U 504 INSERM, Université de Paris Sud XI, 94807 Villejuif, France, 2UMR 1599 CNRS, Cytogénétique, IGR, 94805 Villejuif, France, and 3Hôpital de Pontoise, 95300 Pontoise, France
Received on November 29, 1999; revised on February 1, 2000; accepted on February 12, 2000.
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Abstract |
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Key words: CD15/chromosome location/embryo-fetal development/fucosyltransferase/Lewis x
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Introduction |
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We have previously shown, a differential expression of three categories of 3-fucosyltransferase activities able to synthesize the Lex antigen in different organs. The three groups of enzymes had different transfer profiles when examined using synthetic oligosaccharide acceptors. We called these activity profiles myeloid, plasma, or Lewis (Mollicone et al., 1990
). The myeloid-like
3-fucosyltransferase activity is mainly found in leukocytes and is characterized by the use of type-2 N-acetyllactosamine (Galß1,4GlcNAc) and H-type-2 (Fuc
1,2Galß1,4GlcNAc) acceptors to make Lex or Ley antigens, respectively. The plasma-like
3-fucosyltransferase activity is mainly found in adult plasma and liver and is able to use in addition, sialyl-type-2 N-acetyllactosamine (NeuAc
2,3Galß1,4GlcNAc) to make either of Lex, Ley, or sialyl-Lex. The Lewis-like
3-fucosyltransferase activity is mainly found in adult exocrine secretions and is able to use, in addition, type-1 acceptors such as type-1 N-acetyllactosamine (Galß1,3GlcNAc), H-type-1 (Fuc
1,2Galß1,3GlcNAc), and sialyl-type-1 N-acetyllactosamine (NeuAc
2,3Galß1,3GlcNAc), with higher efficiency than the type-2 acceptors, in order to make, respectively, Lea, Leb, or sialyl-Lea.
Six human genes encoding 3/4-fucosyltransferases have been cloned and registered in the Genome Data Base (GDB) as FUT3 (Kukowska-Latallo et al., 1990
), FUT4 (Goelz et al., 1990
; Lowe et al., 1991
), FUT5 (Weston et al., 1992a
), FUT6 (Weston et al., 1992a
), FUT7 (Natsuka et al., 1994
; Sasaki et al., 1994
), and FUT9 (Kaneko et al., 1999a
). We have previously assigned FUT4 to 11q21 (Reguigne et al., 1994
), the cluster FUT6-FUT3-FUT5 to 19p13.3 (Reguigne-Arnould et al., 1995
) and FUT7 to 9q34.3 (Reguigne-Arnould et al., 1996
).
During the early embryonic stage (before eight weeks of gestation) we only detected a myeloid-like 3-fucosyltransferase activity in all the organs tested. Then, in each organ, we demonstrated a switch from this myeloid-like activity pattern to adult forms of
3-fucosyltransferases, as a function of the maturation of the organ. In each organ we observed a concomitant decrease of the myeloid-like activity and the appearance of plasma- and/or Lewis-like activities after the 10th week of development (Mollicone et al., 1992
). In the early embryonic period the main oligosaccharide epitopes expressed are precursors of ABH and Lewis antigens, Lex is preferentially found on proliferating cells in areas showing progression of organ buds in mesenchyma (Candelier et al., 1993
).
In order to elucidate the genes encoding the enzymes with myeloid activity responsible for the stage-specific expression of Lex on embryonic cells, we examined, by Northern blot and reverse-transcriptase-PCR (RT-PCR), the expression of 3-fucosyltransferase transcripts (FUT3, FUT4, FUT5, FUT6, FUT7, and FUT9) during the first 2 months of human development. We made different cDNA libraries using human mRNA from 40- to 65-day-old embryos and we cloned a new FUT9 transcript by rapid amplification cDNA ends-PCR (RACE-PCR), and confirmed the gene localization on the chromosome band 6q16.
The sequence of the FUT9 human cDNA described in this article has been submitted to GenBank/EBI with the accession number AJ238701.
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Results |
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Reverse transcriptase polymerase chain reaction (RT-PCR)
Three types of mRNA matrix were reverse transcribed with oligo-dT or random primers and used for RT-PCR analyses: (1) a mixture of poly-A+ corresponding to a pool of 10 embryos (cDNA libraries) of 4065 days old; (2) poly-A+ from isolated 38 d, 50 d, and 60 d embryos treated and transcribed as described in Materials and methods; (3) or a pool of Marathon-Ready cDNA reversed transcribed by Clontech, from human poly-A+ mRNA of 8-week-old embryos.
Using distinct sets of specific primers (hF4-U and hF4-L; hF4-8151s and hF4-8150as; hF9-s and hF9-as; see Table I), FUT4 and FUT9 transcripts are reproducibly amplified at this stage of development, in all the cDNA tested.
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Taken together, Northern blots and RT-PCR results illustrate that FUT4 and FUT9 are the earliest transcripts of 3-fucosyltransferase expressed in human embryos and tend to decrease in the majority of adult tissues. During the embryonic period FUT3, FUT6, and FUT5 start to appear, but are only detected by PCR. FUT7 is not yet expressed.
Identification of the 5' and 3' cDNA ends of the embryonic FUT9 transcript
To verify the presence of the FUT9 cDNA in our libraries, we first cloned a fragment of the human embryonic FUT9 transcript with mouse primers (Table I) (Kudo et al., 1998). Then we used this human sequence to design the specific RACE-PCR primers, required to amplify the 5' and 3' cDNA ends of this embryonic FUT9 transcript.
We performed a RACE-PCR to isolate the 5' end of the FUT9 cDNA. A first PCR, using the cDNA-plasmids of the different libraries as templates, was done with the primers: hF9-2as and pCDM8-2118s or hF9-2as and pCDM8-2659as. Then a nested 5' RACE-PCR was performed using the internal primers: hF9-365as and pCDM8-2202s or hF9-365as and pCDM8-2604as (Table I). Several repetitive PCRs on the three cDNA libraries with the above combinations of primers, always gave the same unique PCR product of 466 bp. After gel purification the fragment was integrated in pCR3.1, and 10 clones were selected by sequencing. One 5' end-RACE, error-free, and sense-orientated clone was chosen to construct the entire FUT9 cDNA transcript of 2501 bp (Figure 4).
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Characteristics of the embryonic FUT9 cDNA transcript
This FUT9 cDNA transcript of 2501 bp (AJ238701) has a regular poly-A tail (A16), with eight RNA stabilization sites (ATTTA) and four polyadenylation sites (AATAAA) and is therefore different from the stomach transcript of 3019 bp (AB023021), which has not this poly-A tail (Kaneko et al., 1999b). The first 8 positions of the 5' UT of the embryo FUT9 transcript are different from the equivalent positions of the stomach transcript. One position is different and four nucleotides are missing in the 3' UT region. However, the two transcripts encode the same protein, since only three punctual nucleotide differences, giving no change at the level of the protein sequence were found in the cds (T342
C, T354
A, T357
C). The tissue distribution observed with our transcript is similar to mouse (Kudo et al., 1998
), with regard to expression in brain and kidney. Our FUT9 transcript was not detected in stomach and this is at variance with the stomach localization described for the human adult FUT9 transcript (Kaneko et al., 1999a
), but this could be related to poor quality of the stomach RNA in the commercial blots used. Compared to the mouse 2170 bp cDNA, our transcript has 70% homology in the 5' UT portion, 92% homology in the cds and 55% homology with the mouse 3' UT side and the putative N-glycosylation site is at the same position. This gives an identity of 99% at the level of the two translated proteins, the 1% difference corresponds to the same three amino-acid substitutions described previously (Kaneko et al., 1999a
): mouse Val-37 to Ile; mouse Thr-237 to Ala and mouse Phe-292 to Tyr.
Identification of the 5' and 3' cDNA ends of the embryonic FUT4 transcripts
We first verified the presence of the FUT4 transcripts in our libraries using the internal specific primer combinations: hF4-8150as and hF4-8151s or hF4-69s and hF4-8150as (Table I), which give the 319 and 1055 bp fragments, respectively. They were cloned in pCR3.1 and sequenced. They correspond to the already described FUT4 gene (Lowe et al., 1991). The same system of 3' RACE-PCR performed for FUT9 was performed for this gene, using FUT4 specific sets of primers (hF4-1050s for the first PCR and hF4-1206s for the second PCR) in association with the already described pCDM8 primers. Two fragments corresponding to the 2.3 kb (FUT4-short) and the 3 kb (FUT4-long) FUT4 cDNA poly-A tails were found (Goelz et al., 1990
). We never obtained sequences corresponding to the 6 kb transcripts seen by Northern blot. The FUT4 transcript is one of the genes, together with FUT7, having the largest proportion of GC in its sequence (more than 70% in the first 550 pb of the 5' region). These kind of sequences are difficult to reverse transcribe, because strong secondary structures are generated by the large proportion of GC. The FUT4 clone used in the following transfections and fucosyltransferase assays is similar to the 2.3 kb FUT4 transcript (Goelz et al., 1990
).
Expression of 3-fucosyltransferases in transfected COS7 cells
After 48 h, COS7 cells transfected with pCR3.1 alone or with pCR3.1-FUT9 or pCR3.1-FUT4 constructs were subjected to cell membrane immunofluorescence (Table II). Cells transfected with either of FUT9 or FUT4 constructs expressed Lex epitopes, but FUT4 gave consistently higher percentages of positive cells than FUT9. Both enzyme activities have a myeloid-like 3-fucosyltransferase activity profile, as reported for the orthologous mouse FUT9 sequence (Kudo et al., 1998
).
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Kinetic studies
Apparent Km were calculated for GDP-fucose (range of concentrations 1230 µM) from Lineweaver-Burk plots of initial rate data, obtained at 300 µM concentration for H-type-2 (FUT4, FUT6, FUT9) or H-type-1 (FUT3). The apparent affinities for GDP-fucose of the enzymes encoded by FUT9 and FUT4 (21 µM and 40 µM) were weaker than those encoded by FUT6 and FUT3 (6 µM and 12 µM; Table IV). The apparent affinities were also calculated for H-type-2 at saturation of GDP-fucose for all the enzymes. For FUT6 (plasma-like profile) the apparent Km for H-type-2 (17 µM) appeared stronger than those obtained for FUT4 and FUT9 enzymes (130 µM and 68 µM). The high Km value of 1330 µM for H-type-2 is expected for the FUT3 enzyme, because H-type-2 is not a good acceptor for FUT3, as compared to H-type-1. The Km for H-type-1 (40 µM) is more than 30-fold better. The values of Vmax for the different acceptors were similar for FUT9, FUT6 and FUT3 (H-type-2). The values of Vmax for FUT3 (GDP-fucose and H-type-1) are intermediate and the Vmax values of FUT4 are the best under our experimental conditions (Table IV).
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Chromosomal localization of the FUT9 gene
The presence of the FUT9 gene was tested by PCR on 53 genomic DNAs from human-rodent somatic hybrid cell lines. The correlation between the presence of each human individual chromosome, investigated by classical cytogenetic techniques (Nguyen van Cong et al., 1986; Couillin et al., 1991
), and the FUT9-PCR product of 808 bp is shown in Table V. Twenty-three clones, of which 22 retained the chromosome 6, expressed the human-specific FUT9-PCR product. The remaining 30 somatic hybrid clones, negative by PCR, lacked the chromosome 6. Many positive and negative discrepancies were observed for all the other chromosomes including 9, 11, 14, and 19, where we have previously assigned the other members of the fucosyltransferase gene family (Reguigne-Arnould et al., 1996
; Costache et al., 1997
). These results support a chromosome 6 localization of the human FUT9 gene (Table V). The single discrepant clone (CH.BL1) can be explained by the high sensitivity of PCR. This clone may have retained the chromosome 6 in a low percentage of cells or only a chromosome fragment not detected by classical cytogenetic techniques.
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Southern blots with human genomic DNA restricted with EcoR1 were probed with three different regions of the FUT9 cDNA (Figure 4). As illustrated in this figure, the entire FUT9 cDNA of 2501 bp has three EcoR1 restriction sites at positions: 20, 1529, and 2221. The 5' UT probe of 133 bp revealed a single band at about 2.5 kb in the human genomic DNA restricted with EcoR1. The cds probe detected a single restriction DNA fragment of 1.5 kb, which is expected because this probe detects the fragment situated between the two restriction sites at positions 20 and 1529. When we hybridized with the 3' UT probe, the two expected bands of 1.5 kb and 0.7 kb were found, showing that the 3' end of the gene is contiguous to the cds and is included in the same DNA fragment (Figure 7). Other Southern blots (data not shown) were made with the same genomic DNA restricted with different enzymes or association of enzymes: BalI alone, BalI/EcoRI, EcoRI/HindIII, and BalI/HindIII. The DNA digested with BalI alone or double digested with BalI/EcoRI and probed with the 5' UT of FUT9, detected one band at about 1.5 kb with the BalI digestion and one reduced band of 1.3 kb with the BalI/EcoRI DNA. This difference in size corresponds to the distance between EcoRI and BalI sites, in the cDNA (Figure 4). The Southern blot containing the BalI/EcoRI, BalI/HindIII, and EcoRI/HindIII DNA restrictions was probed with the cds of FUT9, and in each type of restriction profile we detected one fragment with the expected size. A reduced band of 1.3 kb is found on the BalI/EcoRI digest, a reduced band of 1.1 kb for the BalI/HindIII digest and finally a band of 1.35 kb for the EcoRI/HindIII sample. All these bands have the expected sizes if the restriction site positions in a single DNA exon are considered (Figure 4).
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Discussion |
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We have now identified two 3-fucosyltransferase genes susceptible to control the biosynthesis of the Lex antigen during the embryonic stage of human development. FUT4 and FUT9 are the earliest
3-fucosyltransferase genes strongly expressed during the first two months of human embryo-fetal development. At this stage the transcripts of FUT6 and FUT3 genes are weakly expressed, FUT5 was only irregularly and faintly amplified and the FUT7 transcript was not detected at all. These results are in good agreement with our previous data (Mollicone et al., 1992
), since both FUT4 and FUT9 genes encode for
3-fucosyltransferases with a myeloid-like activity profile, mainly able to synthesize the Lex antigen on the type-2 N-acetyllactosamine precursor and the Ley on the H-type-2 structure. Other enzymes with this activity profile are expected in human adult brain, since a myeloid-like
3-fucosyltransferase activity resistant to Co++ was described in this tissue (Mollicone et al., 1992
). We have confirmed this finding in adult and 7-week-old brain, but neither of the 5- or 6-week-old brain samples or any of the cloned variants of FUT4 or FUT9 enzymes kept its enzymatic activity in the presence of Co++.
We detected by Northern blot two transcripts of FUT9: one around 2 kb ubiquitously expressed in tissues from embryonic and fetal stages with a decrease of expression in the adult and the other of about 12 kb with tissue specific distribution, which is expressed strongly in brain, moderately in kidney and weakly in muscle in the fetal period. In the adult it is expressed in pancreas, placenta, and kidney; decreases in brain; disappears from muscle; and was not detected in any of the other tissues tested. The existence of different size transcripts with differential tissue expression, suggests that each transcript may have particular regulatory sequences contributing to the tissue specificity of enzyme expression, as already suggested for the different transcripts observed in FUT3, FUT5, and FUT6 (Cameron et al., 1995).
We have cloned an embryonic FUT9 cDNA of 2.5 kb and located the gene in 6q16. Its tissue expression is similar to the mouse FUT9, with regard to the expression in kidney and brain (Kudo et al., 1998). With regard to the utilization of the H-type-2 and type-2-N-acetyllactosamine acceptors, the FUT9 enzyme has a substrate specificity pattern similar to FUT4. But, in contrast to FUT4, this FUT9 enzyme does not transfer efficiently onto the lac-di-NAc substrate. In presence of MnCl2 there is no transfer on to lac-di-NAc for FUT9 and therefore, this acceptor can be used with MnCl2 to follow the FUT4 activity in tissues. In fact, our previous enzymatic results (Mollicone et al., 1992
), showing a myeloid-like activity pattern in all tissues before 8 weeks, were obtained in presence of 20 mM of MnCl2 and at the optimum pH for FUT4. Therefore, they mainly reflected the expression profile of the FUT4 enzyme, since the FUT9 activity is partially inhibited under these conditions.
During embryo-fetal development there is an early derepression of monomorphic genes such as FUT4 and FUT9, which are the oldest genes of the 3-fucosyltransferase family (Dupuy et al., 1999
) and encode myeloid-like
3-fucosyltransferases. Then, a late expression of adult polymorphic genes as FUT3, FUT6, and FUT7 (Bengston et al., 1999
), which have a specific tissue distribution in the adult (Mollicone et al., 1990
; Cameron et al., 1995
), is observed.
In evolution, FUT8 is the oldest of the family of 6- and
2-fucosyltransferase genes (Costache et al., 1997
; Oriol et al., 1999
) and FUT9 is the oldest of the
3-fucosyltransferase gene family (Kaneko et al., 1999a
). It is interesting that they both seem to be the more conserved fucosyltransferases, since between mouse and human proteins there is only 1% difference for FUT9 and only 5% difference for FUT8, while more than 20% difference is found for FUT4 (Gersten et al., 1995
) and FUT7 (Smith et al., 1996
) between mouse and human proteins. The nucleotide sequences of FUT8 and FUT9 genes have in addition, a high proportion of AT in their cds (the AT/GC ratios are 1.4 for FUT9, 1.2 for FUT8, and ~0.6 for the other types of vertebrate fucosyltransferase genes).
The functional significance of the Lex antigen expressed on the proliferating cells in areas showing progression of organ buds in mesenchyma, is not known yet. We hypothesize that the Lex carbohydrate could be involved in induction mechanisms permitting the mesenchyme to stimulate the organ buds to grow and branch. The Lex could be involved as receptor or ligand in signal transduction pathways, promoting cellular proliferation, since sialyl-Lex has been demonstrated to be a signal transduction molecule for leukocyteplatelet or leukocyteendothelial cell interactions (Wadel et al., 1995).
In lower animal species such as zebrafish, two 3-fucosyltransferase genes zFT1 and zFT2, orthologous homologous of human FUT9, are transiently expressed during embryogenesis (Kageyama et al., 1999
). These two transcripts are separately transcribed before and after hatching, respectively, and they may play distinct roles in zebrafish embryo development. In mouse embryos an
3-fucosyltransferase gene orthologous to the human FUT4 gene is expressed during the first divisions of the embryo (Liu et al., 1999
). A recent work has shown that the presence, in mouse eggs, of an
3-fucosyl residue on structures as Lex or
Gal-Lex, appears to be necessary to bind sperm with high affinity (Johnston et al., 1998
).
In conclusion, the presence of Lex synthesized by early expressed myeloid-like 3-fucosyltransferases may be important in cellcell interactions during embryogenesis.
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Materials and methods |
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RNA isolation and Northern blot analysis
Total RNA was extracted from 10 whole embryos with guanidine isothiocyanate and purified by cesium chloride gradient centrifugation. Embryonic poly-A+ mRNA were double purified using oligo(dT)-cellulose (sigma Type-3 oligo-dT cellulose) chromatography. Poly-A+ RNA (4 µg/lane) from embryos 4070 d old were denatured and fractionated with 1.2% formaldehyde agarose gel electrophoresis and transferred to Hybond-N membranes (Amersham-Pharmacia-Biotec). After transfer and immobilization on the membrane, the blots were hybridized for 16 h at 42°C, under the same conditions as the Southern blots, with 250 µg/ml denatured salmon sperm DNA and 10% dextran sulfate, with the cds-FUT9 probe (Figure 4), the cds-FUT4 probe, the cds-FUT5 probe, and the cds-FUT7 probe. The blots were first washed at low stringency: 3 x 5 min in (2x SSC, 0.1%SDS) at room temperature, followed by one 15 min (2x SSC, 0.1% SDS) wash at 50°C and autoradiographed. A last wash at high stringency was then performed (15 min at 65°C in 0.1x SSC, 0.1% SDS) and a new autoradiography was made. The tissue distribution of FUT4 and FUT9 transcripts was studied on fetal and adult tissues with commercial mRNA blots from Invitrogen, mRNA REALTM Blots (mRNA, Equal Amounts Loaded).
Construction of human embryo cDNA libraries
A mixture of 6 µg poly-A+ mRNA covering a range of ages of 40 to 65 d were reversed transcribed using either oligo-dT or random hexamer primers included in the Amersham kit "cDNA Synthesis System Plus" (RPN-1256Y) and complementary DNAs were synthesized according to the manufacturers recommendations. Hexamer or oligo-dT cDNAs libraries were constructed by inserting size fractionated double strand cDNAs (more than 1 kb) into the expression vector pCDM8, using BstXI adaptors. We obtained about 1.3 x 106 and 6 x 106 independent clones in the hexamer and oligo-dT cDNAs libraries, respectively. A maxi-preparation of the plasmids from each type of library was made and 200 ng of these plasmid cDNAs were used as templates for 5' and 3' RACE-PCR to clone human embryonic FUT4 and FUT9 transcripts. These libraries were also used as DNA templates for RT-PCR.
RT-PCR analysis
Poly-A+ RNAs (1 µg) from single human embryos (38 d, 50 d, or 60 d), were reverse transcribed at 42°C using oligo-dT or random hexamer primers to initiate first strand cDNA synthesis. Two transcriptases were used (1) Marathon cDNA amplification kit from Clontech with the MMLV-RT (Moloney Murine Leukemia VirusReverse Transcriptase), or (2) SUPERSCRIPT-II RT system from Life Technologies Inc. Contaminating DNA was removed from the poly-A+ RNAs by digestion with RNAse-free DNAseI (10 U/µg of RNA from Boehringer Mannheim for 15 min at room temperature, followed by 15 min at 70°C) and poly-A+ were purified by phenol/chloroform extraction.
The PCR reactions were carried out with primers specific for FUT3, FUT4, FUT5, FUT6, FUT7, and FUT9 genes (Table I), the KlenTaq mixture and 1 µl of cDNA templates diluted 1:50 for the first PCR and 1 µl of the first PCR product diluted 1:10 for the second PCR. The same PCR program described for RACE (see below) with the Advantage cDNA amplification mix (Clontech) was used.
cDNA cloning of FUT4 and FUT9 transcripts using 5' and 3' RACE-PCR
All PCRs were performed in 50 µl KlenTaq buffer containing 0.2 µM of each primer, 1 unit of KlenTaq DNA Polymerase (Clontech) and 0.2 mM dNTP with the touchdown-RACE program: initial denaturation 94°C 90 s, followed by 5 cycles of 94°C 30 s and 72°C 4 min, 5 cycles 94°C 30 s and 70°C 4 min, and 25 cycles 94°C 30 s and 68°C 4 min. For 5' and 3' RACE analysis 200 ng of plasmids from the libraries were used as template in the first PCR amplification. Because of the bi-directional orientation of the cDNA inserts in the pCDM8, we used in the first PCR a specific cds gene primer in association with primers designed in the 5' pCDM8-2118s and 3' pCDM8-2659as regions flanking the polylinker. The nested PCR was performed with nested gene specific primers in combination with 5' pCDM8-2202s and 3' pCDM8-2604as vector primers.
FUT9
The first PCR for the 5' and the 3' RACE was performed with the reverse cds specific primer hF9-2as or the sense cds specific primer hF9-1s, in association with the primers hybridizing in the vector: 5' pCDM8-2118s or the 3' pCDM8-2659as. For the nested PCR, we used as template 1 µl of the first 10 times diluted 5' and 3' RACE-PCR reactions and the nested reverse hF9-365as or sense hF9-2s specific cds primers, with the nested vector primers: 5' pCDM8-2202s or 3' pCDM8-2604as.
FUT4
Following the same scheme we used the same pCDM8 primers for the amplification of the FUT4 transcripts. For the 5' RACE, hF4-614as (first PCR) and hF4-473as (nested PCR). For the 3' RACE, hF4-1050s (first PCR) and hF4-1206s (nested PCR).
DNA sequencing
All the RACE-PCR products were cloned into the expression vector PCR3.1 (Eukaryotic TA Cloning Kit from Invitrogen) and sequenced in both directions by the dideoxy chain termination method with the T7 DNA Polymerase (Kit Amersham-Pharmacia-Biotech) and the adapted primers. At least 10 clones were sequenced for each RACE-PCR product.
Southern blot analysis
Triplicates of EcoRI, duplicates of BalI/EcoRI, EcoRI/HindIII, and BalI/HindIII and one BalI digested genomic DNA samples (10 µg/lane) were run in 0.8% agarose electrophoresis for 14 h at 1.25 V/cm in 0.5X TBE. After denaturation and neutralization of the gel, the samples were transferred on Hybond-N membrane (Amersham-Pharmacia-Biotech), in 20x SSC for 48 h. The DNA on the filter was immobilized by baking at 80°C for 2 h. Prehybridization and hybridization were performed for 16 h at 42°C in a buffer containing 50% formamide, 5x SSC, 1x PE, 150 µg/ml denatured salmon sperm DNA, and 5% dextran sulfate. Each blot containing digested DNA with EcoRI, was hybridized with three probes: (1) 5' UT cDNA-FUT9 probe; (2) the cds-FUT9 probe; (3) and the 3' UT cDNA-FUT9 (Figure 4). BalI and BalI/EcoRI digests were hybridized with the 5'UT probe and all the double digested samples were tested with the cds probe. After hybridization with the different probes, all the blots were washed three times for 5 min in 2x SSC, 0.1% SDS at room temperature, followed by one 15 min (2x SSC, 0.1% SDS) wash at 50°C. At this step, the blot with the 5'UT FUT9-probe was autoradiographed for 48 h. The other blots were subjected to a final wash of 10 min at 65°C in (0.1x SSC, 0.1% SDS) and autoradiographed.
Cell membrane indirect immunofluorescence staining
COS7 cells were transfected by the DEAE-dextran method (Davis et al., 1986) with the
3-fucosyltransferase cDNA integrated in PCR3.1. After 48 h (Costache et al., 1997
), the carbohydrate: Lex, sialyl-Lex, Lea, sialyl-Lea, Cdw65 or Vim2, FH6 (sialyl-di-Lex) and B blood group related glycotopes were detected using monoclonal antibodies: 80H5, 82H5 and VIM2 from Immunotech-Coulter company; C-7798 from Sigma; KM93 from Kamiya Biomedical Co.; 7LE and CA-19.9 were gifts from J.Bara (INSERM U 482, Paris, France); FH6 was a gift from H.Clausen (Royal Dental School, Copenhagen, Denmark) and 9W4 was characterized at the II International Workshop on Antibodies against Blood group antigens, Lund, Sweden 1990. The secondary antibodies were fluorescein (FITC)-labeled sheep anti-mouse Ig (H + L) (Pasteur Diagnostics, Marne-la-Coquette, France). Stained cells were mounted under cover slides for observation with an epifluorescence microscope and positive (bright green) cells were counted.
3-Fucosyltransferase assays
Transfected cells were homogenized on ice in 1% Triton X-100 and the concentration of protein was measured by the BCA Protein Assay Reagent Kit, from Pierce. Each enzyme assay in a volume of 35 µl, contained 12.5 µg of the cell protein homogenate, 25 mM Cacodylate buffer pH 6.5 (FUT3, FUT5, FUT6 and FUT9) or 25 mM Tris/HCl pH 7.4 (FUT4), 4 mM ATP, without MnCl2 (FUT9) or 20 mM of MnCl2 (all other 3-fucosyltransferases), 10 mM of L-fucose, 4 µM GDP-(14C)-fucose (300 mCi/mmol, Amersham-Pharmacia-Biotech) and 5 µl of a 1 mg/ml solution of synthetic acceptors with the 8-methoxycarbonyloctyl aglycone (Alberta Research Council, Edmonton, Canada). The mixtures were incubated at 37°C for 2 h (FUT7 and FUT9) or for 1 h (all others). The reactions were stopped by addition of 3 ml of water and then centrifuged, and the supernatant was applied to a conditioned Sep-Pak C18 reverse chromatography cartridge (Waters, Milford) (Palcic et al., 1988
; Fernandez-Mateos et al., 1998
). Enzyme kinetic parameters were determined for FUT3, FUT4, FUT6, and FUT9 enzymes under conditions corresponding to initial velocity with respectively, 20, 12.5, 40, or 25 µg protein aliquots and 15 (FUT3), 30 (FUT4), or 60 (FUT6, FUT9) min incubation at 37°C. Apparent Km were calculated for GDP-fucose (range of concentrations, 4250 µM), H-type-2 (20890 µM for FUT4, FUT6, and FUT9 or 0.14.4 mM for FUT3), H-type-1 (20890 µM for FUT3) from Lineweaver-Burk plots of initial rate data. When Km was calculated for GDP-fucose, H-type-2 or H-type-1 concentrations were 300 µM. When Km was calculated for H-type-2 or H-type-1 the concentration for GDP-fucose was 230 µM for FUT4 and FUT9 or 120 µM for FUT3 and FUT6.
PCR on genomic DNA of hybrid cell lines for chromosome localization of the FUT9 gene
A total of 53 humanrodent interspecific hybrid cell lines were tested. Ten monochromosomal lines were obtained from the ATCC. Twenty-four were from a panel (Nguyen van Cong et al., 1986) and the remaining 19 clones were prepared by P.Coullin (Couillin et al., 1994
; Reguigne-Arnould et al., 1995
, 1996). The PCR for the detection of FUT9 gene was performed with 200 ng of each hybrid genomic DNA in a 50 µl reaction volume containing: 1x PCR buffer and 1 unit of Taq Polymerase from MBI-Fermentas, 200 µM of dNTPs, 2 mM MgCl2, 0.2 µM of each FUT9 primer (hF9-s and hF9-1260as; Table I). The PCR program comprises: initial denaturation 94°C 5 min, followed by 38 cycles of 94°C 45 s, 52°C 45 s, 72°C for 2 min 30 s, and final extension of 72°C 8 min. A FUT9 specific PCR product of 808 bp is obtained when hybrids have the FUT9 gene.
Cytogenetic localization
Slides using standard cytogenetic methods were prepared from lymphocytes of healthy donors. Biotinylated probes from BACs: B601c3 and H330e11 were obtained by nick-translation using the kit proposed by GIBCO BRLR. Regional assignment was performed by simultaneous visualization of FISH signals and the R-banding pattern generated by PRINS (Coullin et al., 1999). Briefly, preparations were first treated by PRINS and then used for FISH. The slides were denatured in 70% formamide at 70°C for 2 min and PRINS reactions were conducted (Coullin et al., 1997
) using the Alu S sequence (5'GCCACTGCACTCCAGCCTGGG3') as primers and digoxigenin-11-dUTP as labeled nucleotide. After denaturation (80°C for 5 min) and the competition phase (37°C for 1 h 30 min), the hybridization mixture containing the probe (40 ng/8 µg human Cot-1TM DNA) was deposited onto the PRINS pretreated slides. FISH procedures were performed directly on these slides (Goguel et al., 1996
). The PRINS staining was revealed during the last post-hybridization treatment using anti-digoxigenin-rhodamine. The BAC probe signal was revealed with avidin-FITC. The preparation was counterstained with DAPI and examined under an epifluorescence microscope (Zeiss Axiophot). Numerized pictures were obtained using a tri-CCD camera and the Vysis computer program for image storing.
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