©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of a Human Genomic Region Containing the H Blood Group (1,2)Fucosyltransferase Gene and Two H Locus-related DNA Restriction Fragments
ISOLATION OF A CANDIDATE FOR THE HUMAN SECRETOR BLOOD GROUP LOCUS (*)

(Received for publication, November 8, 1994)

Sylvie Rouquier (1)(§) John B. Lowe (2) (3)(¶) Robert J. Kelly (2) Anne L. Fertitta (1) Gregory G. Lennon (1)(**)(§§) Dominique Giorgi (1)(**)(¶¶)

From the  (1)Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550 and the (2)Howard Hughes Medical Institute and the (3)Department of Pathology, University of Michigan Medical Center, Medical Sciences Research Building I, Ann Arbor, Michigan 48105-0650

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have used the human H blood group alpha(1,2)fucosyltransferase (FUT1) cDNA to screen chromosome 19 cosmid libraries in a search for the human Secretor (Se) blood group gene (FUT2). One cosmid has been isolated that contains two distinct segments that cross-hybridize with FUT1. We have assembled a 100-kilobase (kb) cosmid contig, localized to 19q13.3, encompassing FUT1 and the two FUT1-related sequences, termed Sec1 and Sec2, for Secretor candidate 1 and 2. Sec1 and Sec2 are separated by 12 kb and are 65.5 kb and 35 kb apart, respectively, from the FUT1 gene. We used a cosmid-dependent direct cDNA selection method to clone a cDNA corresponding to a transcript that emanates from Sec2. This cDNA detects a 3.35-kb transcript in human tissues known to express the Se locus. Together with sequence and expression data reported in the accompanying article (Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B.(1995) J. Biol. Chem. 270, 4640-4649), these data demonstrate that Sec2 corresponds to the human Se blood group locus (FUT2). Our results furthermore define the physical relationship between the H and Se loci and confirm a hypothesis that these two loci represent distinct but closely linked alpha(1,2)fucosyltransferase genes.


INTRODUCTION

Mammalian cells display a complex variety of carbohydrate antigens on their surface. The structures of these carbohydrate moieties are determined largely by the glycosyltransferases responsible for oligosaccharide synthesis. The human H blood group oligosaccharide determinant serves as an essential precursor for the action of specific glycosyltransferases that construct the A and B blood group antigens(1, 2, 3) . The H gene locus (or FUT1) encodes an alpha(1,2)fucosyltransferase (^1)that catalyzes the synthesis of the H determinant via a transglycosylation reaction that transfers L-fucose from the substrate GDP-fucose to the beta-D-galactose residue at the nonreducing terminus of glycoconjugates to form H-active Fucalpha(1,2)Gal-beta moieties. Genetic and biochemical studies indicate that the human genome encodes at least one other distinct alpha(1,2)fucosyltransferase activity, thought to be encoded by a second locus termed the Secretor blood group locus (Se or FUT2)(4, 5, 6) . The Secretor locus-determined alpha(1,2)fucosyltransferase, like the H locus-encoded enzyme, can also synthesize blood group H determinants. Experimental evidence also indicates that the H and Se loci express their cognate alpha(1,2)fucosyltransferases, as well as the corresponding cell surface and soluble H-active oligosaccharide products, in characteristic tissue-specific expression patterns(4, 5, 6) . It has been shown, for example, that expression of the H locus is largely restricted to tissues derived from mesoderm (like erythrocyte progenitors) or ectoderm (epidermis) (reviewed in (7) ), whereas expression of the Se locus is apparently restricted to epithelia derived from embryonic endoderm, including those epithelial cells that line the stomach, intestine, and salivary glands(4, 5, 7) .

Fucosyltransferases have often been used as genetic markers in linkage analyses. Indeed, the first human autosomal linkage group described was that of the ABH Secretor locus and the Lutheran blood group locus LU(8) . Later work established that these loci lie on chromosome 19q(9, 10, 11) . It was subsequently demonstrated that FUT1 and FUT2 are very closely linked(12, 13) , suggesting that these genes may have evolved by gene duplication and subsequent divergence(4) . To date, only the FUT1 gene-encoded alpha(1,2)fucosyltransferase has been cloned(6, 14) . Mapping studies using somatic cell hybrids have assigned FUT1 to chromosome 19(14) . We have recently localized FUT1 to 19q13.3, distal to the APOE and APOC2 loci, using fluorescence in situ hybridization approaches(15) . Molecular analysis of the FUT1 and FUT2 fucosyltransferase loci should serve to elucidate their physical gene map relationship, which is beyond the resolution of meiotic mapping using linked markers.

Because these two alpha(1,2)fucosyltransferases exhibit similar yet distinct catalytic properties, and because their corresponding loci are closely linked, we considered that these genes might be sufficiently related at the primary sequence level to allow isolation of the FUT2 locus by a cross-hybridization approach. We report here the isolation of two novel human DNA sequences that cross-hybridize with the FUT1 locus and localize with the FUT2 locus to the same 100-kb cosmid ``contig'' (group of overlapping clones). One of these two sequences is transcribed and corresponds to a novel alpha(1,2)fucosyltransferase gene characterized in the accompanying report and assigned to the human Se locus (FUT2) (16) . These results provide molecular confirmation of a hypothesis that the H and Se loci correspond to tightly linked and structurally related alpha(1,2)fucosyltransferase genes with distinct tissue-specific expression patterns.


MATERIALS AND METHODS

cDNA, Cosmid, YAC, and BAC Probes

The FUT1 coding sequence probe was a PCR product of the FUT1 cDNA from position 1 to 1133 (14) subcloned in plasmid pTZ (Pharmacia Biotech Inc.). Fifty ng of the gel-purified PCR product were labeled by random-priming to a specific activity of >10^8 cpm/µg, according to (17) . Cosmid insert probes were prepared as described (18) . YAC and BAC probes were generated by Alu-PCR. For the YACs, primers PDJ33, PDJ34, and PDJ66 (19, 20) were used independently or in combination, two by two as described(18) . BAC probes were generated from 150 ng of an alkaline lysis minipreparation by single primer Alu-PCR as described above except that primers ALE1 and ALE3 (21) were also used. Labeling of the PCR products was performed as described(18) .

Chromosome 19 Cosmid Library Screening

Two cosmid libraries (F and R) were generated from human chromosome 19 (22) flow-sorted from the Chinese hamster-human hybrid cell line (5HL9-5B). This hybrid contains a chromosome 19 as its only human material(23) . The libraries were constructed in the cosmid vectors Lawrist 5 (library F) (22) or Lawrist 16 (library R), that are modifications of the LORIST series of vectors originally described in (24) . These vectors contain a double cos site, two unique SfiI sites, and bacteriophage promoters (T7 and SP6 for the F library or T7 and T3 for the R library) flanking the insert. A total of about 19,100 individual clones, corresponding approximately to an 8-fold coverage of chromosome 19, were arrayed in microtiter dishes and spotted in high density arrays onto Nylon membranes (Hybond N) using a Beckman Biomek 1000 workstation (1536 clones/membrane)(25) . Filter hybridizations were performed in duplicate according to standard procedures (26) with a labeled probe concentration of 10^6 cpm/ml. For suppression of repetitive sequences, the probes were prehybridized in the presence of sheared human placental DNA as described(27) . Following hybridization, filters were briefly washed in 2 times SSC, 0.1% SDS at room temperature; once in 2 times SSC, 0.1% SDS at 65 °C (30 min); and once in 0.1 times SSC, 0.1% SDS at 65 °C (30 min). For low stringency hybridizations used to detect imperfectly homologous sequences within the same species, filters were washed using low stringency conditions (rinsed in 2 times SSC, 0.1% SDS at room temperature, followed by one 30-min wash in 2 times SSC, 0.1% SDS at 50 °C).

YAC and BAC Library Screening

High density filters containing 18,000 clones from the Imperial Cancer Research Fund (ICRF) YAC library (28) were processed for colony hybridization as described in (29) . The filters were washed as described above for the cosmid filters. High density filters containing 15,000 clones from the California Institute of Technology (Pasadena, CA) BAC library (30) were hybridized according to standard procedures(26) .

Cosmid, YAC, and BAC DNA Preparation

Cosmid DNA samples were isolated from cultures of 5 ml of LB medium (26) containing 20 µg/ml kanamycin, by alkaline lysis and purification on Qiagen tip-20 columns, following the procedure recommended by the manufacturer (Qiagen, Inc. Chatsworth, CA). Yeast DNA was isolated in 100-µl agarose blocks as described(18) . BAC DNA was isolated from 5 ml of LB broth medium cultures containing 12.5 µg/ml chloramphenicol by a standard alkaline lysis procedure(31) .

Cosmid Clone Analysis, Contig Assembly, and Restriction Mapping

Assessment of contig integrity, contig size, cosmid overlap, and fine EcoRI mapping was carried out either by total restriction enzyme digestion or by partial digestion followed by Southern blot analysis (26) of selected cosmids. For total digestion, cosmid DNAs were single-digested with SfiI, EcoRI, or PstI, or double-digested with EcoRI-SfiI and EcoRI-PstI. Restriction digests were fractionated by electrophoresis through 0.4% and 1.2% agarose gels. The size of each cosmid was then determined by summing the sizes of restriction fragments visualized on the gels under ultraviolet irradiation after ethidium bromide staining. The gels were alkali-blotted as described (32) onto nylon membrane (GeneScreen Plus, DuPont NEN) and used for hybridization as described in ``cosmid screening.'' For partial digests, 6 µg of each cosmid were first digested to completion with SfiI to excise the insert from the Lawrist vector. The buffer was then adjusted to 50 mM NaCl and 100 mM Tris, pH 7.5, to be compatible with EcoRI, and two series of four 15-µl aliquots, each containing 0.5 µg of the SfiI digest, were partially digested using 0, 0.05, 0.15, and 0.5 unit of EcoRI for 45 min at 37 °C followed by an incubation at 68 °C for 10 min to inactivate the enzyme. These samples, and complete SfiI-EcoRI digests, were fractionated through a 0.4% agarose gel in 0.5 times TBE for 20 h at 30 V. The gels were blotted as described above and hybridized separately for 12 h at 37 °C with 10 pmol of [P]ATP end-labeled (26) oligonucleotides (T3 and T7, for the R clones; Sp6 and T7 for the F clones), in 6 times SSC, 5 times Denhardt's solution, 0.05% sodium pyrophosphate, 0.1% SDS. The filters were then subjected to two, 1-h 37 °C washes, in the same buffer without Denhardt's solution. The positions of the EcoRI sites relative to both vector-insert junctions were deduced from the autoradiograms by measuring the sizes of the successive partial EcoRI fragments hybridizing at one end with the radioactive T3, T7, or Sp6 primers. Similarly, the PstI restriction map of the 18.10-kb EcoRI fragment from cosmid 16240 was established by digesting cosmid DNA to completion with EcoRI, followed by a PstI partial restriction, and hybridization with labeled Sp6 primer. To determine the size of the cosmid contig and to establish the extent of overlaps between cosmids, each cosmid was digested completely with EcoRI and was subjected to Southern blot analysis with the SfiI insert of each cosmid. This allowed us to determine which EcoRI fragments are unique to each cosmid, which are shared by adjacent cosmids, and which correspond to vector/insert junction fragments.

YAC and BAC Clone Analysis

Undigested YAC clones were analyzed by pulsed field gel electrophoresis using a contour-clamped homogeneous electric field apparatus (Bio-Rad DRII) as described(18) . After ethidium bromide staining, gels were destained in water for 30 min prior to alkali blotting on nylon membranes (GeneScreen Plus, DuPont NEN). For restriction analysis, 100-µl YAC blocks were enzyme-digested and analyzed as described(18) . We isolated the YAC ends by an inverse PCR method described in (18) and (33) , using two sets of restriction endonucleases (set 1: Sau3AI, TaqI, or HaeIII; set 2: AluI, HhaI, or HaeIII), each set pertaining to one end of the YAC vectors. Yeast DNA was digested, ligated, and amplified by PCR to obtain human sequence from each end of the YAC, using two primer sets: YAK5-U and YAK5-R for the left arm (set 1 ligated DNA), YAK3-U and YAK3-R for the right arm (set 2 ligated DNA), and PCR conditions described in (33) . PCR fragments corresponding to the amplified YAC ends were isolated by agarose gel electrophoresis, labeled by random priming, and used as probes.

Insert BAC DNA was separated from the vector by a NotI digestion (Life Technologies, Inc.). Separation of the digested DNA was carried out by pulsed field gel electrophoresis on a Bio-Rad contour-clamped homogeneous electric field Mapper apparatus for 20 h, at a field strength of 6 V/cm in a 1% agarose gel in 0.5 times TBE, at 16 °C, with a linear pulse from 5 to 15 s. After ethidium bromide staining, the gel was alkali-blotted as described above.

cDNA Selection Using Magnetic Bead Capture (``Hybrid Selection'')

cDNA sequences were isolated using a modified procedure of the hybrid selection method described in (34, 35, 36, 37, 38) , using magnetic beads (Dynabeads M-280, Dynal, Oslo). Double-stranded cDNA was synthesized from 200 ng of polyadenylated human fetal brain RNA (Clontech), blunt-ended with T4 DNA polymerase. A UNI-Amp adaptor was then ligated to both ends of 2 ng of the cDNA molecules (UNI-Amp Plus kit, Clontech). The UNI-Amp primer was used to amplify one-fourth of the ligation reaction. One-tenth of this first amplification was submitted to a second PCR reaction with the same primer. One µg of this double-amplified cDNA was prehybridized overnight with 5 µg of Cot1 DNA, to block human repetitive sequences within the cDNA, and then hybridized for 24 h with 100 ng of biotinylated cosmid DNA (BioNick, Life Technologies, Inc.), following hybridization conditions recommended by the manufacturer (Dynal). After hybridization, cosmid-cDNA hybrids were separated from unbound cDNA using streptavidin-coated magnetic beads. The biotin-streptavidin reaction, the washes to remove unspecifically bound cDNA, and the elution of specific cDNAs were performed as described in (36) . The eluate was then desalted by chromatography over a Sephadex G-50 (Pharmacia) spin column. For secondary cycles of enrichment, approximately 1 µg of amplified, eluted cDNA was recycled through the above process, including the repeat blocking step. After the second round of enrichment, the eluted cDNAs were amplified again by PCR and subcloned directly in the TA vector (Invitrogen). The recombinant clones were analyzed by PCR. All the PCR reactions were performed with UNI-Amp primer for 35 cycles, following the conditions of the manufacturer (Clontech). Specificity for cDNA origin of hybrid-selected products was confirmed using the UNI-Amp primer and cosmid DNA (instead of cDNA) template.

Northern and Genomic Southern Blot Analysis

Samples of human colon, small intestine, kidney, and liver were obtained through an agreement with the Cooperative Human Tissue Network (Columbus, OH). Total RNA was prepared from these tissues by guanidine isothiocyanate lysis and cesium chloride gradient ultracentrifugation procedures (26) . Polyadenylated mRNA was prepared by two rounds of oligo(dT)-cellulose affinity chromatography(26) . Five µg of polyadenylated mRNA from each tissue were fractionated through MOPS-buffered agarose gels, transferred to nylon hybridization membranes, and hybridized with radiolabeled DNA fragments, using procedures for Northern blotting described elsewhere(6, 26, 39) . After hybridization, blots were rinsed in 2 times SSC, 0.1% SDS at room temperature, were then washed for 30 min at 65 °C in 0.2 times SSC, 0.1% SDS, and were subjected to autoradiography. EcoRI-digested genomic DNA (10 µg/lane) was subjected to Southern blot analysis as described(26, 39) .

DNA Sequence Analysis

The cDNA insert in a TA cloning vector (pCRII, Invitrogen) was sequenced by the method of Sanger et al. (40) using the T7 DNA polymerase kit (Pharmacia) and M13 forward and reverse primers and 17-mer oligonucleotide primers synthesized according to the sequence of the cDNA insert. The 8.2-kb and 18.5-kb EcoRI fragments of cosmid 31553 were digested with PstI. The 1.1-kb EcoRI-PstI and 1.3-kb PstI-PstI fragments that cross-hybridized with the FUT1 cDNA probe were subcloned in Bluescript KS (Stratagene) and sequenced(16) .

Fluorescence in Situ Hybridization

Cosmids 27513 and 31553 were localized to chromosome bands by fluorescence in situ hybridization (FISH) analysis to metaphase chromosome spreads as described previously(41, 42, 43) . Two-color fluorescence in situ hybridization was performed to map these cosmids relative to each other or relative to other chromosome 19 cosmids whose localization to chromosome bands has been reported previously(44) . With this approach, markers must lie at least 1 megabase pairs apart for their order to be determined(45) . (^2)These cosmids were chosen from 19q13.2-13.3 to 19q13.3-13.4 and include 10384, 16236, 18618, 15743, 19556, 16957, 20019, and 18887(44, 46) . The physical distance between cosmids 27513 and 31553 was measured in interphase chromatin, in 200 randomly selected interphase nuclei, as an estimate of the distance between them on the linear DNA molecule (47, 48) . Chromosomal assignment of YAC clones was done using Alu-PCR products as probes(49) . Two hundred ng of each biotinylated PCR product were blocked for repetitive sequences with human Cot1 DNA (Life Technologies, Inc.) (49) and then hybridized to metaphase chromosome spreads as described above. The slides were labeled with fluorescein-conjugated detection reagents, and the YAC PCR products were mapped as described above.


RESULTS

Molecular Cloning of Two Human Genomic DNA Segments Cross-hybridizing with the H Blood Group Antigen alpha(1,2)Fucosyltransferase cDNA (FUT1)

Biochemical and genetic evidence indicates that the human genome contains two loci, termed FUT1 and FUT2, that encode distinct alpha(1,2)fucosyltransferases(4, 5, 6) . Genetic analyses indicate that these two genes are tightly linked on chromosome 19q(12, 13) . A cDNA corresponding to the FUT1 locus has recently been cloned(6, 14) . Since genetic and biochemical evidence suggest that the enzymes encoded by FUT1 and FUT2 maintain similar catalytic properties, it seemed possible that they would maintain sufficient primary sequence similarity to allow isolation of the FUT2 locus, using cross-hybridization methods and a probe derived from FUT1. Low stringency Southern blot hybridization experiments indicated that the coding region of the FUT1 cDNA detects a strongly hybridizing human DNA EcoRI restriction fragment of 6.4 kb (specific for the H gene), as well as a weakly hybridizing 8.2-kb EcoRI fragment(6) . To ascertain the nature of this cross-hybridizing fragment, and to attempt to isolate candidate sequences for the FUT2 locus, we screened two chromosome 19 cosmid libraries at low stringency with the FUT1 cDNA probe. A total of 13 hybridization-positive cosmid clones were isolated from the 8-fold redundant libraries. Southern blot analysis of an EcoRI digest of these cosmid clones allowed them to be placed into different groups, based upon their restriction patterns, and the size(s) of the fragment(s) that hybridized with the FUT1 probe. Twelve cosmids contained a 6.4-kb EcoRI fragment that hybridized strongly with the FUT1 probe, and that corresponds to the H locus (6) . The remaining cosmid(31553) contained a weakly hybridizing 8.2-kb EcoRI fragment (corresponding to the fragment detected by the FUT1 probe on human genomic DNA Southern blots, (6) ) and a second, very weakly hybridizing 18.5-kb EcoRI hybridizing fragment which was not evident on low stringency human genomic Southern blots. We chose cosmid 27513 as the reference clone containing the 6.4-kb EcoRI (FUT1) fragment. The 6.4-kb EcoRI fragment (from cosmid 27513), and the 8.2-kb and 18.5-kb EcoRI fragments (from cosmid 31553) were gel-purified and used to probe Southern blots containing EcoRI-digested human genomic DNA. As shown in Fig. 1, each probe hybridizes to a single human genomic EcoRI fragment corresponding to its own size. This result demonstrates that these three different cloned DNA fragments accurately represent their counterparts within the human genome.


Figure 1: Genomic Southern blot hybridization. EcoRI-digested human genomic DNA (10 µg/lane), prepared from blood samples, was run on an 0.8% agarose gel in 0.5 times TBE at 1 V/cm for 10 h, and subjected to Southern blot analysis (see ``Materials and Methods''), using as probes: lane 1, 6.4-kb EcoRI fragment (FUT1) from cosmid 27513; lane 2, 8.2-kb EcoRI fragment (Sec1) from cosmid 31553; lane 3, 18.5-kb EcoRI fragment (Sec2) from cosmid 31553. The sizes, indicated in kilobases at the right of the panel, were determined according to a /HindIII marker (Life Technologies, Inc.) run in parallel.



Fluorescence in Situ Hybridization Localizes the FUT1 Cross-hybridizing Sequences to 19q13.3

Given the genetic linkage data between FUT1 and FUT2, we sought to use fluorescent in situ hybridization (FISH) procedures to determine if cosmid clones 27513 and 31553 were physically linked on chromosome 19. Both cosmids localize to 19q13.3 when hybridized to metaphase chromosome spreads (Fig. 2). Cosmids 27513 and 31553 are too close to be mapped relative to each other (i.e. less than 1 megabase pair, see ``Materials and Methods''), using two-color metaphase ordering (Fig. 2). The closest flanking markers are cosmid 18618 on the proximal side and cosmid 15743 on the distal side. These have been previously FISH-mapped on q13.3 and q13.3-13.4, respectively(44) , confirming the chromosome banding results. The distance between cosmids 27513 and 31553 was also found to be too small to be measured precisely when FISH mapping to interphase nuclei was used, indicating that these two cosmids are separated by less than 100 kb (47, 48) on 19q13.3.


Figure 2: Fluorescence in situ hybridization mapping of cosmids 27513 and 31553. Two-color FISH on metaphase chromosomes of cosmid 27513 and 31553 probes using avidin-Texas red (red signals) and digoxygenin-FITC (green signals). A double-exposure photograph of probes provided localization of both signals: a 15-s exposure through a two-color filter permitted detection of Texas red and fluorescein isothiocyanate labels, followed by a short automatic exposure through a separate filter for visualization of DAPI-stained metaphase. A discrete signal is discernable on all four chromosome 19 chromatids for each cosmid. The band location of these signals (q13.3) is determined relative to 4&cjs1227;,6-diamidine-2-phenylindole dihydrochloride/actinomycin bands.



Characterization of YACs and BACs Spanning the Region Containing the FUT1 Cross-hybridizing Sequences

Based on the FISH results, we screened the ICRF YAC library and the Caltech BAC library (see ``Materials and Methods'') in an attempt to isolate one cloned DNA molecule containing FUT1 and FUT1-related sequences. SfiI inserts of cosmids 27513 and 31553 were radioactively labeled and used separately as probes to screen these two libraries.

The YAC library screening yielded a single 450-kb YAC (clone F117A10) that hybridized only with cosmid 27513. FISH and Southern blot analyses and end probe mapping studies (28) demonstrate that YAC F117A10 is chimeric on its left end (data not shown). No YACs were obtained with cosmid 31553.

Two BACs (28F10, 152C3) were isolated using cosmid 31553, while none were obtained with cosmid 27513. Both BACs contain inserts of approximately 120 kb. EcoRI digestion of these two BACs yields the same pattern, suggesting that they contain the same DNA segment. Southern blot analyses show that both BACs contain sequences that cross-hybridize at high stringency with cosmid 31553, but not with cosmid 27513. Southern blot analyses also show that cosmid 31553 identifies the 8.2-kb and 18.5-kb EcoRI fragments found in cosmid 31553, indicating that both BACs contain the two FUT1-related sequences. Only 28F10 was used for further experiments.

Construction of a Cosmid Contig and EcoRI Map of the Region Spanning FUT1 and Its Cross-hybridizing Sequences

To establish a single cosmid contig containing the region between FUT1 and the FUT1-related sequences, we screened the two cosmid libraries separately with the SfiI inserts of cosmids 27513 and 31553. Twenty-five cosmids hybridized to cosmid 27513 (including cosmid 27513 and the other 11 isolated in the original screen with the FUT1 probe). Nine cosmids hybridized to cosmid 31553. Two unique cosmids (29493 and 16240) hybridized both to cosmid 27513 and to cosmid 31553. These data, and Southern blot analyses, indicate cosmids 16240, 27355, and 29493 bridge the gap between cosmid 27513 and cosmid 31553, ordered as 27513-29493-16240-27355-31553, and yield the physical map shown in Fig. 3A. The YAC F117A10 and the BAC 28F10 have been positioned relative to the cosmid contig by hybridizing radiolabeled Alu-PCR products derived from each, to the two cosmid libraries, and to a blot containing the EcoRI-digested DNAs derived from cosmids 27513 through 31553. The YAC F117A10 overlaps cosmids 27513, 29493, 16240, and the BAC 28F10 overlaps cosmids 16240, 27355, and 31553 (Fig. 3A). Additional Southern blot analyses (see ``Materials and Methods'') yield a fine EcoRI map of a five-cosmid contig that encompasses approximately 100 kb (Fig. 3A) and support the results of the FISH analyses presented above. Additional cosmids adjacent to this contig, and overlapping it, have also been analyzed and confirm the structure of this region (data not shown).


Figure 3: Physical map of the region encompassing the FUT1 and FUT1-related sequences on 19q13.3. A, physical map of the FUT1 and FUT1-related region. The region is covered by a set of overlapping cosmids numbered and represented by solid lines. The YAC F117A10 and the BAC 28F10 are represented above the cosmid contig: the shaded lines are the portions belonging to the FUT region. The YAC dashed line indicates the chimeric portion. The boxed R indicates the right arm of the pYAC4 vector. This YAC overlaps the cosmids 27513, 29493, and 16240 and extends the cosmid contig on the left side; the BAC overlaps the cosmids 16240, 27355, and 31553 and extends the cosmid contig on the right side. The precise overlaps between the YAC, the BAC, and cosmid 16240 were not determined. The interrupted solid lines in the YAC and BAC indicate they are not drawn to scale. The partial EcoRI digest strategy of the 100-kb encompassing cosmid contig was used to order the EcoRI fragments (see ``Materials and Methods''). EcoRI sites are indicated by vertical lines. SfiI sites are positioned. The vector primer sequences (T3, T7, or Sp6) are boxed at each cosmid end. The position of FUT1 (H), Sec1, and Sec2 (boxed) are placed on the genomic map; sizes of the corresponding EcoRI fragments are indicated in kilobases. B, PstI restriction map of the 18.10-kb EcoRI fragment of cosmid 16240. The PstI restriction map was established by a partial PstI digestion. The FUT1 cDNA probe hybridizes at low stringency (see ``Materials and Methods'') the 1.3-kb fragment (Sec2). The hatched rectangle represents the 500-bp cDNA.



Assignment of the FUT1-related Sequences to Specific EcoRI Fragments within the Cosmid Contig

Fig. 3A displays the position of the H locus within the 6.4-kb EcoRI fragment of cosmid 27513 and the positions of the two FUT1-related sequences within the 8.2-kb and 18.5-kb EcoRI fragments in cosmid 31553. To determine if other FUT1-related sequences might be present within the region bounded by the cosmid contig, we prepared a Southern blot of EcoRI-digested DNA derived from the five cosmids representing this contig and probed it at low stringency using the FUT1 cDNA probe. As shown in Fig. 4, only the three previously identified cross-hybridizing EcoRI fragments are identified (the 6.4-kb fragment of FUT1, the 8.2-kb fragment, and the 18.5-kb fragment), indicating that no other FUT1-related sequences are present within this contig. (The 18.5-kb fragment is slightly truncated to 18.1 kb in cosmid 16240; see Fig. 3, A and B). After high stringency washes, both 8.2-kb and 18.5-kb signals disappeared confirming the heterologous nature of that hybridization. Because these latter EcoRI fragments are closely linked to and cross-hybridize with the FUT1 locus, they represent candidates for the human Secretor blood group locus (FUT2). We therefore termed them Sec1 (for Secretor candidate 1; 8.2-kb fragment) and Sec2 (for Secretor candidate 2; 18.5-kb fragment). The Sec1 and Sec2 EcoRI fragments are separated by 12.0 kb and are 65.5 kb and 35.0 kb apart, respectively, from the 6.4-kb EcoRI fragment corresponding to FUT1 (Fig. 3A).


Figure 4: Low stringency Southern blot analysis of the cosmid contig using the FUT1 fucosyltransferase cDNA probe. The cosmids were EcoRI-digested (1 µg) and analyzed by electrophoresis, on a 0.5% agarose gel. The gel was blotted, and the membrane was hybridized with radiolabeled FUT1 cDNA probe using low stringency wash conditions. The 8.2-kb Sec1 fragment is contained only in cosmid 31553. The 18.50-kb EcoRI Sec2 fragment is contained in cosmids 16240, 27355, and 31553. The sizes, indicated in kilobases at the right of the panel, were determined by comparison to a /HindIII and 1-kb ladder standards (Life Technologies, Inc.) run in parallel.



Direct Selection of cDNAs Derived from the Cosmid Contig

To determine if Sec1 and/or Sec2 are transcribed, we used a hybrid selection method in which a cloned genomic sequence rescues corresponding transcribed sequences from a population of cDNAs(34, 35, 36, 37, 38) . Double-stranded cDNA prepared from human fetal brain mRNA was subjected to hybrid selection (see ``Materials and Methods'') with cosmid 27513, 29493, or 31553, to represent the minimal spanning set of the contig. An intense 500-bp PCR product was obtained after the second round of selection with cosmid 31553 (Fig. 5A). This product hybridizes strongly with cosmid 31553 (Fig. 5B), suggesting that it corresponds to a transcribed segment of cosmid 31553. Discrete products were also obtained with cosmid 27513, but not with cosmid 29493 (Fig. 5A). The product derived from cosmid 27513 hybridizes to cosmid 27513 and to a FUT1 cDNA probe (data not shown), suggesting that it rescued FUT1 cDNAs from the fetal brain cDNA preparation. This sequence was not analyzed further.


Figure 5: Direct selection of cDNA using magnetic bead capture. A, gel electrophoresis pattern of selected products after first round (lanes 1, 3, and 5) and second round (lanes 2, 4, and 6) of selection-amplification, utilizing cosmids 27513 (lanes 1 and 2), 29493 (lanes 3 and 4), and 31553 (lanes 5 and 6) and using Uni-Amp primers for PCR amplification (see ``Materials and Methods''). One-fifth of the PCR reaction was loaded on each lane. B, a gel with the second round of selection-amplification (one-tenth of the PCR reaction) of cosmids 27513 (lane 1), 29493 (lane 2), and 31553 (lane 3) was transferred to a nylon membrane and hybridized with the P-labeled SfiI insert of cosmid 31553. Relative mobilities of molecular size markers (1-kb ladder (Life Technologies, Inc.)) are indicated, in kilobases, at the right of each picture.



The Cosmid 31553-selected cDNA Corresponds to the Sec2 Sequence and to a 3.35-kb Transcript in Human Intestine and Lung

The 500-bp cosmid 31553-derived hybrid selected cDNA hybridizes to the 18.5-kb EcoRI fragment (containing Sec2) on the cosmid contig (Fig. 6A) and to a single 18.5-kb EcoRI fragment on a human genomic DNA Southern blot (Fig. 6B). These results demonstrate that this 500-bp cDNA does not contain significant amounts of repetitive sequence and suggest that it corresponds to the Sec2 sequence within the 18.5-kb EcoRI fragment encompassed by cosmids 16240, 27355, and 31553 (Fig. 3A).


Figure 6: Southern blot hybridization of the hybrid selected cDNA. A, the cosmids composing the contig described in Fig. 3A were EcoRI-digested (1 µg) and analyzed by electrophoresis on a 0.5% agarose gel. The gel was blotted, and the membrane was hybridized with the Sec2-selected cDNA piece. B, EcoRI-digested human genomic DNA (10 µg/lane), prepared from blood samples, was run on a 0.8% agarose gel in 0.5 times TBE at 1 V/cm for 10 h and subjected to Southern blot analysis using the same probe. The sizes, indicated at the right of each panel, were determined according to a /HindIII marker (Life Technologies, Inc.) run in parallel.



This cDNA identifies a 3.35-kb transcript in colon and small intestine and a less abundant yet similarly sized transcript in lung (Fig. 7). Transcripts corresponding to this cDNA were not detected in kidney or liver. Since the Secretor locus determines expression of H blood group determinants in intestinal, colonic, and pulmonary epithelia, but not in hepatocytes or in the kidney(4) , these results are consistent with the possibility that this cDNA and its transcribed locus within Sec2 correspond to the human Secretor, or FUT2, locus.


Figure 7: Northern blot analysis of human mRNA. Northern blots were prepared with 5 µg of polyadenylated mRNA isolated from human tissues (colon, lane 1; small intestine, lane 2; lung, lane 3; liver, lane 4; kidney, lane 5) (see ``Materials and Methods''). Blots were probed with the radiolabeled Sec2 cDNA isolated by hybrid selection using cosmid 31553 (see text and (16) ), using hybridization and washing conditions described under ``Materials and Methods'' and in (39) . The migration positions of RNA molecular size standards, in kilobases, are indicated at the left.



The sequence of this cDNA and corresponding genomic DNA (16) indicates that it is within the Sec2 sequence in the 18.5-kb EcoRI fragment (Fig. 3A), lies 3` to the termination codon of a novel human alpha(1,2)fucosyltransferase gene characterized in the accompanying paper (16) and thus represents the 3`-untranslated region of this locus.

Thus, the sequence of the hybrid selected cDNA, and its genomic counterpart, unambiguously assign its cognate transcript to the human Secretor blood group alpha(1,2)fucosyltransferase locus described in the accompanying article(16) . When considered together with previously published genetic data linking the FUT1 and FUT2 loci, the chromosomal localization and mapping data presented here provide a molecular confirmation of a hypothesis that the H and Secretor blood group loci represent closely linked, structurally similar alpha(1,2)fucosyltransferase genes with distinct tissue-specific expression patterns.


DISCUSSION

Genetic and biochemical evidence indicates that the human genome encodes at least two alpha(1,2)fucosyltransferase activities thought to represent the products of two different loci (H or FUT1 and Se or FUT2) closely linked on chromosome 19(4, 5, 6, 12) . A third distinct alpha(1,2)fucosyltransferase activity may also be expressed by human cells(50) . To date, only the FUT1 fucosyltransferase gene has been cloned(6, 14) . All known human alpha(1,2)fucosyltransferase activities can utilize structurally identical substrates, suggesting that they maintain substantial amounts of primary sequence similarity. We therefore sought to isolate additional alpha(1,2)fucosyltransferase genes, including perhaps the FUT2 locus, by cross-hybridization with the FUT1 cDNA. We were encouraged by the success of similar approaches that have allowed the isolation of a family of alpha(1,3)fucosyltransferase genes (51, 52, 53) and by our observation that the FUT1 cDNA cross-hybridizes with an 8.2-kb EcoRI human DNA restriction fragment.

A low stringency hybridization screen of two chromosome 19 specific cosmid libraries with the FUT1 cDNA yielded one cosmid clone (31553) containing the 8.2-kb EcoRI fragment. We have termed the cross-hybridizing sequence in this fragment Sec1. We also found that cosmid 31553 contains a second, distinct cross-hybridizing sequence that we have termed Sec2. This sequence is found on an 18.5-kb EcoRI fragment. This 18.5-kb EcoRI fragment was not detectable on human genomic Southern blots probed at low stringency with the FUT1 cDNA.

To determine if either of these two sequences were transcribed, we used a hybrid selection strategy (34, 35, 36, 37, 38) capable of isolating cDNAs corresponding to nonabundant transcripts emanating from genes whose boundaries fall within cosmid 31553. This approach yielded an 500-bp cDNA that hybridizes only with the Sec2-containing 18.5-kb EcoRI fragment. Northern blot analyses demonstrate that this cDNA detects mRNA transcripts in small intestine, colon, and lung, but not in liver or kidney. Sequence analysis of this cDNA, and of the corresponding genomic DNA region of Sec2, indicates that this cDNA is derived from the 3`-untranslated region of a novel human alpha(1,2)fucosyltransferase gene. Since previous observations indicate that the Secretor alpha(1,2)fucosyltransferase gene is expressed in secretory epithelia(5) , these results are consistent with the possibility that Sec2 corresponds to the human Secretor (FUT2) blood group locus. Data in the accompanying paper (16) confirm this notion, by demonstrating that Sec2 encodes an alpha(1,2)fucosyltransferase activity with catalytic properties virtually identical with that ascribed to the human Secretor alpha(1,2)fucosyltransferase.

By contrast, we have been unable to detect transcripts corresponding to Sec1, and we did not isolate any cDNAs corresponding to this sequence by the hybrid selection approach. These observations suggest that Sec1 might represent a pseudogene. This conclusion is supported by DNA sequence data reported for Sec1 in the accompanying manuscript(16) .

We have used a physical mapping strategy to position the FUT1 (H) gene and the two FUT1-related sequences along a 100-kb region of human chromosome 19. This map indicates that FUT1 and the Sec2 locus are separated by a distance of roughly 35 kb. Since previous linkage analyses (4, 5, 6, 12) have established that the H and Secretor loci are closely linked on 19q, the close physical proximity of the FUT1 and Sec2 loci is also consistent with the studies here, and in the accompanying paper(16) , that assign Sec2 to the Secretor locus.

The positions of these sequences is also consistent with biochemical studies suggesting that the H locus arose via a gene duplication from an ancestral Secretor-type alpha(1,2)fucosyltransferase gene(5) . This situation is analogous to that observed for members of human alpha(1, 3)fucosyltransferase genes, that cluster on the short arm of chromosome 19(51, 52, 53, 54, 55) . These observations suggest the possibility that additional alpha(1,2)fucosyltransferase genes, including perhaps one corresponding to a third distinct alpha(1,2)fucosyltransferase activity(50) , might exist in close physical linkage to the FUT1 and Sec2 loci. Efforts to isolate such sequences may be assisted by our isolation of additional cosmids positioned beyond the boundaries of the cosmid contig described here (data not shown), through our use of YAC and BAC cloning and mapping strategies.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant 1R01HL48859 and was performed under the auspices of the U.S. Department of Energy at Lawrence Livermore National Laboratory under Contract No. W-7405-ENG-48. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Fellowship ARC/ATIPE from the CNRS. Present address: CRBM, CNRS UPR 9008, B.P. 5051, 1919 Route de Mende, 34033 Montpellier Cedex, France.

Associate Investigator of the Howard Hughes Medical Institute.

**
Co-equal senior authors.

§§
To whom correspondence and reprint requests should be addressed: Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94550. Tel.: 510-422-5711; Fax: 510-422-2282.

¶¶
Full-time investigator of the CNRS. Present address: CRBM, CNRS UPR 9008, B.P. 5051, 1919 Route de Mende, 34033 Montpellier Cedex, France.

(^1)
The abbreviations used are: alpha(1,2)fucosyltransferase, GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase; alpha(1,3)fucosyltransferase, GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-fucosyltransferase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); MOPS, 4-morpholinepropanesulfonic acid; FISH, fluorescence in situ hybridization.

(^2)
B. Trask, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Hans Lehrach from the ICRF in London for providing the YAC library and Drs. Melvin Simon and Hiroaki Shizuya from California Institute of Technology (Pasadena, CA) for the BAC library. We acknowledge Anne Bergmann for technical help in FISH experiments.


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