(Received for publication, April 14, 1995; and in revised form, May 24, 1995)
From the
The human (1,3)-fucosyltransferase genes FUT3, FUT5, and
FUT6 form a cluster on chromosome 19p13.3. Expression was studied using
reverse transcriptase-polymerase chain reaction, rapid amplification of
cDNA ends, and Northern analyses. FUT3 and FUT6 were expressed at high
levels, while FUT5 expression was lower and restricted to fewer cell
types. Alternatively spliced transcripts were identified for FUT3 and
FUT6 in kidney, liver, and colon. A 2.37-kilobase pair (kb) FUT3
transcript, detected at high levels in kidney and colon, was absent in
liver. FUT6 expression was characterized by a 3.5-kb transcript present
in kidney and liver, and a 2.5-kb transcript in colon and liver. Two
polyadenylation sites were shown for FUT5, but absence of consensus
sequences suggests reduced efficiency for cleavage and polyadenylation.
Two polyadenylation sites were also shown for FUT6, with the
alternatively spliced downstream signal in tissues expressing high
levels of FUT6. In these tissues, additional splicing results in
isoforms with catalytic domain deletions. No detectable
(1,3)- or
(1,4)-fucosyltransferase activity was found in assays of cells
transfected with FUT6 isoform cDNAs. Thus, tissue-specific
post-transcriptional modifications are associated with expression
patterns of FUT3, FUT5, and FUT6.
(1,3)- and
(1,4)-fucosylated
(2,3)-sialylated
lactosaminoglycans are components of ligands for the cell adhesion
receptors E-selectin and P-selectin(1) . Cell surface
expression of sialyl Lewis x, sialyl Lewis a, and related fucosylated
antigens varies during development and malignant
transformation(2, 3, 4, 5, 6, 7) .
Enzymatic data imply that corresponding
(1,3)- and
(1,4)-fucosyltransferase activities also change during these
dynamic processes(8, 9, 10) . Recent studies,
for example, have examined expression of sialyl Lewis x and
(1,3)-fucosyltransferase activities during hepatic
inflammation(11, 12) . Transition from an
``embryonic''
(1,3)-fucosyltransferase activity to
``adult'' forms (Lewis and plasma type) during renal
development has also been recently described(13) .
Most
non-hematopoietic malignancies in adults involve malignant
transformation of epithelial cells of a particular organ. One example
of these highly prevalent diseases is adenocarcinoma of the colon,
where studies have correlated metastasis and invasion with tumor cell
expression of fucosylated lactosaminoglycans(14, 15) .
Interpretation of these results and the underlying mechanisms involved
is difficult due to the complex and changing array of (1,3)- and
(1,4)-fucosyltransferase activities found in normal epithelial
cells of gastrointestinal, genitourinary, and gonadal
organs(16) . In general, Lewis and plasma fucosyltransferase
activities have been highest in normally differentiated epithelium, but
it is not known how expression of
(1,3)-fucosyltransferases is
controlled.
Molecular cloning studies to date have resulted in the
isolation of five human genes encoding
(1,3)-fucosyltransferases(17, 18, 19, 20, 21, 22, 23, 24, 25) .
The
(1,3)-fucosyltransferase genes FUT3 (encoding the Lewis
(1,3/1,4)-fucosyltransferase), FUT5 (an unspecified type of
(1,3)-fucosyltransferase), and FUT6 (the plasma
(1,3)-fucosyltransferase) form a cluster on human chromosome
19p13.3 (26) . It has been postulated that these tandemly
arranged homologues are regulated in a manner analogous to
developmental switching of globin genes(27) . Surveys of human
leukemia and epithelial carcinoma cell lines suggest that FUT3 and FUT6
transcripts are expressed at high levels in transformed
epithelia(24, 28) . Given these data, we sought to
study expression of FUT3, FUT5, and FUT6 at the transcript level in
normally differentiated human tissues. Our analyses show that
(1,3)-fucosyltransferase expression patterns in kidney, liver, and
colon depend, at least in part, on differential processing of FUT
transcripts.
Figure 5: Schematic and sequence of alternatively spliced and polyadenylated FUT6 transcripts. A, genomic structure and position of exons A-G are shown in the top line of the figure. All transcript structures are confirmed by standard RT-PCR, extended length RT-PCR, 5`-RACE, and 3`-RACE (``Experimental Procedures''). Primers used for 5`-RACE and RT-PCR experiments are shown (U1, U2, L1, and L2 sequences are in Table 2). Major transcripts are detectable on Northern analyses and in at least three cDNA clones obtained by hybridization (Major I, six clones; Major II, five clones; Minor I, two clones; Minor II, two clones; Minor III, one clone). The 3.5-kb FUT6 transcript visible on Northern blot analyses (e.g. kidney and liver) is mostly comprised of the Major I species. The 2.5-kb transcript (e.g. colon and liver) corresponds to Major II. B, sequences of exons A-G. Splice consensus sequences (intronic) are in lower case. Dotted underlining in exon G denotes highly repetitive sequences similar to human Alu motifs (61). Start and stop codons are shown in bold type. The coding region is as previously published(22) . 3`-RACE experiments and genomic sequence comparisons confirm use of two polyadenylation signal sequences (proximal, underlined; distal, double-underlined) and two downstream cleavage sites (TA&cjs1219; and CA&cjs1219;, respectively).
Figure 7: FUT6 isoform schematic, sequence, and expression. A, schematic representation of FUT6 isoforms. Numbers in parentheses refer to previously published codon numbers(22) . Relative to FUT6 wild type sequence, splicing within the coding region results in the loss of 12 (Isoform I) and 82 (Isoform II) carboxyl-terminal amino acid residues. Consensus sequences for both splice events are shown. Asterisks refer to the stop codon present in the open reading frame of the isoforms. Restriction enzyme sites used to create isoform expression constructs are noted (``Experimental Procedures''). B, amino acid and nucleotide sequence of FUT6 isoforms. Numbers and residues in parentheses refer to previously published FUT6 sequence (22) . The italicized subsequence in Isoform II is identical to the sequence of Isoform I. C, RT-PCR was performed as described under ``Experimental Procedures'' using primers noted in the schematic. cDNAs were generated with L1 (from exon G) and amplified with U/L3. For colon, a second cDNA synthesis was performed with a lower strand primer from exon F (L2), followed by the same PCR amplification with U/L3. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. The amplified fragments are 1140 bp (wild type, highest levels), 1045 bp (isoform I), and 865 bp (isoform II). Isoform I is expressed at higher levels than isoform II (>10-fold) in kidney (K). Liver (L) shows lower expression of both isoforms than kidney. For colon (C), amplifiable levels of cDNA are not generated with the L1 primer from exon G (no wild type or isoform fragments in first C lane). When colon cDNAs are synthesized with the exon F lower strand primer (L2), high levels of 1140-bp wild type fragment are amplified, but no isoform transcripts are detectable (second C lane).
Figure 1:
Northern blot analyses of human tissues
using coding region probes. 20 µg of poly(A) RNA
was prepared from a single pathologically reviewed specimen for each
tissue type, divided into 5 µg/lane, electrophoresed, and
transferred to a single membrane. Each resulting blot was then
subdivided into strips, and hybridized with a
P-labeled
coding region probe specific for FUT3, FUT5, FUT6, or
-actin
(``Experimental Procedures''). Autoradiographic films were
exposed for 10-14 days (except for
-actin lanes, A,
which were exposed for 12-15 h). Numbers refer to RNA size in kb.
The major transcript size noted for FUT3 (lane 3) was 2.37 kb.
For FUT6 (lane 6), 2.5- and 3.5-kb transcripts are noted. No
hybridizing signal was detected for FUT5 in the tissues studied (data
not shown).
Figure 2:
PCR analyses of human tissues and cells
using coding region primers. 0.2 µg of poly(A) RNA
was reverse-transcribed with a common lower strand primer (except
-actin; see Table 1) and used for PCR analyses as described
under ``Experimental Procedures.'' FUT3 and FUT6
amplifications were performed for 30 cycles, FUT5 for 35 cycles, and
-actin for 25 cycles. Control amplifications with no reverse
transcriptase are not shown. PCR products were electrophoresed in 1.2%
agarose gels and stained with ethidium bromide. Numbers refer to sizes
of amplified fragments (bp). Lane 1, kidney; lane 2,
liver; lane 3, lung; lane 4, colon; lane 5,
small intestine; lane 6, stomach; lane 7, T
lymphocytes; lane 8, bladder; lane 9, uterus; lane 10, ovary; lane 11, testicle; lane 12,
salivary gland; lane 13, brain; lane 14, HL-60
cells.
High levels of FUT3 transcript were found in colon, stomach, small intestine, lung, and kidney (Fig. 1). Lesser amounts were seen in salivary gland, bladder, uterus, and liver (Fig. 2). The predominant FUT3 transcript present in most tissues is 2.37 kb. FUT5 transcripts were not detectable by hybridization techniques (data not shown). By RT-PCR, FUT5 expression was restricted in quantity and distribution to liver, colon, and testicle (Fig. 2). Trace amounts of FUT5 message were also detected in unselected T lymphocytes, HL-60 cells, and brain. For FUT6, a 3.5-kb transcript was seen in kidney, liver, and small intestine; a 2.5-kb transcript was present in liver, colon, salivary gland, bladder, and uterus (Fig. 1). Because of the tissue-specific differences in FUT3 and FUT6 transcripts shown here, kidney, liver, and colon were chosen for further study.
Figure 3: Schematic, sequence, and expression of alternatively spliced FUT3 transcripts. A, genomic structure and organization of exons A-C are shown in the top line of the figure. Splice acceptor sequences for Major transcripts I and II are denoted B" and B`, respectively (italicized). All transcript structures were confirmed by RT-PCR, 5`-RACE, and 3`-RACE (``Experimental Procedures''). Primers used for 5`-RACE and RT-PCR experiments are shown (U, L1, L2 sequences are in Table 2). For this figure, major transcripts are defined as those present on Northern analyses and in at least two cDNA clones obtained by cross-hybridization (``Experimental Procedures''; Major I, two clones; Major II, four clones; Minor I, one clone; Minor II, one clone). No alternative splicing or polyadenylation events were found in the 3`-UT region of FUT3. The previously published cDNA for FUT3 (17) is depicted as Minor II. The 2.37-kb transcript on Northern analyses is mostly comprised of the Major II species. FUT3 Minor I (with exon B) is found in liver. B, sequences of exons A-C. Positions of B` and B" are shown (/). Intronic splice consensus sequences are in lower case. The start codon is shown in bold type. The mono-exonic coding region is as previously published(17) . 3`-RACE experiments and genomic sequence comparison confirm the polyadenylation signal sequence (underlined) followed by the cleavage site (&cjs1219;) 19 nucleotides downstream (CA, differs from previously published GA, (17) ). C, RT-PCR analyses (``Experimental Procedures'') were performed using lower strand primers from the FUT3 coding region and an upper strand primer from exon A (Table 2). PCR products were electrophoresed in a 1% agarose gel and stained with ethidium bromide. Fragments corresponding to Major I (545 bp), Major II (599 bp), and Minor I (948 bp) are seen in kidney (lane K). In liver, only the Minor I transcript is amplified (lane L). In colon (lane C), the 599-bp fragment (corresponding to Major II) is found at highest levels. Minor transcript II is found at lower levels in colon (409 bp). An additional faint 635-bp fragment, amplified at trace levels in colon, can also be seen in lane C. This minor species was not detectable in most specimens and is not depicted in A.
The 5` exons in Fig. 3were mapped by 5`-RACE and genomic sequence analyses (data not shown). All FUT3 transcripts were shown to start with Exon A. Preferential selection of the 3` splice acceptor for exon B` (Fig. 3) corresponds to the length of the polypyrimidine tract preceding it and correlates with high levels of the 2.37-kb message (e.g. colon, Fig. 1). No alternative splicing or polyadenylation was found by RACE analyses of the 3`-UT region of FUT3 in these tissues. A minor 3`-UT sequence difference with the A431 transcript (17) was noted, with the major transcript polyadenylation cleavage site CA predominating in normal tissues (Fig. 3). G/T clusters are present in genomic sequence immediately downstream of this site (data not shown).
Figure 4: Schematic and sequence of FUT5 transcripts. A, genomic structure and position of exons A and B are shown in the first schematic. Transcript structures were confirmed by RT-PCR, 5`-RACE, and 3`-RACE (``Experimental Procedures''). Primers used for 3`-RACE and RT-PCR experiments are shown (U1, U2, L1, L2 sequences are in Table 3). FUT5 transcripts were not detectable by Northern blot analyses but were isolated by cross-hybridization screenings (``Experimental Procedures''; Minor I, one clone; Minor II, one clone). Minor transcript I was present at higher levels than Minor transcript II in all PCR assays (>10-fold, data not shown). No alternative splicing events were found in the 5`-UT or 3`-UT regions of FUT5. B, sequences of exons A and B. Splice consensus sequences (intronic) are in lower case. The start and stop codons are shown in bold type. The mono-exonic coding region is as previously published(21) . 3`-RACE experiments and genomic sequence comparisons confirm that the 3`-UT is colinear with genomic sequence until nucleotide 1723. Cleavage and polyadenylation are directed by two previously unpublished signal sequences (underlined). Terminal GA and CA sequences (respectively) are depicted (&cjs1219;). The proximal cleavage and polyadenylation events are found in most liver and colon transcripts.
Figure 6:
Northern blot analysis using FUT6 5`-UT
region exonic probes. 15 µg of poly(A) RNA was
prepared from a single pathologically reviewed specimen for each tissue
type, divided into 5 µg/lane, electrophoresed, and transferred to a
single membrane. Each resulting blot was then divided into three
strips, hybridized with
P-labeled 5`-UT probes, and washed
at high stringency (``Experimental Procedures'').
Autoradiographic films were exposed for 10 days, except for
-actin
lanes, which were for 12 h. Probe 2, containing sequence from exon C
(bp 1-330), recognizes both major transcripts (3.5 and 2.5 kb),
while probe 1, which contains exon A sequence (bp 1-285),
hybridizes only with the 3.5-kb transcript. Kidney (lane K)
contains only the 3.5-kb transcript, liver (lane L) contains
both, and colon (lane C), the 2.5-kb
species.
In the FUT6 3`-UT region, alternative splicing of two exons containing disparate polyadenylation sites is shown. The proximal signal, ATTAAA, which is reported to occur in 12% of mRNAs compiled from many species(32) , reduces 3` maturation to 4% of control in comparative studies(33) . This low efficiency signal is responsible for polyadenylation of 3`-UT sequence that is contiguous with the coding region (Fig. 5). Cleavage/polyadenylation occurs 12 bp downstream of this signal at a TA terminus. The distal site contains two overlapping consensus signal sequences for polyadenylation, AATAAA, but exists on a separate 245-bp exon (Fig. 5, exon G). Here, cleavage/polyadenylation occurs 21 bp downstream of the poly(A) signal at a CA terminus. Both signals are flanked by G/T-clusters (data not shown). The proximal, less efficient polyadenylation event (e.g. colon) and the exon G splice event (bringing in the distal, more efficient signal, e.g. kidney) appear to be competitive post-transcriptional modifications(34, 35, 36) . In liver, these disparate events occur with equal frequency (Fig. 6).
Immunohistologic and biochemical surveys of non-diseased
human organs containing a large proportion of epithelial cells suggest
that at least two (1,3)-fucosyltransferase genes are expressed at
high levels (FUT3, corresponding to Lewis
(1,3/1,4)-fucosyltransferase activity and FUT6, corresponding to
plasma
(1,3)-fucosyltransferase activity). Coexistence of other
fucosyltransferase activities is frequently noted, however(9) .
Molecular analyses have become useful in defining the relative roles of
these enzymes.
Transcript surveys of leukemia and epithelial
carcinoma cell lines (28) have shown the presence of multiple
fucosyltransferase transcripts in a given line, with highest expression
levels of FUT3 and FUT6 transcripts in transformed epithelia, and much
lower levels of these messages in leukemia cells. Conversely, the
(1,3)-fucosyltransferases FUT4 and Fuc-TVII are expressed at high
levels in normal and transformed leukocytes(24) . FUT5, which
is highly homologous to FUT3 and FUT6 (21, 22) and
part of the 19p13.3 locus(26) , appears to have lower levels of
expression in both epithelial and hematopoietic cell lines. FUT4, which
appears to be expressed earlier in development(27) , is less
homologous and non-syntenic(26) . When considered together,
this genomic organization and expression pattern suggest that control
at transcriptional and/or post-transcriptional levels may underlie
developmental and tissue-specific fucosyltransferase expression
patterns.
We studied expression of the 19p13.3
(1,3)-fucosyltransferase genes in normal human tissues to find
underlying molecular determinants for this specificity. Our results
correlate transcript levels with the high FUT3 and FUT6 enzyme
activities observed in epithelial tissues of specific types, confirm
lower overall expression levels of FUT5, and suggest that RNA
processing serves as a point of control for
(1,3)-fucosyltransferase expression, particularly for FUT6. Large
differences in mature transcript structure were found in full-length
kidney, liver, and colon cDNAs.
In kidney, high levels of Lewis x and sialyl Lewis x are found on glomerular podocytes and proximal tubular cells(27, 37) . Transitional epithelia of the calyx, ureter, and bladder appear to have lower levels of fucosylated glycan expression. The functional significance of this distribution is not known. Our results are consistent with high renal parenchymal expression of sialyl Lewis x and confirm that FUT3 and FUT6 are responsible for this biosynthesis. Our results also correlate with enzymatic data showing fully differentiated kidney to possess high levels of Lewis (calyceal) and plasma (tubular) fucosyltransferase activities(13, 27) , with developing kidney having a myeloid-like pattern. In situ hybridization with FUT3 and FUT6 riboprobes composed of flanking exons will be required to verify this distribution.
Normal human liver tissue contains both plasma and
Lewis fucosyltransferase
activities(9, 12, 38) . Previous studies have
correlated increased (1,3/1,4)-fucosyltransferase activity with
bile duct proliferation, while
(1,3)-fucosyltransferase activity
was markedly increased in hepatic parenchymal
injury(12, 39) . Enzymatic assays performed on
cellular fractions suggest that hepatocytes are the source of
approximately 75% of
(1,3)-fucosyltransferase activity and 25% of
(1,3/1,4)-fucosyltransferase activity detected in whole tissue
extracts(38) . Normal hepatocytes, however, do not express high
levels of sialyl Lewis x on their surface(11, 40) .
Increased levels of surface-expressed sialyl Lewis x have been shown in
hepatic necro-inflammatory lesions and hepatocellular
carcinoma(11, 40) . High level expression of both the
2.5- and 3.5-kb FUT6 transcripts are found in biopsy specimens with
hepatocellular inflammation (
)and in transformed
hepatocytes(28) . It is not yet clear, however, whether changes
in FUT6 message levels underlie these enzymatic and immunohistochemical
observations.
In contrast, normal colon contains high levels of the
FUT6 2.5-kb transcript and virtually no 3.5-kb transcript. Other
gastrointestinal mucosa do not contain high amounts of FUT6 message,
but instead show strong expression of the FUT3 2.37-kb transcript (e.g. stomach, Fig. 1). Our results correlate with
recent enzymatic analyses of plasma and Lewis fucosyltransferase
activities in benign and malignant gastrointestinal
mucosa(41) . Normal stomach mucosa shows low
(1,3)-fucosyltransferase and high
(1,4)-fucosyltransferase
activity, while normal colon mucosa has high
(1,3)-fucosyltransferase and moderate
(1,4)-fucosyltransferase activity. FUT3 and FUT6 co-expression is
not shown by these enzymatic data nor by the analysis of transcript
levels presented here. Of note, FUT6 2.5-kb transcripts may appear as a
composite signal with 2.37-kb FUT3 transcripts when probes containing
catalytic domain sequence are used(17, 28) . Upstream
probes described in this report allow separation of these messages (Fig. 6).
Another human tissue expressing high levels of FUT6
2.5-kb message was salivary gland. The high level of expression of FUT6 versus FUT3 transcripts was unexpected, given the
concentration of Lewis enzyme activity in
saliva(42, 43) , but was also highly reproducible (n = 8 specimens). Enzymatic assays of salivary gland
and other epithelial tissue extracts do not necessarily correlate with
(1,3/1,4)-fucosyltransferase activity purified from saliva nor
with Lewis blood
type(43, 44, 45, 46, 47) .
Possible explanations for these findings include differential splicing
of FUT3 and/or FUT6 transcripts resulting in species not characterized
or the presence of additional, as yet undescribed enzyme(s) capable of
(1,4)-fucosylation(48) . None of the alternatively spliced
FUT6 transcripts isolated to date were found to direct
(1,4)-fucosyltransferase activity when tested in transfection
assays (Table 4). In addition, our RT-PCR screening assays (Fig. 2) used primers spanning most of the coding region for all
three 19p genes, making additional splice events an unlikely
explanation.
FUT3 and FUT6 undergo extensive post-transcriptional
processing of the 5`-UT region. While their coding regions are highly
homologous(22) , the 5`-UT regions share no homology, are
expressed in a tissue-specific manner, and have different
organizations. Similar splicing complexity restricted to the 5`-UT
region has been described in other human gene families. Diverse mRNAs
encoding tryptophan hydroxylase, for example, are generated from a
single promoter followed by extensive splicing upstream of the
translation start site(49) . FUT3 5`-UT exonic organization (Fig. 3) and upstream genomic sequence are also consistent with
alternative splicing following transcription initiation from a single
site. All FUT3 transcripts start with exon A, i.e. they are alternatively spliced forms originating
from one pre-mRNA species. Exon A ends in a canonical splice donor
sequence, while relative use of 3` splice acceptors within exon B
likely depends on competition(50) . Frequency of 3` splice
acceptor selection approximately corresponds to the length of
polypyrimidine tract and presence of AG in the intronic sequence
preceding the exon(51, 52) . As seen in Fig. 3,
potential splice acceptor competition exists between sites B, B`, and
B" (which are all preceded by AG), but sequence preceding B` has the
longest uninterrupted polypyrimidine tract. This model is consistent
with the predominance of Major II FUT3 transcripts reported here. The
2.72-kb Minor I transcript in liver suggests that tissue-specific
trans-acting factors also influence site selection and/or message
stability.
In the more complex case of FUT6, we cannot presently
rule out transcription initiation from alternative promoters followed
by differential splicing of intervening sequences in the 5`-UT region.
Particularly suggestive of this organization is the presence of exon A
only in the 3.5-kb transcript, exon C in both the 3.5- and 2.5-kb
transcripts, and the apparent absence of a common upstream exon in
full-length cDNAs(53) . Exon B, which does not have strong
splice consensus sequences(51) , is only rarely
skipped(50) , and contains putative initiation site(s) for the
2.5-kb transcript. In contrast, exon C has a very strong
splice donor site and can be followed by the differentially spliced
exons D or E, likely on the basis of the 3` splice acceptor competition
model(51) . In general, mRNAs that maintain such 5`-UT
diversity and length are transcribed from alternative promoters whose
activation may be associated with tissue and developmental stage
specificity (e.g. human insulin-like growth factor
II, (54) ). For FUT6, functional testing of putative promoter
regions will be required to definitively map transcriptional sites.
Despite its highly homologous open reading frame (which encodes a catalytically active enzyme in vitro, (21) and (55) ), low expression levels of FUT5 were seen in differentiated tissues. Colon and liver were found to contain modest but reproducible amounts of FUT5 message, thus allowing isolation of flanking sequences by RACE, PCR cloning, and cross-hybridization methods. Possible explanations for low FUT5 expression include order-dependent transcriptional switching during tissue differentiation(56) , differential splice events at temporally specific developmental stages(54) , lack of message transport from nucleus to cytoplasm(32) , and/or lack of stable message due to degradation(32) . Given the lack of consensus polyadenylation signal sequences in FUT5 mRNAs, the 10-fold excess of the proximally cleaved species, and the low levels of FUT5 message in transformed cells, post-transcriptional transport, and/or degradation mechanisms seem most likely. Site-directed mutagenesis of FUT5 polyadenylation signal sequences (33) and determining the actual rate of FUT5 mRNA synthesis at various stages of development will be required to address this hypothesis.
FUT6 also uses alternative polyadenylation signals, but unlike FUT5, undergoes a pivotal 3`-UT splice event. The exclusive use of the proximal, ``low efficiency'' signal ATTAAA (33) in colon, which has high levels of 2.5-kb FUT6 message, is in sharp contrast to the predominant use of two overlapping AATAAA sites in kidney 3.5-kb transcripts, which contain exon G. 3`-RACE experiments show that liver uses both sequences equally. These data are consistent with a competition model of 3` alternative splicing and polyadenylation (34, 35, 36) , which suggests that in the case of FUT6, the fate of the RNA precursor is mainly determined by competition between the exon G splice event and cleavage/polyadenylation at the proximal signal sequence (36) . Tissue-specific factors influencing post-transcriptional ``choice'' of these two species have not been identified, and it is not known what the actual FUT6 transcription rates are preceding these events in each tissue. Functional analyses will be required to compare the effects of these 3` end modifications(34) .
One notable effect of the exon G splice event is that additional tissue-specific splice events can occur, resulting in isoforms of FUT6 which are catalytically inactive in assays with oligosaccharide acceptors. Sequence comparisons of these full-length transcripts (particularly in the putative stem regions(22) , and upstream exons (this work)) are not consistent with the presence of another highly homologous fucosyltransferase gene or pseudogene. Based on previously defined mutations in FUT6(57) , the particular carboxyl-terminal regions deleted would indeed be expected to result in loss of function (i.e. no detectable fucosyltransferase activity). Single amino acid changes in carboxyl-terminal residues have been shown to have profound effects on fucosyltransferase synthetic capability, while upstream coding region changes appear to have less effect(43, 57) . It is not yet known, however, whether complex carbohydrate acceptors can be fucosylated by FUT6 isoforms.
It is also not clear whether use of
exon G is part of a concerted regulatory process or merely an event
associated with prolonged half-life of FUT6 precursor transcripts. The
tissue specificity of this splice event suggests down-regulation.
On/off gene regulation at the level of splicing has been estimated to
occur in at least 5% of regulatory ``decisions'' in Drosophila(58) , but its frequency in other species is
unknown. Detection of such non-functional mRNA species is frequently
hampered by size and stability of the alternatively spliced
species(58) , but that does not seem to be the case for FUT6,
since the catalytic domain splice events occur only when the consensus
polyadenylation signal in exon G is used. While some examples of this
form of regulation involve a single promoter directing constitutive
expression of a primary transcript, many other isoform families are
generated in the context of tissue specificity(59) ,
alternative promoter use(53) , and diverse splicing
options(60) . Our data for FUT6 show that isoform species are
another post-transcriptional modification associated with
tissue-specific (1,3)-fucosyltransferase gene expression.
DNA sequences have been deposited in GenBank: FUT3 Major I, U27326[GenBank]; FUT3 Major II, U27327[GenBank]; FUT3 Minor I, U27328[GenBank]; FUT5 Minor I, U27329[GenBank]; FUT5 Minor II, U27330[GenBank]; FUT6 Isoform I, U27331[GenBank]; FUT6 Isoform II, U27332[GenBank]; FUT6 Major I, U27333[GenBank]; FUT6 Major II, U27334[GenBank]; FUT6 Minor I, U27335[GenBank]; FUT6 Minor II, U27336[GenBank]; FUT6 Minor III, U27337[GenBank].