected only in the transformant with the Hmat-1 cDNA (data not shown). These observations indicate that the expressed Hmat-1 gene product could complement the temperature-sensitivity for cell growth due to the S.cerevisiae alg1-1 mutation.



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Fig. 6. Complementation of alg1-1 mutation by the Hmat-1 gene. Colonies from the S.cerevisiae alg1-1 mutant (PRY56), YEp352GAP-transformed PRY56 (Vector, mock-transformation), and PRY56 transformed with the Hmat-1 cDNA (Hmat-1) were replated on YPD media (A and C) or SD(Ura-) media (B and D) and incubated at 26°C (A and B) or 36°C (C and D) for 3 days.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The glycosyltransferases involved in the early seven steps of the assembly of lipid-linked oligosaccharides use the nucleotide-sugars UDP-GlcNAc and GDP-Man as donor substrates and react at the cytosolic face of the rough ER membrane (Snider and Robbins, 1982Go; Hirschberg and Snider, 1987Go; Abeijon and Hirschberg, 1990Go, 1992). These steps are essential for growth in yeast as shown by the analysis using tunicamycin (Arnold and Tanner, 1982Go) and various S.cerevisiae Asn-linked glycosylation mutants such as alg1 (Huffaker and Robbins, 1982Go), alg2 (Huffaker and Robbins, 1983Go), and alg7 (Rine et al., 1983Go). Moreover, using yeast mutants defective in these steps, several yeast glycosyltransferase genes have been isolated by the complementation of each mutation and characterized (Barnes et al., 1984Go; Albright and Robbins, 1990Go; Jackson et al., 1993Go). However, in the case of the higher eukaryotes, such conditional lethal mutant cell lines deficient in the early assembly of lipid-linked oligosaccharides have not yet been isolated with the exception of the mouse G258 mutant line (Nishikawa, 1984Go, 1991; Kataoka et al., 1997Go, 1999), making the expression cloning of mammalian glycosyltransferase genes involved in these steps very difficult. Isolation of these genes by the conventional homology cloning method have not yet been reported except for the hamster GlcNAc-1-P transferase gene (Scocca and Krag, 1990Go). This difficulty may have been due to significant differences of nucleotide sequence between yeast genes and their mammalian counterparts. Therefore, we improved the homology screening method for isolating the human mannosyltransferase genes (Takahashi et al., 1997Go); we performed homology searches on the human EST database using as probes the deduced amino acid sequences of the S.cerevisiae mannosyltransferases with the tBLASTn search program (Altschul et al., 1990Go) and identified several human EST clones. By using the nucleotide sequences of these EST clones, we designed gene-specific PCR primers and partially amplified the human counterparts of the yeast mannosyltransferase genes by PCR using a human cDNA library as template. We then screened the same cDNA library with the gene-specific PCR product. By this methodology, we were finally successful in isolating the Hmat-1 gene that can complement the defect in the S.cerevisiae alg1-1 mutant.

MT-I is involved in the first mannosylation step of lipid-linked oligosaccharide synthesis. In yeast, the defect in the function of this enzyme (i.e., alg1-1 mutation) directly resulted in underglycosylation of glycoproteins and cell death (Huffaker and Robbins, 1982Go). Furthermore, expression of the MT-I gene is regulated at the transcriptional level in the cell division cycle (Lennon et al., 1995Go) or morphogenesis (Lee et al., 1997Go). These studies prove that MT-I is one of the key enzymes in the assembly of lipid-linked oligosaccharides.

We have isolated the 1.8 kb Hmat-1 cDNA and determined its nucleotide sequence (Figure 4). It contained both a methionine codon whose surrounding sequence (Figure 4, nucleotides 10–19) matched a Kozak initiation codon consensus sequence (Kozak, 1984Go) and a termination codon. These results suggest that it contains the full-length coding region of the Hmat-1 gene. The predicted Hmat-1 protein was very similar to the S.cerevisiae ALG1 protein and was suggestive of a type II transmembrane protein. However, in this protein, there was no sequence which was homologous to the potential dolichol recognition sequence in the S.cerevisiae ALG1 protein (Albright et al., 1989Go). Alternatively, the Hmat-1 protein had eight highly conserved sequences shared with ALG1 protein and T26A5.4 gene product (Figure 5, 39VLGDXXXSPR48, 130QXPPXXP136, 154IDWHNXXY161, 283STSXTXD­EX­XXILXXA298, 319ITGKGP324, 347WLXXEDYP354, 361DXGXXLHXSXSGXDLPMKXXDXFGXXXPXXA391, and 399ELVXXXXNG407). Moreover, these conserved sequences also exist in MT-I from Dictyostelium discoideum (Lee et al., 1997Go) and Schizosaccharomyces pombe (GenBank accession number Z99753) (data not shown). Some of these conserved regions may function in the topological localization to the rough ER membrane, in the recognition of GDP-Man and/or GlcNAc2-P-P-Dol, and in the catalytic domain of this enzyme. Particularly, the highly hydrophobic N-terminal region containing the 39VLGDXXXSPR48 sequence may play an important role in functional expression of this enzyme, because the truncated Hmat-1 cDNA lacking this region (Figure 4, nucleotides 1–186) could not complement the S.cerevisiae alg1-1 mutation (data not shown).

The fact that the 1.8 kb Hmat-1 cDNA complemented the S.cerevisiae alg1-1 mutation strongly suggests that the Hmat-1 gene encodes human MT-I and that the expressed human MT-I functions normally in yeast. This leads to the possibility that other human glycosyltransferases involved in the early assembly of lipid-linked oligosaccharides should be functionally conserved between yeast and human. This possibility is supported by the successful complementation of the S.cerevisiae alg7 mutation with human GlcNAc-1-P transferase (Eckert et al., 1998Go). If all the glycosyltransferases involved in the biosynthesis of lipid-linked oligosaccharides were functionally conserved between yeast and human, the S.cerevisiae N-glycosylation mutants such as alg2 (Huffaker and Robbins, 1983Go), alg3 (Huffaker and Robbins, 1983Go), alg6 (Reiss et al., 1996Go), alg8 (Stagljar et al., 1994Go), alg9 (Burda et al., 1996Go), and alg10 (Burda and Aebi, 1998Go) might be useful for the isolation of their human counterpart genes by the expression cloning method.

The importance of Asn-linked glycosylation in human has been demonstrated in various ways. Particularly, in the inherited group of diseases, carbohydrate deficient glycoprotein syndrome (CDGS), the Asn-linked oligosaccharides were severely impaired either in number per molecule of glycoprotein (underglycosylation, CDGS type I) or in structure (CDGS type II). It has been demonstrated that CDGS type II is due to the deficiency of N-acetylglucosaminyltransferase II (Jaeken et al., 1994Go; Tan et al., 1996Go). On the other hand, it has been demonstrated that about 80% of patients with CDGS type I have a deficiency of phosphomannomutase (CDGS type Ia) (Van Schaftingen and Jaeken, 1995Go). A deficiency of the enzyme phosphomannose isomerase has also been demonstrated to be responsible for patients showing a CDGS type I phenotype (CDGS type Ib) (de Koning et al., 1998Go; Niehues et al., 1998Go). Genes for the human glycosyltransferases involved in the synthesis of lipid-linked oligosaccharides are potential mutation sites for other subtypes of CDGS type I. Recently, it have been reported that a deficiency of glucosyltransferase I involved in the synthesis of lipid-linked oligosaccharides caused underglycosylation in a CDGS patient (CDGS type Ic or V) (Körner et al., 1998Go; Burda et al., 1998Go). Therefore, genetic analysis of CDGS patients for defects in mannosyltransferase genes involved in the synthesis of lipid-linked oligosaccharides may be clinically useful.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Screening of EST database
The amino acid sequence deduced from the S.cerevisiae ALG1 gene was used to detect homologous human EST clones by searching the human EST database (GenBank) with the tBLASTn program (Altschul et al., 1990Go).

Design of gene-specific PCR primers
Based on the coincident nucleotide sequences among seven homologous human EST clones detected in the homology search described above (Figure 1), we designed two PCR primers, A1-1F and A1-2R (Table I, Greiner Japan). PCR primers, A1-10F and A1-8RS (Table I, Greiner Japan) were designed from the 5' terminus of AA675921 (GenBank accession number) and 3' terminus of HR1-4, respectively (Figure 3). These primers also contained restriction enzyme recognition sequences at their 5' ends, which were used for subcloning into the plasmid vector (Table I, underlined). A1-10FX is the same as A1-10F, except a XhoI site, instead of a BamHI site in A1-10F.

Screening of cDNA library by PCR
Using two primers, A1-1F and A1-2R, we screened a {lambda}gt10 human fetal brain cDNA library (Clontech) by PCR. The PCR reaction mixture was prepared as follows. Phage solution containing about 1010 clones was first heated at 90°C for 10 min. The final PCR reaction mixture contained 10 mM Tris pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTPs, 1 µM of both A1-1F primer and A1-2R primer, and 2.5 U Taq DNA polymerase (Roche Diagnostics) in a total volume of 100 µl. After overlaying 100 µl mineral oil (Sigma), the first round of PCR was performed by 30 cycles as follows: 94°C 15 sec (45 sec at initial cycle), 60°C 30 sec, 72°C 2 min (10 min at the last cycle) using PC-700 thermal cycler (Astek). The second round of PCR was carried out using 5 µl of the first PCR reaction mixture under the same conditions. After the PCR, 10 µl of the PCR reaction mixture was analyzed on 1% agarose gel electrophoresis using {lambda} DNA (Takara) digested with Sty I (Roche Diagnostics) as size marker.

Rescreening of cDNA library
Using the 430 bp PCR product (Figure 2, lane B) obtained from the above PCR as a probe, the same cDNA library was rescreened by plaque hybridization. Digoxigenin (DIG)-labeled probe was prepared by Digoxigenin (DIG) DNA Labeling Kit (Roche Diagnostics).

The cDNA library was diluted with SM solution (50 mM Tris HCl pH 7.5, 0.1 M NaCl, 8 mM MgSO4, 0.01% gelatin). The Escherichia coli C600hfl strain (hflA150[chr::Tn10] lacY1 leuB6 mcrA- supE44 thi-1 thr-1 tonA21) was infected with the diluted cDNA library at 37°C for 20 min, mixed with LB liquid medium containing 0.7% agar, plated onto LB solid medium containing 1.5% agar, and incubated at 37°C for 8 h in order to form about 50,000 plaques per plate. Plaques were transferred onto Colony Plaque Screen nylon membranes (NEN) and DNAs on the membranes were denaturated by 0.5 N NaOH/1.5M NaCl solution for 1 min, neutralized by 0.5 M Tris HCl pH 7.5/1.5M NaCl solution for 1 min, and washed in 2 x SSC solution (0.3 M NaCl, 0.034 M sodium citrate) for 5 min. The membranes were dried for plaque hybridization.

Plaque hybridization was carried out in the mixture containing 5 x SSC (0.75 M NaCl, 0.085M sodium citrate), 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS, 1% blocking reagent (Roche Diagnostics) at 68°C for 20 h. After washing with Solution I (2 x SSC, 0.1% SDS) at room temperature for 15 min twice and then solution II (0.2 x SSC, 0.1% SDS) at 65°C for 30 min twice, positive plaques were detected, using DIG Luminescent Detection Kit (Roche Diagnostics) and Hyper Film-ECL (Amersham).

Each positive plaque was recovered in 100 µl of SM solution and subjected to PCR. Two PCR primers, GT10-F and GT10-R (Table I, Greiner Japan), which were designed based on the flanking sequences of the {lambda}gt10 cloning site, were used for amplification of the cDNA inserts.

Cloning and nucleotide sequencing of cDNAs
The original PCR product (about 430 bp) and the cDNA insert from HR1-3 were digested with BamHI and HindIII (Roche Diagnostics) and ligated into a plasmid vector, pUC118 or pUC119 (Vieira and Messing, 1987Go) previously digested with the same restriction enzymes. The cDNA insert from HR1-4 was digested with BamHI and EcoRI (Roche Diagnostics) and ligated into a plasmid vector, pUC118 or pUC119 previously digested with the same restriction enzymes. All the ligation reactions were carried out with DNA Ligation Kit Ver.2 (Takara). The ligation mixtures were used to transform E.coli JM109 strain (recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 {Delta}(lac-proAB)/F'[traD36 proAB+ lacIq lacZ{Delta}M15]) by the calcium chloride method (Cohen et al., 1972Go). Selection of the resultant transformants was performed by the standard method (Sambrook et al., 1989Go).

Cloning of the 1.8 kb Hmat-1 cDNA was performed as follows. Marathon-Ready cDNA (from human brain, Clontech) was subjected to PCR with the primers, A1-10F and A1-8RS. PCR was carried out under the same condition described above except that the total volume of the reaction mixture at the first round of PCR was 50 µl. The PCR product (about 1.8 kb) was digested with BamHI and SalI (Roche Diagnostics), ligated to pUC118 or pUC119 previously digested with the same restriction enzymes. The ligation reaction, transformation of JM109 strain, and selection of the resultant transformants were performed as described above.

Single-stranded DNAs were prepared by infecting the resultant transformants with the helper phage M13K07 (Vieira and Messing, 1987Go) and sequenced by the dideoxy chain termination method (Sanger et al., 1977Go). If necessary, construction of deletion subclones for sequencing was carried out with Deletion Kit for Kilo-Sequence (Takara). The electrophoresis for sequencing with 8% polyacrylamide gel was performed at 15 mA for 4.5 to 9 h. Sequencing reaction and detection of DNA ladders were performed by Sequencing High -Cycle- Kit (Toyobo) and Hyper Film-ECL (Amersham).

Complementation test
The S.cerevisiae PRY56 strain (MATa alg1-1 ura3-52) (Huffaker and Robbins, 1982Go), was maintained on nutrient YPD medium at 26°C (permissive temperature for PRY56). The yeast episomal expression vector, YEp352GAP (Yoko-o et al., 1993Go), which contains URA3 gene and GAP promoter, was used for the expression of the Hmat-1 gene in PRY56 strain.

The 1.8 kb Hmat-1 cDNA subcloned into the pUC vector was reamplified by PCR with the primers, A1-10FX and A1-8RS. PCR was carried out under the same condition described above. The PCR product was digested with XhoI and SalI (Roche Diagnostics) and ligated into YEp352GAP previously digested with the same restriction enzymes. The ligation reaction, transformation of JM109 strain, and selection of the resultant transformants were performed as described above. The recombinant plasmid DNA was prepared by the standard method (Sambrook et al., 1989Go) and the PRY56 strain was transformed by the lithium acetate method (Ito et al., 1983Go). The transformants were first selected as uracil-auxotrophs on synthetic SD medium lacking uracil [SD(Ura-) medium] at 26°C, and then the growth of the transformants was examined on YPD and SD(Ura-) media at 36°C (nonpermissive temperature for PRY56).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Prof. P.W.Robbins (Massachusetts Institute of Technology, Cambridge, MA, USA) for kindly providing us with the S.cerevisiae alg1-1 mutant PRY56, Dr. T.Yoko-o (National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, Japan) for providing us with the yeast expression vector YEp352GAP, and Dr. H.Tachikawa (Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan) for advising about the yeast techniques. We also thank Prof. Y.Nagai (Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan) for encouragement and Prof. H. Schachter (Hospital for Sick Children, Toronto, Canada) for critical reading of the manuscript. This study was supported in part by Grants-in-Aid for Scientific Research on Priority Area Nos. 10178104 and 11159213 from the Ministry of Education, Science, Sports and Culture of Japan, and research funds from School of Engineering at Tokai University.


    Abbreviations
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
MT-I, mannosyltransferase I; ER, endoplasmic reticulum; Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine; Dol, dolichol; EST, expressed sequence tags; Asn, asparagine; Ser, serine; Thr, threonine; Dol-P, dolichol phosphate; GlcNAc-1-P, N-acetylglucosamine-1-phosphate; GDP, guanosine diphosphate; PCR, polymerase chain reaction; BLAST, basic local alignment search tool; dNTP, deoxyribonucleoside triphosphate; LB, Luria-Bertani; dUTP, deoxyuridine triphosphate; bp, base pair; kb, kilobase pair; ORF, open reading frame.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Abeijon,C. and Hirschberg,C.B. (1992) Topography of glycosylation reactions in the endoplasmic reticulum. Trends Biochem. Sci., 17, 32–36.[ISI][Medline]

Albright,C.F. and Robbins,P.W. (1990) The sequence and transcript heterogeneity of the yeast gene ALG1, an essential mannosyltransferase involved in N-glycosylation. J. Biol. Chem., 265, 7042–7049.[Abstract/Free Full Text]

Albright,C.F., Orlean,P. and Robbins,P.W. (1989) A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition. Proc. Natl. Acad. Sci. USA, 86, 7366–7369.[Abstract]

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