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
(1,2)fucosyltransferase (
)that catalyzes the synthesis
of the H determinant via a transglycosylation reaction that transfers L-fucose from the substrate GDP-fucose to the
-D-galactose residue at the nonreducing terminus of
glycoconjugates to form H-active Fuc
(1,2)Gal-
moieties. Genetic and biochemical studies indicate that the human
genome encodes at least one other distinct
(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
(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
(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
(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
(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
(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
(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
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
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
SSC, 0.1% SDS
at room temperature; once in 2
SSC, 0.1% SDS at 65 °C (30
min); and once in 0.1
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
SSC, 0.1% SDS at room
temperature, followed by one 30-min wash in 2
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
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
SSC, 5
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
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
SSC, 0.1% SDS at room
temperature, were then washed for 30 min at 65 °C in 0.2
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) . (
)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
(1,2)Fucosyltransferase cDNA (FUT1)
Biochemical and genetic
evidence indicates that the human genome contains two loci, termed FUT1 and FUT2, that encode distinct
(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
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
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
(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
(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
(1,2)fucosyltransferase genes
with distinct tissue-specific expression patterns.
DISCUSSION
Genetic and biochemical evidence indicates that the human
genome encodes at least two
(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
(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
(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
(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
(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
(1,2)fucosyltransferase gene. Since previous observations
indicate that the Secretor
(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
(1,2)fucosyltransferase activity with
catalytic properties virtually identical with that ascribed to the
human Secretor
(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
(1,2)fucosyltransferase gene(5) . This situation is
analogous to that observed for members of human
(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
(1,2)fucosyltransferase genes, including perhaps one corresponding
to a third distinct
(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.