Activity and tissue distribution of splice variants of {alpha}6-fucosyltransferase in human embryogenesis

Ivan Martinez-Duncker2, Jean-Claude Michalski3, Chantal Bauvy2, Jean-Jacques Candelier2, Benoît Mennesson4, Patrice Codogno2, Rafael Oriol2 and Rosella Mollicone1,2

2 Unité de Glycobiologie et Signalisation Cellulaire, INSERM U504, IFR 89, GDR CNRS 2590, Université de Paris Sud XI, 16 Ave Paul Vaillant-Couturier, 94807 Villejuif Cedex France; 3 UMR 8576 CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France; and 4 Hôpital de Pontoise, 95300 Pontoise, France

Received on May 6, 2003; revised on August 12, 2003; accepted on September 8, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The product of the FUT8 gene transfers an {alpha}1-6 fucose on the innermost N-acetylglucosamine of the chitobiose core of N-glycans. Northern blot analysis shows four main transcripts of 3.0, 3.3, 3.9, and 4.2 kb in the embryo. The larger forms around 4-kb decrease in fetus and adult. Fourteen embryo transcripts of FUT8 were cloned. Twelve exons comprising two new 5'untranslated-exons (A and B) and two new 3'UT-ends (L1 and L2) and the complete genomic organization of the FUT8 gene (330 kb) are described. Transcripts starting with the 5'UT-exon A are always associated with exons C and D. Exon B initiates another series of transcripts associated to exon C and D or directly to exon D. A third series of transcripts starts at exon C. The data suggest an expression of FUT8 regulated by three different promoters, starting transcription in exons A, B, or C. The A or C series are better expressed than the B series. After transfection with these cDNA constructs the transcripts with 5'UT-exons A or C have higher expression of FUT8 transcripts and higher {alpha}6-fucosyltransferase activity, whereas the activity of the B series is about two-thirds lower for both parameters, suggesting that exon B reduces the expression of the transcripts.

Key words: {alpha}6-fucosyltransferase / alternative splicing / FUT8 isoforms / genomic organization / human embryo


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Fucosyltransferases catalyze the transfer of GDP-fucose to free oligosaccharides or to oligosaccharides linked to proteins or lipids (Costache et al., 1997bGo). Fucosyl residues are found in five different linkages: {alpha}2-, {alpha}3-, {alpha}4-, and {alpha}6- or directly linked to serine or threonine in the case of protein-O-fucosylation (Wang et al., 2001Go; Becker and Lowe, 2003Go). The human {alpha}2-, {alpha}3-, and {alpha}4-fucosyltransferases are implicated in terminal fucosylations and are responsible of the synthesis of ABH and Lewis histo-blood group antigens (Oriol, 1995Go). The {alpha}6-fucosyltransferase transfers fucose on glycoproteins to the innermost asparagine-linked N-acetylglucosamine (GlcNAc) of the chitobiose disaccharide-core unit (Oriol et al., 1999Go).

The human fucosyltransferases and their genetic expression are developmentally regulated (Mollicone et al., 1992Go; Candelier et al., 2000Go). Some of their final products (Sia-Lex and Sia-Lea) are implicated in selectin-dependent leukocyte recruitment or lymphocyte homing (Lowe, 2001Go). Most of the {alpha}2- and {alpha}3/4-fucosyltransferase activities are contributed by several enzymes encoded by different genes, whereas the {alpha}6-fucosyltransferase is encoded by a single gene. As all the other Golgi glycosyltransferases, the {alpha}6-fucosyltransferase is a globular type II transmembrane protein with a catalytic domain in the luminal part of the trans-Golgi vesicles (Paulson and Colley, 1989Go). This protein has 575 amino acids, has no N-glycosylation site, and is involved in the first steps of the biosynthesis of animal N-glycoproteins (Costache et al., 1997bGo). This core {alpha}6-fucosylation is widely distributed in many glycoproteins (Aronson and Kuranda, 1989Go) and is particularly abundant in brain tissue (Shimizu et al., 1993Go). It is also present in insect cells, in which fucose can be linked in {alpha}1,6 or {alpha}1,3 on the chitobiose unit (Staudacher and Marz, 1998Go; Staudacher et al., 1999Go). Alternatively, in plants {alpha}6-fucosylation is missing and core fucosylation is always {alpha}1,3 linked on the same innermost GlcNAc of core chitobiose (Fitchette-Lainé et al., 1997Go).

Changes in the pattern of protein glycosylation may interfere with cellular functions and may thus lead to health disorders (Aebi and Hennet, 2001Go; Jaeken, 2003Go). The level of these fucosyl-N-glycan chains on {alpha}-fetoprotein are useful for the differential diagnosis of hepatocellular carcinomas (Breborowicz et al., 1981Go; Sato et al., 1993Go; Takeda et al., 1993Go). In certain serum proteins (ceruloplasmin and transferrin), the content of {alpha}6-fucosyl-N-glycans is increased in malignancy (Yamashita et al., 1993Go; Noda et al., 2002Go). In the case of early hepatocellular carcinomas, this increase is observed more than 6 months prior to clinical diagnosis (Shiraki et al., 1995Go). This {alpha}6-fucosylation has also been found expressed in several human cancer cell lines (Miyoshi et al., 1997Go).

The {alpha}6-fucosyltransferase was initially characterized from porcine liver extracts (Longmore and Schachter, 1982Go), cultured human skin fibroblasts (Voynow et al., 1991Go), and platelets after blood clotting (Koscielak et al., 1995Go; Kaminska et al., 1998Go). Recently, purification of the {alpha}6-fucosyltransferase from porcine brain and the human gastric cancer MKN45 cell line allowed the cloning of the porcine (Uozumi et al., 1996Go) and the human fucosyltransferase FUT8 cDNAs (Yanagidani et al., 1997Go). This gene has been also identified and cloned in bovines (Javaud et al., 2000Go) and mice (Hayashi et al., 2000Go). Using the porcine sequence of FUT8, we obtained 37 overlapping human expressed sequence tags (ESTs) that could be aligned in a single contig containing the whole mRNA with the 3'untranslated (UT)-polyA sequence, and we revealed the existence of a new retina splice variant of the {alpha}6-fucosyltransferase. These EST sequences allowed us to map the human FUT8 gene to chromosome 14q23 (Costache et al., 1997aGo). Recently, we performed a new chromosome localization of this gene by FISH and localized it to the interval 14q23.2–24.1 (Couillin et al., 2002Go).

In this article, we report the presence of 14 splice variants of the {alpha}6-fucosyltransferase gene expressed early in human embryos. With these new transcripts, we have found two additional 5'UT sequences (exons A and B), completed the 5'UT-exon C, and found two distinct 3'UT terminus (L1 and L2). We report the tissue distribution of these new {alpha}6-fucosyltransferase mRNA transcripts in embryo, fetal, and adult stages. This work allowed us to propose the complete genomic organization for this gene and the cloning of two retina splice variants. In addition, we show that the presence of the 5'UT exon B diminishes the amount of FUT8 transcript and decreases the {alpha}6-fucosyltransferase activity by two-thirds, as compared to FUT8 transcripts starting with exons A or C.


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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Alternatively spliced embryonic FUT8 transcripts
From the three Marathon-adaptor-ligated double-strand cDNA libraries we made with human polyA+ mRNA of 38-day-, 50-day- and 60-day-old embryos, we generated FUT8 5'- and 3'-rapid amplification of cDNA ends (RACE) fragments and cloned the 14 full-length cDNA transcripts reported in Figure 1.



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Fig. 1. Schema of the genomic organization of the Homo sapiens FUT8 gene. Exons (A to L) are represented by rectangles with their relative sizes. A1, B1, B3, B5, B6, C1, D1, and retina1 contain the short 3'UT end L1, and A2, B2, B4, C2, D2, and retina 2 contain the long 3'UT end L2. The introns are represented by a V-shaped line. The cds exons are gray, and the exons flanking the cds are black. With the exception of the retina splice variants, all the transcripts use the ATG1 start codon. EspI, PpuMI, and MluI are the restriction sites used for the reconstruction of the full-length transcripts. Position numbers under exons A and B correspond to the starting points of different transcripts. The human retina sequence starts in the 3' side of the exon H (ATG2) and ends with the K and L1 or L2 terminus, but an alternative splice event occurred, shunting exons I and J. The same TAA stop codon is present in all the different splice variants of FUT8.

 
The 5'UT-exons were mapped by a compilation of several 5'UT-RACE-polymerase chain reaction (PCR) sequence analysis reported in Material and methods. All the FUT8 transcripts (except retina) started either with exons A, B, C, or D. In embryos, the transcripts initiated by the 322-bp full-length exon A are well expressed and always associated with exons C and D at the 5'UT side and with the L1 or the L2 3'UT terminus. The transcripts initiated by exon B are associated with exons C and/or D giving FUT8 transcripts with three types of 5'UT-BCD associations. They start at positions 1530 and 1296 of the exon B. The transcripts having the 5'UT-BCD association starting at position 1296 are well expressed, whereas the transcripts starting at position 530 of exon B are weakly expressed in our embryo libraries. We also found a minor FUT8 transcript (B5) with the exon B starting at position 530 directly linked to exon D and associated to the L1 terminus only. Another minor transcript, with a full-length exon B of 1400 bp and a L1 3'UT terminus was designated B6. All these types of FUT8 transcripts were generated by alternative splicing events and by using two polyadenylation/cleavage sites in the same exon L, to give the L1 or L2 isoforms (Figure 1). To confirm that these different types of 5'UT ends are associated with L1 and/or L2 at the 3'UT side, we amplified all the FUT8 full-length cDNA, and the retina splice variants, by reverse transcriptase (RT)-PCR to obtain the short (L1) and the long (L2) isoforms for each type of FUT8 transcript. Figure 2 shows an example of RT-PCR obtained with primers specific for A1, A2, B1, B2, C1, C2, and for the short and long retina splice variants.



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Fig. 2. Evidence for the expression of FUT8 full-length cDNA transcripts in our human embryo libraries. The RT-PCR with specific combinations of primers revealed full-length transcripts starting with exon A (lanes 1 and 2), exon B (lanes 3 and 4), exon C (lanes 5 and 6) and the retina splice variant (lanes 7 and 8). For each kind of transcript, antisense primers were selected to amplify either the L1 terminus (lanes 1, 3, 5, and 7) or the L2 terminus (lanes 2, 4, 6, and 8). Lanes 9 and 10 are negative controls without reverse transcriptase or PCR template, respectively. On the left side are indicated the sizes of the control DNA markers run on the lane L of the 1% agarose gel.

 
Expression of FUT8 {alpha}6-fucosyltransferase mRNA transcripts in various tissues
By northern blot analysis with poly-A+mRNA from entire embryos of 40–70 days, we detected a FUT8 profile of four bands, hybridizing with a FUT8 specific cds probe. Two abundant and broad transcript bands were detected at 3.0 and 3.3 kb. Two other faint bands were seen at 3.9 and 4.2 kb (Figure 3A). During this period the same FUT8 mRNA profile is expressed in all the embryos with similar intensity.



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Fig. 3. (A) Northern blot made with poly-A+ (4 µg/lane) extracted from whole embryos of 40–70 days of age. The blots were hybridized with the FUT8-cds probe. The migration of the DNA size control markers are shown on the left side, and the approximate sizes of the 4 detected FUT8 transcript bands are shown on the right side. (B and C) Commercial (Invitrogen) northern blots (mRNA REAL blots), loaded with pools of poly-A+ (2 µg/lane) from human fetal and adult tissues. (B) contains fetal tissues of different ages: brain (20 weeks), liver (24 weeks), lung (37 weeks), muscle (28 weeks), hearth (12 weeks), kidney (28 and 32 weeks), skin (20 weeks), small intestine (28 weeks), and adult lung. (C) shows adult tissues. The commercial blots were hybridized with the same FUT8-cds probe used for the embryo tissues and contain the same 3.0- and 3.3-kb transcripts seen in embryos, but only a very faint band at about 4 kb is seen in the area corresponding to the 3.9–4.2-kb embryo transcripts. ß-Actin was added in fetal and adult tissue blots as a control of mRNA integrity.

 
The tissue distribution of FUT8 transcripts was also studied with northern blots containing fetal and adult tissue mRNAs (Figure 3B and C) and revealed with the same FUT8-cds probe used for the embryo profile. During the fetal period the eight tissues tested expressed FUT8 transcripts, mainly as the doublet of mRNA of 3.0 and 3.3 kb but with different profiles and intensities. This doublet is abundant in skin and small intestine, moderately expressed in heart and kidney, and weakly or faintly expressed in lung, muscle, and liver. A stronger expression of the upper band (3.3 kb) was observed in fetal brain. In all other tissues the two mRNA bands are equivalent. The fetal large size FUT8 transcripts (3.9 and 4.2 kb) appeared only as a single weak band of approximately 4 kb, weaker than the corresponding embryo bands.

All the adult tissues express the two major FUT8 transcripts of 3.0 and 3.3 kb. For each tissue, the adult and the fetal profiles are qualitatively similar, but they have quantitative expression differences. Large amounts of the FUT8 transcript doublet are found in brain, placenta, lung, stomach, and jejunum. Moderate expression of this doublet was found in pancreas, uterus, kidney, and urinary bladder. A weak expression was noted in heart, ileum, colon, and spleen; a faint expression was observed in rectum and muscle; and it was not detected in liver. In the intestinal tract we observed a clear craniocaudal decrease of the expression of the FUT8 doublet, as previously shown with FUT4 transcripts at the same stage (Cailleau-Thomas et al., 2000Go). For some tissues the level of the expression of FUT8 increases from fetal to adult stages (brain, lung, kidney). In adult brain, expression of both bands is increased. On the contrary, expression decreases or disappears in heart, muscle, and liver. Again, the high molecular weight FUT8 mRNAs of about 4 kb are faintly or not expressed at all in adult tissues (Figure 3B and C).

In addition, we hybridized the same northern blots with probes specific for 5'UT exons A, B, or C. The exon C probe follows the same expression profile as the FUT8-cds probe but weaker. The exon B probe detected only a weak band at about 4 kb in fetal tissues and a faint band of the same size in adult tissues. The expression of these large exon B transcripts decreases or disappears in the adult. This probe is able to detect transcripts with full-length exon B (B6) or with exon B starting at the position 530 (B3, B4, or B5) (Figure 1). The exon A probe detected all the FUT8 transcripts starting with the 5'UT exon A. This probe detected only one broad and strong band of approximately 3.3 kb, with ubiquitous distribution at the fetal stage. The expression of these transcripts decreases slightly in the adult but remains moderately expressed.

Influence of the 5'UT-region of the FUT8 isoforms on the {alpha}6-fucosyltransferase activity
We transfected the different FUT8-cDNA constructs and the empty vector into COS7 cells. The comparative {alpha}6-fucosyltransferase activities (Table I) were measured in the cellular homogenates, using the biotinylated biantennary asialo-agalacto-glycoasparagine glycan as acceptor substrate (see Materials and methods).


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Table I. {alpha}6-Fucosyltransferase activity of the native constructs, and the construct vector ratio of activities

 
The best activities were observed with the constructs having the cds of FUT8 with no additional 5'UT sequence (FUT8-cds) or with a short 5'UT exon C sequence (C1) or having the 5'UT exon combination ACD (ACD-L1) (Table I). When we expressed the FUT8-cDNA isoforms having the 5'UT exon combination BCD (B3 or B6) or BD (B5), we found a two-thirds reduction of the {alpha}6-fucosyltransferase activity. This reduction was independent of the length of the exon B (B3 or B6) or the presence of exon C (B5). The results suggest that the presence of exon B influences the translation rate of the {alpha}6-fucosyltransferase (Table I). These results were confirmed at the transcription level by dot-spot quantification. Five micrograms of total RNA were hybridized with the FUT8-cds and ß-actin probes. Strong expression of FUT8 transcripts can be seen in dots 3, 4, and 5 (C1 and A1 clone 1 and 2) and a significant reduction of expression is observed in dots 6, 7, and 8 (B3, B5, and B6). All dots are strongly positive with the control ß-actin probe (Figure 4).



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Fig. 4. Dot-blots of total RNA hybridized with the cds-FUT8 probe (line A) or the ß-actin probe (line B). Each dot was charged with 5 µg RNA from native COS7 cells (1), COS7 cells transfected with PCR3.1 alone (2), COS7 cells transfected with the transcripts C1 (3), A1 clone 1 (4), A1 clone 2 (5), B3 (6), B6 (7), and B5 (8).

 
Influence of the GFP tag on activity and intracellular traffic of the {alpha}6-fucosyltransferase
To define if the green fluorescent protein (GFP) tag influences the {alpha}6-fucosyltransferase activity, we expressed into COS7 cells the FUT8-fusion protein tagged with the 25 kDa GFP in the NH2 or in the COOH terminus (Table II). After transient expression into COS7 cells, we measured the {alpha}6-fucosyltransferase activities associated to the GFP-tagged fusion proteins compared with the untagged FUT8 enzymes. For each construct series several clones were transfected, and we tested the {alpha}6-fucosyltransferase activity with the same biotinylated N-glycan conjugate acceptor. When the GFP was linked at the COOH terminus the activity (790 ± 65 pmol/h/mg) was similar to the nontagged FUT8-cds protein (740 ± 10 pmol/h/mg). Unlike this, the fusion protein with the GFP linked at the NH2 terminus had an average reduction of activity of one-third (525 ± 100 pmol/h/mg) (Table II).


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Table II. {alpha}6-Fucosyltransferase activity of the GFP-tagged constructs on the amino or the carboxy terminus and the construct vector ratio of activities

 
To define if the activity difference observed between the COOH-tagged and the NH2-tagged series were due to a quantitative difference of protein translation, we did comparative western blots with the transfected FUT8-GFP-tagged protein extracts (Figure 5). When revealed with an anti-GFP antibody, the amount of enzyme was lower in the COS7 cells transfected with the NH2-tagged clone 1 (lane 2) and clone 2 (lane 5) compared with the COOH-tagged clone 1 (lane 3) and clone 2 (lane 4). These results correlate well with the fucosyltransferase activity differences found between the two series of GFP-recombinant enzymes. In general, the extracts with lower protein expression (Figure 5) had lower fucosyltransferase activity (Table II). A dot-blot made with total RNA from COS7 cells transfected with the same constructs gave similar levels of transcript expression for the untagged and the COOH-tagged constructs, whereas the expression of the NH2-tagged construct was always reduced in good agreement with the enzyme activity and the western blot results.



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Fig. 5. Western blot analysis of the 90-kDa recombinant GFP-tagged {alpha}6-fucosyltransferase expressed in COS7 cells after transfection with chimeric constructs of FUT8, generated by fusion of GFP protein to the NH2 terminus (clone 1, lane 2 and clone 2, lane 5) or to the COOH terminus (clone 1, lane 3 and clone 2, lane 4). Lane 1 contains a negative control with the extract of COS7 cells transfected with the vector alone. The blot was revealed with a rabbit anti-GFP antiserum (1:10,000) and visualized with the chemiluminescent reagents from the ECL-Plus western blot detection kit.

 
By confocal immunofluorescence microscopy, the COOH-tagged fusion protein (green) colocalized with giantin (blue), in the Golgi apparatus (Figure 6A), but not with the endoplasmic reticulum (ER) calnexin (red) (Figure 6B). The action of the brefeldin A on the cells transfected with COOH-tagged FUT8 constructs confirms the localization of the FUT8 protein in the Golgi apparatus and has a dramatic effect on the Golgi morphology by inducing a redistribution of Golgi proteins in the ER (Lippincott-Schwartz et al., 1988Go). In this case all the markers (calnexin in red, giantin in blue, and the COOH-tagged protein in green) colocalized in Golgi and ER (Figure 6C). When the GFP was linked at the NH2 terminus, the fusion protein was distributed in the ER and in the Golgi apparatus of the transfected cells (Figure 6D). The enzyme fraction situated in the Golgi colocalized with the giantin revealed with AMCA. The fraction retained or delayed in the ER colocalized with the ER marker (calnexin). This situation is somehow similar to the picture obtained with the fusion FUT8 protein tagged at the COOH-terminus on brefeldin A–treated cells (Figure 6C). The treatment with brefeldin A on the cells transfected with NH2-tagged constructs did not change the diffuse repartition of the NH2-tagged FUT8 fusion protein (data not shown).



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Fig. 6. Detection of recombinant {alpha}6-fucosyltransferase expression by confocal microscopy in COS7 cells after transient transfection with COOH- GFP-tagged constructs (A, B, C), or NH2-GFP-tagged constructs (D). In (A), (C) and (D) the Golgi apparatus was stained with mouse anti-giantin and revealed with AMCA-conjugated anti-mouse Ig (blue). In (B) and (C) the ER was labeled with rabbit polyclonal anti-calnexin (1:200) and revealed with Cya3 labeled anti-rabbit Ig (red). In (A) the Golgi of all the cells is stained in blue with giantin and the Golgi of about 30% of the cells is also stained green with the COOH-tagged FUT8 illustrating colocalization of the two markers in the transfected cells. In (B) the ER is stained in red with calnexin and the same green fluorescence of COOH-tagged protein illustrates different localization of the two markers (the [A] and [B] pictures were taken on the same field with appropriate wavelength selection to visualize the green and blue (A) or of the green and red (B) fluorescence). In (C) the Golgi apparatus was disrupted by pretreatment of cells with brefeldin A (5 µg/ml), and they were stained with: COOH-tagged FUT8 (green), anti-giantin (blue), and anti-calnexin (red). The picture illustrates the partial redistribution of the Golgi proteins, giantin, and COOH-tagged FUT8 in the ER compartment, where they colocalize with calnexin. In (D) the green stain of NH2-tagged fusion protein is distributed in two locations, some colocalize with the giantin protein in the Golgi (blue and green) and some are diffuse in the cytoplasm, probably in the ER (green). The white bar corresponds to 10 µm.

 
Genomic organization of FUT8
The cloning of the 5'UT exons A and B gave the complete organization map of the FUT8 gene. This map resulted from the analysis of a compilation of several genomic and transcript sequences. Figure 1 shows the position of the 12 exons of the FUT8 gene. We named the 5'UT exons A, B, and C, following their order of discovery and their relative expression at the mRNA level. By RACE-PCR the first amplification gave a majority of cDNA clones with the 5'UT exon A and a few clones with the 5'UT exon B. After amplifications with nondigested genomic DNA and ExB-s and ExA-225as primers (Table III), in a PCR reaction using the Long Expand Template PCR system, we only amplified a 1.2-kb genomic fragment, indicating that the 5'UT exon B is the first exon found in the sense 5' to 3' on the DNA. This fragment gave the 3'exon–intron boundary of the exon B and the full-length intron sequence (899 bp) between exon B and exon A (Table IV). A blast search in EMBL with the 1.2-kb fragment containing the B–A intron identified a genomic sequence of 180 kb (AL355840). This sequence confirmed the order and the relative positions of the 5'UT exons: B, A, and C, the B–A intron size of 899 bp; and gave the size of the intron situated between exons A and C (40,405 bp) (Table IV). This sequence revealed additional information on the intron–exon boundaries of the 5'side of the exon B and confirmed the sizes of the B, A, and C exons (Table IV).


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Table III. Oligonucleotide primer sequences used in this study for {alpha}6-fucosyltransferase amplifications and sequencing

 

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Table IV. Identification and size of the FUT8 exons and introns and the intron/EXON..EXON/intron boundaries. Uppercase letters are exonic sequences and lower case letters are intronic sequences

 
Using the genome walker system from Clontech with specific primers (Table III) as described in Materials and methods, we amplified a 2-kb fragment that gave the 3'exon–intron boundary of exon C and three DNA fragments of 4 kb, 1 kb, and 1.4 kb, respectively, containing the 5'exon–intron boundary sequence of exon D. We sequenced 1 kb in 5' from exon D of the three genomic sequences, and we made a BLAST search of the EMBL database that retrieved a 148-kb sequence (AL161871) that contained exons D, E, F, and G. The search of the EMBL database with the A1 FUT8 cDNA identified the same two chromosome 14 sequences (AL161871 and AL355840) and two new ones: AL109847 of 186 kb (containing exons E, F, G, H, I, J, K, and L) and AL359236 of 182 kb (containing exons A and B). The comparison of the different cDNA sequences of FUT8 isoforms with the nucleotide sequence of the identified clones illustrate that they contain the complete FUT8 gene organization. It spans 330 kb and contains the 12 exons ranging from 98 to 1400 bp and the 11 introns ranging from 117 bp to 104,616 bp (Table IV). All the intron–exon boundaries followed the AG/GT rule (Breathnach and Chambon, 1981Go). We also found five microsatellite sequences with more than 15 GT repeats. Two flanked the 5' side of exon E and three were localized on the 3' side of exon G.


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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In the present study, we clone 14 complete splice variants of the human {alpha}6-fucosyltransferase (Figure 1) expressed during the first 2 months of human embryo development. The expression of the different splice variants of {alpha}6-fucosyltransferase (with the exception of the retina variants) is observed in all tissues during the embryo-fetal development and decreases or disappears in several tissues at the adult stage. The A1 and A2 splice variants are very well expressed in all tissues and correspond to the upper band (3.3 kb) of the doublet of mRNA detected with the FUT8-cds probe and with the specific exon A probe. After transfection of the A splice variants, the {alpha}6-fucosyltransferase activity is as strong as the activity found with the shorter splice variants C1, D1, or FUT8-cds.

On northern blots, the C1 and the C2 transcripts are associated to the lower band of the doublet of FUT8 mRNA (3.0 kb). The exon C probe detects in addition the upper band of the doublet (3.3 kb), when the 5'UT exon C is included in the ACD association. This probe weakly detects the exon C in the transcripts with the 5'UT-BCD association. The splice variants with full-length exon B or with exon B starting at position 530 are not well expressed compared with the other isoforms. The specific exon B probe detects two weak bands at approximately 3.9 and 4.2 kb during embryonic and fetal stages (Figure 3), corresponding to the B3, B4, B5, and B6 transcripts. The low expression of these transcripts in vivo is also seen in vitro, because transfections of cDNA with 5'UT exon A or exon C generate a stronger expression of FUT8 transcripts and higher enzyme activities, than transfections with transcripts containing the 5'UT exon B (Table I and Figure 4).

After tagging on the NH2 side, retention in the ER of a part of the fusion protein was observed (Figure 6), and this correlated with a decrease of FUT8 transcript expression (data not shown) and of the {alpha}6-fucosyltransferase activity (Table II). These experiments suggests that tagging on the COOH terminal side is less deleterious than tagging on the NH2 terminal side.

Retina-1 and -2 splice variants are not detected with the FUT8-cds probe, probably due to the tissue specificity of the transcript and its low representation when we compare the weight of the retina tissue to the other organs and tissues. The only way to reveal them is through RT-PCR with the specific primers used in the present work. The retina isoforms do not have the exons I and J involved in the recognition of the acceptor substrate and are inactive. We do not know if these molecules can act as regulators of the GDP-fucose concentration in the retina or elsewhere in the organism because they have the conserved motif III, which was proposed to be involved in the GDP-fucose recognition (Oriol et al., 1999Go).

Recently the genomic organization of the bovine fut8b gene has been defined. The coding region of the gene consists of five exons spanning a 37-kb genomic region, and each exon encodes a domain homologous to other proteins, in good agreement with the theory of Gilbert (1987)Go, proposing that the polyexonic ancestral genes have evolved by combinations of single exons encoding elementary peptide domains. In fact, the first exon of fut8b has homology with cytoskeleton proteins, the second presents a proline-rich region with a sequence similar with the ligand of the SH3-domain, the third exon encodes a gyrase-like domain and the last exon shows a domain homologous to the SH3 conserved motif of the SH2-SH3 family (Javaud et al., 2000Go). A PSI-BLAST search with each human peptide exon sequence identified a similar phenomenon with (1) cytoskeleton proteins for the D, E, and F exons; (2) a proline-rich region for the 3'-end of the exon I; (3) a sequence of DNA-gyrases for exon J; (4) a domain homologous to glucosamine-fructose 6-phosphate aminotransferase for exon K, and (5) an SH3 domain in exon L.

In this study we found the A and B exons and completed the genomic organization of the human FUT8 gene. A partial genomic structure and promoter analysis of the human FUT8 gene was previously reported using SKOV3 cells (Yamaguchi et al., 2000Go). This last work reported that the FUT8 gene has at least nine exons spanning a genomic area of more than 50 kb, with a coding region sequence divided into eight exons. The translation initiation codon assigned at exon 2 and the stop codon at exon 9. Exon 1 encoded only 5'UT sequences and corresponds to 31 bases of the 5'UT exon C described herein. Exon 9 (Yamaguchi et al., 2000Go) corresponds to a fusion of the exons K and L1, described in the present article.

The present analysis shows that the exon C is 98 bp long and contains an initiation transcription site located 15 nucleotides downstream a typical TATA-box consensus but no CAAT-motif. The embryo cDNA show the presence of two additional 5'UT sequences, which we have called exon B and exon A. CAAT and GC boxes are present in the 5' flanking regions of exons B and A, but there is no TATA box. Therefore, the data suggest that the expression of FUT8 is regulated by at least three different promoters that can initiate transcription at either exons A, B, or C giving proteins with the same cds. The work suggests that 5'UT exons regulate transcription and translation expression rates.


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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
{alpha}6-Fucosyltransferase acceptor preparation
The desialylated biantennary glycoasparagine was obtained by exhaustive pronase digestion of a pool of human plasma glycoproteins followed by fractionation on Bio-Gel P4 (Bio-Rad, Hercules, CA). Afterward, it was desialylated by formic acid hydrolysis (formic acid solution pH 2.0, 1 h, 80°C) and degalactosylated by hydrolysis with Escherichia coli recombinant ß-galactosidase from Sigma (St. Louis, MO: 10 U/mg substrate in 1 ml 0.1 M phosphate buffer, pH 7.0, 24 h, at 37°C). The asialo-agalacto-glycoasparagine was linked to EZ-link sulfo-NHS-LC-LC-Biotin from Pierce (Rockford, IL). Efficiency of biotinylation was assessed by matrix-assisted laser desorption/ionization mass spectrometry analysis in a Voyager Elite DESTR Pro instrument (Perseptive Biosystem, Sramingham, MA) equipped with a nitrogen UV laser ({lambda} = 337 nm) (Figure 7). The m/z at 1792 [M+H]+ and 1814 [M+Na]+ correspond to the biotinylated conjugate biotin-Lc-AsnGlcNAc4Man3, and the m/z at 1453 [M+Na]+ corresponds to the unconjugated glycoasparagine. The biotinylated product (Figure 8) was separated from the unconjugated form by Sep-Pak C18 (Waters, Milford, PA) reverse chromatography (Palcic et al., 1988Go).



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Fig. 7. MALDI-MS analysis of the biotin labeled asialo-agalacto-glycoasparagine used as acceptor substrate for {alpha}6-fucosyltransferases. The m/z at 1792 [M+H]+ and 1814 [M+Na]+ correspond to the biotinylated conjugate; the m/z at 1453 [M+Na]+ corresponds to the unconjugated glycoasparagine molecule.

 


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Fig. 8. Chemical structure of the biotinylated N-glycan conjugate acceptor substrate (Biotin-Lc-Asn-GlcNAc4-Man3) retained with the Sep-Pak C18 separation method (Palcic et al., 1988Go), and used for detection of {alpha}6-fucosyltransferase activity. The 14C-fucose is linked by the enzyme in {alpha}1,6 orientation, onto the internal N-acetylglucosamine.

 
{alpha}6-Fucosyltransferase assays
Transfected COS7 cells were homogenized on ice in 2% Triton X-100, and protein was measured (Biorad, Bradford Protein micro-assay). Each enzyme assay was performed in 50 µl with 50 µg of protein cell extract, 70 mM cacodylate buffer (pH 7.25), 10 mM L-fucose, 6 µM GDP-(14C)-L-fucose (25.104 cpm/test at 300 mCi/mmol, Amersham Pharmacia Biotech, Little Chalfont, U.K.), and 5 µg biotinylated glycoasparagine acceptor GlcNAc2-Man3-GlcNAc2-Asn-LNS-Biotin (Figure 8). The mixtures were incubated at 37°C for 4 h, and their activity revealed by Sep-Pak C18 reverse chromatography (Palcic et al., 1988Go; Cailleau-Thomas et al., 2000Go). The transfer of (14C)-fucose is expressed in pmol/h/mg of protein.

RNA isolation, northern blot, and RNA dot-blot analysis
Embryos aged 40–70 days were obtained from legal abortions and used to make the three embryo cDNA libraries (Cailleau-Thomas et al., 2000Go). Total RNA was extracted from human embryos or transfected COS7 cells by the guanidine isothiocyanate method and submitted to digestion with RNase free-DNase I (10 U/µg RNA from Roche, Meylan, France) for 15 min (Cailleau-Thomas et al., 2000Go). Embryonic poly-A+ mRNAs were double-purified using oligo(dT)-cellulose (Type 3; Sigma) chromatography, denaturated, and fractionated with 1.2% phosphate-agarose gel electrophoresis. RNA was transferred to Hybond-N membranes (Amersham Pharmacia Biotech) and immobilized by baking at 80°C for 2 h.

Prehybridization and hybridization of northern blots were performed for 16 h at 42°C in a buffer containing 50% formamide, 5x saline sodium citrate (SSC), 1x polyvinylpyrrolidone EDTA (PE), 250 µg/ml denatured salmon sperm DNA, and 10% dextran sulfate, using the probes specific for cds-FUT8 of 998 bp (ExD-12s and F8-10as); exon A of 152 bp (ExA-111s, ExA-262as); exon B of 740 bp (ExB-AI6s, ExB-40as), and exon C of 98 bp (ExC-1 s, ExC-98as), which were obtained by PCR amplification of the A1 clone.

In addition, the tissue distribution of FUT8 transcripts was studied on fetal and adult tissues, with commercial mRNA blots from Invitrogen (Carlsbad, CA) loaded with 2 µg/lane (mRNA REAL Blots, mRNA equal amounts loaded). The blots were first washed at low stringency: 2x 15 min in 2x SSC, 0.1% sodium dodecyl sulfate (SDS) at 42°C, followed by one 15-min wash (2x SSC; 0.1% SDS) at 50°C and autoradiographed. A last 15-min wash in (0.5x SSC; 0.1% SDS) at 60°C was performed, and another autoradiography was done. The films were revealed after 3 days at -80°C or 10 days for the exons B– and C–specific probes.

After COS7 transfections with FUT8 transcripts, 5 µg total RNA was spotted on a Hybond membrane (Davis et al., 1986Go) and hybridized with the cds-FUT8 probe of 998 bp or the ß-actin probe. These RNA dot-blots were washed, and autoradiographies were revealed after 4 h contact of the film at -80°C.

Construction of embryo cDNA libraries
Poly-A+ mRNAs (1 µg) from single embryos (38, 50, or 60 days) were reverse transcribed at 42°C for 90 min using the cDNA-oligo-dT-synthesis primer (52mer) from the Clontech (Palo Alto, CA) Marathon cDNA amplification kit to initiate the first strand cDNA synthesis. Two reverse transcriptases were used to produce the different embryonic adapter-ligated double-stranded cDNA libraries: (1) the Moloney murine leukemia virus-reverse transcriptase from the Marathon cDNA amplification kit (Clontech) or (2) the Superscript-II RT system (200 U) from Invitrogen.

The PCR amplifications were carried out with FUT8 specific primers (Table III), the Klentaq mixture, and 1 µl cDNA templates from the different embryo libraries diluted 1:50 for the first PCR and 1 µl of the first PCR product diluted 1:10 for the second PCR. The same amplification program described for RACE (see later description) with the Advantage cDNA amplification kit mix (Clontech) was used. All PCRs were performed in 50 µl, with 1X Klentaq buffer, 0.2 µM of each primer, 1 unit of Klentaq DNA Polymerase and 0.2 mM dNTP with the touchdown-RACE program: initial denaturation 94°C 90 s, followed by five cycles of 94°C 30 s and 72°C 4 min, five cycles of 94°C 30 s and 70°C 4 min and 25 cycles of 94°C 30 s and 68°C 4 min.

Cloning of FUT8 transcripts with RACE-PCR
The 3' end of FUT8 cDNA was obtained after a double PCR with primers ExD-12 s and AP1 for the first PCR and nested primers F8-1 s and AP2 for the second PCR (Table III). The final PCR products were cloned in the TA cloning vector PCR3.1 (Invitrogen), and 20 clones were sequenced. The same procedure was followed to identify the 5'UT end of the FUT8 transcripts. Several associations of primers were used to obtain the specific final full-length 5' cDNA ends of the transcripts A1, A2, B1, B2, B3, B4, B5, B6, C1, C2, D1, D2, and the two retina transcripts. In the PCR we used the primers: (1) F8-9as and AP1 for the first amplification and the combination of nested primers F8-10as and AP2 or F8-Retas and AP2 for the second round of PCR, or (2) F8-10as and AP1 followed by two types of combinations of nested primers: F8-2as and AP2 or F8-5'Ras and AP2. The full-length 5'UT exon B was amplified from the libraries reversed transcribed with the superscript II-RT (Invitrogen) and the association of ExB-as and AP1 and ExB-40as and AP2 or ExB-as and AP1 and ExB-F8as and AP2 in a double PCR.

To confirm the full-length of the clones with exon A, the association of primers ExA-262as and AP1 and ExA-206as and AP2 were used for the first and the second amplifications, respectively. Before the reconstitution of the different FUT8-cDNA isoforms we made an RT-PCR to verify their expression in the libraries as full-length transcripts. We used the KlenTaq DNA polymerase and the following associations of primers in a double PCR with the program described: A1, ExA-111 s, and F8-L1as; A2, ExA-111 s, and F8-L2as; B1, B3, B5, and B6, ExB-s and F8-L1as; B2 and B4, ExB-s, and F8-L2as; B3 and B5, ExB-AI6 s, and F8-L1as; B4, ExB-AI6 s, and F8-L2as; B6, ExB-1 s, and F8-L1as. C1 and C2 are cDNA having exon C and L1 or L2 ends, ExC-1s and F8-L1as for C1, ExC-1s and F8-L2as for C2 transcripts. D1 and D2 are cDNA starting with exon D and having L1 or L2 terminus; ExD-1s and F8-L1as were used for D1 and ExD-1s and F8-L2as for D2 transcripts; Retina-1, F8-Ret-s, and F8-L1as; Retina-2, F8-Ret-s, and F8-L2as. The PCR fragments were fractionated in 1% sea plaque GTG-agarose gel (Ozyme, St. Quentin en Yvelines, France).

Construction of the full-length FUT8 cDNA-transcripts
The cDNA constructs A1 (ACD-L1) and A2 (ACD-L2) (Figure 1) were built with the 3'UT-RACE product amplified with the primers F8-1s and AP2 (Table III) and the 5'UT-RACE fragment amplified with the primers F8-5'Ras and AP2. The 3'UT-RACE fragments start on the ATG codon and ends in the 3'UT-polyA corresponding to L1 or L2. The fragment containing the 5'UT-ACD exon association overlaps with the 3'UT fragment on the 72 nucleotides downstream of the ATG translation start-codon in the FUT8 cds. This overlapping region contains the PpuMI restriction site. Among the 20 plasmids sequenced for each RACE-PCR fragment (5' or 3'), we selected two plasmids containing the L1 or the L2 fragment and one clone with the complete 5'UT exon association ACD without PCR errors. The resulting A1 and A2 full-length FUT8 cDNA plasmids were obtained via restriction fragment interchange procedures. The vectors containing the inserts with the cds and L1 or the cds and L2 are restricted with the NheI and PpuMI enzymes (Figure 1). The NheI enzyme cuts 67 bp upstream from the insert integration site, and PpuMI cuts at position 54 of the insert, downstream of the ATG codon. The small NheI–PpuMI fragments were discarded and replaced by the NheI–PpuMI fragment isolated from the clone containing the complete 5'UT-ACD exon association. The two final full-length cDNA-constructs were called A1 and A2.

The cDNA constructs having the different 5'UT-BCD and 5'UT-BD exon associations were built the two final full-length constructs A1 or A2. We replaced the 5'UT-ACD exon association by the respective clones containing the distinct 5'UT-BCD- and 5'UT-BD-RACE fragments, amplified with F8-2as or F8-10as and AP2 primers. The BCD isoforms corresponding to the constructs B1 and B2 were made by digestion of the A1 and A2 constructs and the 5'UT-BCD-RACE clone, starting at position 1296 in the exon B (Figure 1) with MluI (unique restriction site located at position 248 downstream of the ATG codon for all the clones) and NheI restriction enzymes. We replaced these small released fragments from A1 and A2 with the homologous (MluI–NheI) fragment from the 5'UT-RACE-BCD clone obtained after amplification with the F8-10as and AP2 primers (position 1296 of the exon B, Figure 1).

Representative plasmids containing the 5'UT-BCD association with L1 or L2 poly-A-tails, were designated as constructs B1 and B2, respectively. We then built the definitive full-length B3 and B4 constructs using the complete 5'UT-BCD-RACE fragment containing the exon B initiating at position 530 (Figure 1) and the B1 and B2 constructs. The full-length B5 cDNA was built using the B3 construct and the 5'UT-BD-RACE fragment amplified with the primers F8-2as and AP2. These fragments contain the unique EspI restriction site at position 1312 in exon B and the MluI restriction site at position 248 after the ATG codon (Figure 1). We cut B3 with EspI and MluI enzymes and replaced this fragment encompassing the BCD exon association boundary with the homologous EspI–MluI fragment containing only the BD exon association from the 5'UT-RACE clone.

The B6 construct was obtained using the B3 clone and the full-length 5'UT-RACE-PCR fragment of 1364 bp amplified with the primers ExB-as and AP2, containing the EspI restriction site. We cut the B3 clone and the 5'UT-exon B fragment with EspI and NheI restriction enzymes and we replaced this fragment with the homologous EspI–NheI fragment containing the 1364 bp exon B from the 5'UT-RACE. From the 5'UT-RACE-PCR obtained with F8-10as and AP2 primers, we obtained several clones starting either with full-length 5'UT exon C or with exon D. The C1, C2, D1, and D2 constructs were built from A1 and A2 by digestion with NheI and PpuMI and replacement of the small fragment by the NheI–PpuMI fragment isolated from the 5'RACE clones with the 5'UT-CD exon association or exon D (Figure 1).

DNA sequencing
Sequencing was performed on the RACE-PCR products cloned into the PCR3.1 expression vectorm and they were sequenced in both strands by the dideoxy chain termination method with the T7 DNA polymerase (Amersham Pharmacia Biotech). At least 20 clones were sequenced for each RACE-PCR product.

Transient expression of FUT8 cDNA isoforms and GFP-tagged-FUT8 in COS-7 cells
All the full-length cDNA transcripts shown in Figure 1 were inserted into PCR3.1 (Invitrogen, TA-cloning System). The tagged FUT8 enzymes, with a carboxy terminus or an amino terminus GFP of 25 kDa, were prepared by PCR with the respective primer associations: ExD-12 s with F8-20as, and F8-21s with F8-9as (Table III). The PCR products were inserted into the mammalian pcDNA3.1-GFP vector (TOPO-GFP cloning kit, Invitrogen). The COS7 cells were transiently transfected with these different constructs, by the DEAE-dextran method (Cailleau-Thomas et al., 2000Go). After 48 h, the cells were washed twice with phosphate buffered saline (PBS) and harvested for enzyme activity, western blot, or RNA dot-blot analysis.

SDS–PAGE and western blot
Transfected cell pellets were washed twice in cold PBS and homogenized by sonication in 50 mM cacodylate buffer (pH 7.2) containing 2% Triton X-100. Proteins (50 µg) were resolved by 10% SDS–polyacrylamide gel electrophoresis (Laemmli, 1970Go) in a protean III apparatus (BioRad) and transferred onto a nitrocellulose membrane (BIOBOND-NC, Whatman) for immunoblots. The membrane was blocked for 1 h at room temperature with 5% (w/v) nonfat dry milk in TTBS buffer (20 mM Tris–HCl [pH 7.5], 500 mM NaCl and 0.1% Tween-20, w/v) and then incubated overnight at 4°C with a rabbit anti-GFP antiserum (Invitrogen) diluted at 1:10,000 in dilution buffer (TTBS plus 1% nonfat dry milk, w/v). The blot was developed with the chemiluminescent reagents from the ECL-Plus Western blot detection kit (Amersham Biosciences RPN-2132, U.K.).

Immunofluorescent localization of the GFP-tagged FUT8 fusion proteins
Cells (2 x 105) were seeded on glass cover slips in 35 mm cell culture petri dishes 24 h before transfection. Three micrograms of the GFP-tagged cDNA constructs were used to transfect COS7 cells. After 48 h, they were washed with PBS. The cells were fixed for 15 min in 2% paraformaldehyde in PBS, then washed with PBS and incubated for 20 min in 50 mM NH4Cl in PBS. Thereafter they were permeabilized with 0.075% saponin, 0.1% bovine serum albumin (BSA) in PBS for 15 min followed by overnight or 1-h incubations, with primary antibodies. The GFP-tagged FUT8 fusion proteins were visualized by confocal microscopy. Double or triple immunofluorescence steps were made with a monoclonal primary antibody against a Golgi protein marker (anti-giantin) (Linstedt and Hauri, 1993Go; used at 1:1000 in PBS-BSA 1%), a rabbit polyclonal antibody against an ER marker protein (anti-calnexin from StressGen Biotechnologies (Victoria, BC, Canada), used at 1:200 in PBS-BSA 1%), and the GFP-tagged-FUT8 constructs. After washing the cells three times in PBS-BSA 0.1%, the anti-giantin was revealed with secondary antibodies: anti-mouse Ig-AMCA (blue fluorochrome, dilution 1:20) and the anti-calnexin with an anti-rabbit Ig-Cya3 (red fluorochrome, dilution 1:200). The secondary antibodies were incubated for 1 h and the reaction was stopped by washing three times in PBS. The cover slips with the labeled cells were mounted on slides with Mowiol, and the fluorescence was analyzed in a Biorad MRC-500 confocal laser scanning microscope system (Richmond, CA). When required, the transfected cells were treated with 5 µg/ml brefeldin A (Sigma) for 1 h at 37°C.

PCR amplification of the A, B, C, and D exon–intron boundaries of FUT8
Normal human genomic DNA was digested with one of several endonucleases (EcoRV, ScaI, DraI, PvuII, SspI) and ligated to the Marathon adapters from the Clontech genome walker kit. The intron–exon bnoundaries around exons A, B, C, and D were isolated by PCR amplification with APW1 and APW2 upstream primers and specific FUT8 downstream primers (Table III). We used the Advantage Genomic PCR kit from Clontech containing the Polymerase Mix (K-1906-1) or the Expand Long Template PCR System (Boehringer Mannheim). Native embryonic genomic DNA was also used to amplify the B–A intron sequence with the primers: ExA-s and ExB-as or ExB-s and ExA-225as in a double PCR reaction. The sequences around exon C and exon D were amplified by double PCR with APW1 and APW2 in association with the sense (ExC-1s and ExC-13s) or the antisense primers (ExC-98as and ExC-40as) for exon C, and with the sense (ExD-1s and ExD-34s) and antisense primers (F8-5'Ras and ExD-59as) for exon D. The PCRs for the boundary regions were made with Clontech polymerase mix with a program of 94°C for 1 min followed by seven cycles of 94°C for 25 s, 72°C for 5 min, and 32 cycles of 94°C for 25 s, 67°C for 5 min, and 6 min at 67°C. PCR amplification was performed in 50 µl by using 1 µl of the 50x DNA polymerase Tth mix, 1x buffer, 1.1 mM of Mg(OAc)2, 0.2 mM of each dNTP, 0.2 µM of each primer, and 100 ng genomic DNA. Gene fragments were amplified using the Expand Long template PCR system contained in a 50-µl reaction mixture: 0.2 µM of each primer, 2.6 U DNA polymerase, 0.25 mM of each dNTP, 50 ng DNA, 2.25 mM MgCl2. Amplifications were performed with 1 cycle of 94°C for 2 min followed by 35 cycles of 30 s at 65°C and 20 min at 68°C.

Accession numbers: human FUT8 cDNA sequences deposited at the EMBL databank: A1, Y17976; A2, Y17977; B1, Y17978; B2, YI7979; B3, AJ536056; B4, AJ536054; B5, AJ536055; B6, AJ536056; C1, AJ539535; C2, AJ539536; retina short, AJ514324; retina long, AJ514325.


    Acknowledgements
 
A. Cailleau-Thomas contributed in the early phases of the work. The work was partially supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), and Association de la Recherche contre le Cancer (ARC) grant RM-5348.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: mollicone{at}vjf.inserm.fr Back


    Abbreviations
 
BSA, bovine serum albumin; EST, expressed sequence tag; GFP, green fluorescent protein; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PE, polyvinylpyrrolidone EDTA; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; UT, untranslated


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
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