©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Human Chromosome 19p (1,3)-Fucosyltransferase Genes in Normal Tissues
ALTERNATIVE SPLICING, POLYADENYLATION, AND ISOFORMS (*)

(Received for publication, April 14, 1995; and in revised form, May 24, 1995)

H. Scott Cameron (1)(§) Dorota Szczepaniak (1)(§) Brent W. Weston (1) (2) (3)(¶)

From the  (1)Department of Pediatrics, Division of Pediatric Hematology/Oncology, the (2)Lineberger Comprehensive Cancer Center, and the (3)Center for Thrombosis and Hemostasis, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human alpha(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 alpha(1,3)- or alpha(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.


INTRODUCTION

alpha(1,3)- and alpha(1,4)-fucosylated alpha(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 alpha(1,3)- and alpha(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 alpha(1,3)-fucosyltransferase activities during hepatic inflammation(11, 12) . Transition from an ``embryonic'' alpha(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 alpha(1,3)- and alpha(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 alpha(1,3)-fucosyltransferases is controlled.

Molecular cloning studies to date have resulted in the isolation of five human genes encoding alpha(1,3)-fucosyltransferases(17, 18, 19, 20, 21, 22, 23, 24, 25) . The alpha(1,3)-fucosyltransferase genes FUT3 (encoding the Lewis alpha(1,3/1,4)-fucosyltransferase), FUT5 (an unspecified type of alpha(1,3)-fucosyltransferase), and FUT6 (the plasma alpha(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 alpha(1,3)-fucosyltransferase expression patterns in kidney, liver, and colon depend, at least in part, on differential processing of FUT transcripts.


EXPERIMENTAL PROCEDURES

Human Tissue Collection, Cell Culture, and RNA Isolation

Human tissues were obtained through the Cooperative Human Tissue Network (CHTN), which provides pathologic review and accompanying tissue block specimens by protocol. Tissues were snap-frozen less than 30 min from harvest. Specimens with documented fibrosis, malignancy, or inflammation were excluded. Approximately 70 samples meeting these criteria were processed for the studies reported. To facilitate comparison with other published results(24, 28) , human HL-60 cells and unselected peripheral blood T lymphocytes were also prepared. HL-60 cells were grown in RPMI 1640 medium with 10% fetal calf serum. For T lymphocyte preparations, peripheral blood buffy coat was washed with 1% fetal calf serum. Lymphocytes were stimulated for 2 days in 20% fetal calf serum and 1% phytohemagglutinin. The T cell population was then expanded by placing the cells in 20% fetal calf serum, 10% T-cell conditioned medium(29) , 50 units/ml interleukin-2, and 1% sodium pyruvate in RPMI 1640 medium. Total cellular RNA was extracted from homogenized cells and tissues with guanidine isothiocyanate and purified by cesium chloride gradient centrifugation(30) . Poly(A) RNA was prepared using oligo(dT)-cellulose chromatography(30) .

Northern Blot Analysis

Poly(A) RNA (20 µg, divided into 5 µg per lane) was denatured and fractionated with 1.2% formaldehyde agarose gel electrophoresis (30) and transferred to Hybond-N membranes (Amersham). The membranes were cut into four separate strips, which were then prehybridized (19) for 2-4 h at 42 °C. Gene-specific probes were amplified using previously described PCR (^1)conditions (22) and the following primer sets: FUT3 lower strand (L) base pairs (bp) 287-316 and upper strand (U) bp 157-186(17) ; FUT5 L bp 253-282 and U bp 139-167(21) ; and FUT6 L bp 207-236 and U bp 86-115(22) , corresponding to fragment sizes of 160, 144, and 151 bp, respectively. Hybridization to beta-actin message was used as control(28) . Probes were labeled with [P]dCTP by random priming and purified from unincorporated isotope at a specific activity of 1 10^9 cpm/µg or higher(22) . Hybridization (19) was carried out at 42 °C for 16-20 h. The final wash was carried out at 65 °C with 0.5 SSC and 0.2% SDS for 30 min. Autoradiography was performed with an intensifying screen at -80 °C for 10-14 days. Autoradiograms were scanned on a Pixelcraft Pro Imager scanner (Oakland, CA). The images were sized, grouped, and labeled using Adobe Photoshop 3.0 (Mountain View, CA).

RT-PCR Analysis of Coding Regions

First strand cDNA was prepared using 0.2 µg of poly(A) RNA. Synthesis of cDNA was carried out in a 30-µl reaction volume with 13 units of Moloney murine leukemia virus reverse transcriptase and 125 ng of the common lower strand primer (denoted L in Table 1). This primer was chosen to ensure that FUT catalytic domain sequence was present in all amplified fragments(22) . PCR consisted of 30 cycles for FUT3 and FUT6, while FUT5 required 35 cycles. Amplifications were performed using the cDNA primer and gene-specific upper strand primers (denoted U in Table 1) according to the following temperature profile: denaturation 94 °C, 1.5 min; annealing 70 °C, 1.5 min; extension 72 °C, 2 min; and final extension 72 °C for 8 min. To control for genomic contamination, parallel amplifications of all samples with no reverse transcriptase were performed (data not shown). RT-PCR of human cytoplasmic beta-actin was performed with previously reported primers (28) to verify the quality/quantity of RNA and further exclude the possibility of genomic contamination (25 cycles). PCR products were cloned into the pCRII vector (Invitrogen) and sequenced by the dideoxy chain termination method. Sequence analysis and alignments were performed with the MacVector (International Biotechnologies, Inc.) and Entrez (NLM) sequence analysis programs.



Human cDNA Library Screening

Approximately 1 10^6 recombinant phages from normal human kidney and liver cDNA libraries (insert sizes ranging from 0.6 to 4.5 kb in gt10, Clontech) were screened by plaque hybridization as described previously (19) using probes derived from the FUT6 sequence. These probes included a 1.2-kb HindIII fragment (22) spanning the FUT6 coding region (recognizes all three FUT genes) and a 330-bp fragment derived from the FUT6 5`-UT sequence (recognizes only FUT6, see Fig. 5, exon C, bp 1-330). Filters were processed as described above. Twenty-six cDNA clones were isolated (sixteen FUT6, eight FUT3, and two FUT5), characterized by Southern blot analysis(22) , cloned into the pcDNA-1 vector (Invitrogen), and sequenced as described above.


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).





Rapid Amplification of 5` and 3` cDNA Ends

Rapid amplification of cDNA ends (RACE) was performed with the 5`-AmpliFINDER RACE system (Clontech) on kidney, liver, and colon poly(A) RNA (the same specimens used in Northern and RT-PCR analyses). 5`-RACE first strand cDNA was generated with gene-specific lower strand primers (L1, Table 2) followed by PCR amplification with nested gene-specific lower strand primers (L2, Table 2). 3`-RACE was performed as two separate PCR amplifications with gene-specific upper strand primer sets (U1 and U2, Table 3). Following 30 cycles, 2 µl of a one-tenth diluted primary PCR product was applied to the secondary nested amplification of 40 cycles. Where genomic and cDNA sequences were colinear, parallel amplifications of samples with no reverse transcriptase were performed (data not shown). PCR products were cloned and sequenced as above. Flanking exons in 5`- and 3`-RACE clones were verified by RT-PCR assays and Northern blot analyses.



Extended Length PCR of cDNA Contigs

Extended length PCR (XL-PCR, Perkin Elmer) was performed according to the manufacturer's protocol in a 100-µl reaction volume in the GeneAmp PCR System 9600 (Perkin Elmer). The reaction mixture consisted of 2 µl of kidney, liver, and colon cDNAs (oligo-dT primed poly(A) RNA), 125 ng of each primer, 200 µM dNTPs, 10 reaction buffer, and 4 units of rTth DNA polymerase XL. Parallel amplifications of samples with no reverse transcriptase added were performed as above. Hot start PCR (30) and 30 cycles of amplification were employed with the following temperature profile: denaturation 94 °C, 1 min; annealing/extension 72 °C, 4 min; and final extension 72 °C, 10 min. cDNA contigs were amplified with the following primer sets: FUT3 L (Table 3) and U (Table 2); FUT5 L1 (distal poly(A), Table 3) and U (Table 2); FUT6 L1 (distal poly(A), Table 3) and U1 (3.5 kb, Table 2).

Genomic Mapping of FUT Flanking Exons

XL-PCR (above) and Southern blot analyses (22) were performed on total human genomic DNA using exonic probes and primers listed in Tables I-III. In addition, overlapping genomic clones encompassing the 19p13.3 FUT locus (26) and selected subclones (data not shown) were used to verify splice sites, map exons, and provide intronic sequence.

Subcloning of FUT6 Isoform Constructs

FUT6 isoform transcripts were amplified using the primers listed in Table 3and Fig. 8. The resulting fragments were cloned into the pCRII vector (Invitrogen) and sequenced to exclude PCR errors. To construct expression vectors consisting largely of the coding sequences of the isoforms, the catalytic domain from the FUT6 ``wild type'' clone, pcDNA1-Fuc-TVI(22) , was replaced with the truncated catalytic domain from each of the isoform clones. The first isoform expression plasmid was constructed by BstXI digestion of pcDNA1-Fuc-TVI and replacement of the released fragment with the BstXI fragment from the pCRII Isoform I clone. (Fig. 7A; second BstXI site in pCRII polylinker). The second isoform construct was made by PstI digestion of pcDNA1-Fuc-TVI and replacement of the released fragment with the PstI fragment from the pCRII Isoform II clone (Fig. 7A; second PstI site in pCRII polylinker). Representative plasmids containing the inserts in the sense orientation with respect to the pcDNA-1 cytomegalovirus promoter were verified by sequence analysis and designated pcDNA1-Fuc-TVI-I (encoding Isoform I) and pcDNA1-Fuc-TVI-II (encoding Isoform II).


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).



Transfection of COS-7 Cells with FUT6 Isoform Constructs and Preparation of Cell Extracts

COS-7 cells, cultured as described (17) , were transfected with plasmid DNAs (pcDNA1, Invitrogen; pcDNA1-FucTVI, (22) ; pcDNA1-Fuc-TIII, (17) ; pcDNA1-Fuc-TVI-I, this work; or pcDNA1-Fuc-TVI-II, this work), using a previously described DEAE-dextran procedure(17) . Cell extracts containing 1% Triton X-100 and 25% glycerol were prepared from sonicated transfected COS-7 cells after a 72-h incubation period(17) .

Fucosyltransferase Assays

Fucosyltransferase assays with low molecular weight acceptor substrates were performed as described previously(21, 22) . Acceptor substrates were added to a final concentration of 20 mM (LacNAc, N-acetyllactosamine, Galbeta14GlcNAc and LNB-I, lacto-N-biose I, Galbeta13GlcNAc, and sialyl LacNAc, alpha(2,3)sialyl-N-acetyllactosamine, NeuNAcalpha23Galbeta14GlcNAc). Control assays with no added acceptor were performed using the same conditions. Reactions were incubated at 37 °C for 1 h(22) . In addition, longer incubations were performed to detect possible trace activities (typically 4 h). Product separations for assays with neutral acceptors (LacNAc and LNB-I) were performed using columns with Dowex 1X2-400, formate form(17) . For assays with sialyl LacNAc, separations were performed with Dowex 1-X8 (PO4) columns, equilibrated as described(31) . Flow-through fractions and elutions were collected, pooled, and counted to measure product formation. Product identifications were performed as described(22) .


RESULTS

FUT3 and FUT6 Transcripts Are Expressed at High Levels in Several Normal Tissues, while FUT5 Expression Is Lower and Restricted in Distribution

Biochemical surveys of human tissues and cells have suggested the presence of at least three alpha(1,3)-fucosyltransferase activities in epithelia(9) . Lewis alpha(1,3/1,4)-fucosyltransferase activity is co-expressed with two other alpha(1,3)-fucosyltransferase activities in some instances(8) . Given these data, we performed semiquantitative studies of fucosyltransferase transcripts in normally differentiated tissues ( Fig. 1and Fig. 2). For each tissue, Northern blot analyses were performed in parallel with ``stem'' region probes containing divergent coding region sequence (``Experimental Procedures''). For PCR analyses, reverse transcription was carried out with a lower strand primer common to the catalytic domain of all three FUT genes, followed by amplification with gene-specific upper strand primers derived from stem regions ( Table 1and (22) ). These approaches allow comparison of message levels by tissue (Fig. 1) and gene (Fig. 2).


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 beta-actin (``Experimental Procedures''). Autoradiographic films were exposed for 10-14 days (except for beta-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 beta-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 beta-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.

Alternatively Spliced FUT3 Transcripts Show Tissue-specific Expression of 5`-UT Region Exons

To better understand the expression of FUT3, RACE and hybridization screenings of normal human colon, kidney, and liver libraries were performed (``Experimental Procedures''). Fig. 3summarizes the results of these analyses. The predominant FUT3 2.37-kb transcript was designated ``Major II'' because it was isolated in 4 of 8 cDNA clones and showed high levels in colon and kidney (599-bp fragment). This transcript was absent in liver, which contains a 2.72-kb message (948-bp fragment). A previously reported FUT3 cDNA species, originally isolated from A-431 epithelial carcinoma cells(17) , is shown as Minor II in Fig. 3; it is present in colon at low levels (409-bp fragment).


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).

FUT5 Transcripts Use Two Polyadenylation Sites

FUT5, which encodes an enzyme with substrate specificity that closely resembles FUT6(21) , was found to be expressed at low but consistent levels in liver and colon (Fig. 2). Considering the above data showing alternative splicing for FUT3, we postulated that post-transcriptional modifications of FUT5 might affect its expression. Using the same RACE and hybridization screenings described above, no alternative splicing was found in 5`-UT, coding, or 3`-UT regions (Fig. 4). RACE and genomic analyses showed that two polyadenylation signal sequences were used in liver and colon. These putative signals differ from the typical AAUAAA and other published consensus motifs(32) . Minor transcript I, which uses the proximal polyadenylation signal CACAAA, is present at approximately 10-fold higher levels than minor transcript II, which uses GATAAT. Both transcripts contain terminal elements indicative of 3`-UT processing: GA and CA terminal sequences, respectively, and G/T clusters (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.



Alternatively Spliced and Polyadenylated FUT6 Transcripts Are Differentially Expressed in Kidney, Liver, and Colon

As shown in Fig. 1, FUT6 transcripts include a 3.5-kb message present in kidney and liver, and a 2.5-kb species in colon and liver. To determine the structure of these transcripts, RACE and hybridization screenings of colon, kidney, and liver libraries were performed as described above. In addition, extended length RT-PCR was performed to verify the exonic composition of full-length alternative transcripts (``Experimental Procedures''). Fig. 5and Fig. 6summarize these results. The kidney 3.5-kb major transcript begins with exon A and contains an additional 5`-UT region, exon B, compared to the major 2.5-kb transcript from colon. Liver contains equivalent amounts of both major species. Rare transcripts were also found in liver that lacked exon B (Minor III) but contained exons A and C. The low abundance of this spliced species (one cDNA clone isolated by hybridization, confirmed by RACE and extended length RT-PCR) correlates with the absence of splice consensus sequences for exon B (Fig. 5).


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 beta-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).

Tissues That Express High Levels of FUT6 Transcripts Containing Exon G Undergo Additional Splicing within the Coding Region to Form Isoform Transcripts

Further PCR characterization of clones obtained by RACE and hybridization techniques revealed that a subset of kidney and liver transcripts existed with aberrant sizes. These truncated transcripts were only observed when exon G was present and comprised 10-25% of clones containing the distal polyadenylation signal (data not shown). Representative transcripts from kidney and liver were cloned and sequenced, revealing two additional splice events occurring within the FUT6 coding region (Fig. 7A). Sequence analysis of these clones confirmed identity with the FUT6 stem region(22) . Extended length PCR was used to verify the relation of these isoform splice variants to upstream exonic sequence (data not shown). The removal of wild type carboxyl-terminal nucleotides and replacement with isoform residues results in a new putative open reading frame (Fig. 7B). RT-PCR analysis confirms the tissue-specific distribution of isoform transcripts, which correlates with expression level of exon G (Fig. 7C).

Transcripts Encoding FUT6 Isoforms Do Not Direct alpha(1,3)-Fucosyltransferase Activity in Transfected Cells

To determine if the isoform coding regions could direct alpha(1,3)- and/or alpha(1,4)-fucosyltransferase activity, the putative open reading frames were used to construct expression plasmids and transfected into mammalian cells as described(22) . Extracts prepared from transfected cells were tested for alpha(1,3)- and alpha(1,4)-fucosyltransferase activity in assays containing low molecular weight oligosaccharides(17) . Control extracts prepared from pcDNA1-transfected COS-7 cells transferred no detectable fucose to any acceptor. Extracts prepared from cells transfected with pcDNA1-Fuc-TVI and pcDNA1-Fuc-TIII were found to contain fucosyltransferase activities similar to previously published values (17, 22) . Under the same assay conditions, no alpha(1,3)- or alpha(1,4)-fucosyltransferase activity was found in any of the isoform extracts (specific activity less than 1.0 pmol/mg/h). These results suggest that the alternatively spliced isoform transcripts do not encode functional alpha(1,3)- or alpha(1,4)-fucosyltransferases.


DISCUSSION

Immunohistologic and biochemical surveys of non-diseased human organs containing a large proportion of epithelial cells suggest that at least two alpha(1,3)-fucosyltransferase genes are expressed at high levels (FUT3, corresponding to Lewis alpha(1,3/1,4)-fucosyltransferase activity and FUT6, corresponding to plasma alpha(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 alpha(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 alpha(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 alpha(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 alpha(1,3/1,4)-fucosyltransferase activity with bile duct proliferation, while alpha(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 alpha(1,3)-fucosyltransferase activity and 25% of alpha(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 (^2)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 alpha(1,3)-fucosyltransferase and high alpha(1,4)-fucosyltransferase activity, while normal colon mucosa has high alpha(1,3)-fucosyltransferase and moderate alpha(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 alpha(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 alpha(1,4)-fucosylation(48) . None of the alternatively spliced FUT6 transcripts isolated to date were found to direct alpha(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.^2 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.^2 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 alpha(1,3)-fucosyltransferase gene expression.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA01758, the Lineberger Comprehensive Cancer Center, and the Center for Thrombosis and Hemostasis. Tissues were supplied through the Cooperative Human Tissue Network (CHTN) of the National Cancer Institute. 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.

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].

§
Contributed equally to this work.

To whom correspondence should be addressed: University of North Carolina, Dept. of Pediatrics, Div. of Hematology and Oncology, CB 7220 Burnett Womack Building, Chapel Hill, NC 27599-7220. Tel.: 919-966-1178; Fax: 919-966-7629.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; sialyl Lewis x, NeuNAcalpha23Galbeta14[Fucalpha13]GlcNAc; sialyl Lewis a, NeuNAcalpha23Galbeta13[Fucalpha14]GlcNAc; alpha(1,3)-fucosyltransferase, GDP-fucose:beta-D-N-acetylglucosaminide-3-alpha-L-fucosyltransferase; kb, kilobase; bp, base pair; 5`-UT, 5`-untranslated; 3`-UT, 3`-untranslated; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-mediated polymerase chain reaction; XL-PCR, extended length polymerase chain reaction; U and L, upper and lower, respectively.

(^2)
H. S. Cameron, D. Szczepaniak, and B. W. Weston, unpublished data.


ACKNOWLEDGEMENTS

We thank Susan Gidwitz and Kate Senger for technical assistance, Doug Mokaren for expert illustration work, and Bill Marzluff, Gilbert White, and Beverly Mitchell for advice and support.


REFERENCES

  1. Lasky, L. (1992) Science 258,964-969 [Medline] [Order article via Infotrieve]
  2. Bird, J. M., and Kimber, S. J. (1984) Dev. Biol. 104,449-460 [Medline] [Order article via Infotrieve]
  3. Solter, D., and Knowles, B. B. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,5565-5569 [Abstract]
  4. Kannagi, R., Nudelman E., Levery, S. B., and Hakomori, S. (1982) J. Biol. Chem. 257,14865-14874 [Abstract/Free Full Text]
  5. Pennington, J. E., Rastan, S., Roelcke, D., and Feizi, T. (1985) J. Embryol. Exp. Morph. 90,337-361
  6. Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D., and Evans, M. J. (1981) Nature 292,156-158 [Medline] [Order article via Infotrieve]
  7. Feizi, T., and Childs, R. A. (1987) Biochem. J. 245,1-11 [Medline] [Order article via Infotrieve]
  8. Mollicone, R., Gibaud, A., Francois, A., Ratcliffe, M., and Oriol, R. (1990) Eur. J. Biochem. 191,169-176 [Abstract]
  9. Mollicone, R., Candelier, J. J., Mennesson, B., Couillin, P., Venot, A. P., and Oriol, R. (1992) Carbohydr. Res. 228,265-276 [CrossRef][Medline] [Order article via Infotrieve]
  10. Kuijpers, T. (1993) Blood 81,873-882 [Medline] [Order article via Infotrieve]
  11. Okada Y., Usumoto R., Muguruma, M., Shimoe, T., Sunayama, T., and Yamada, G. (1991) J. Hepatol. 10,1-7
  12. Jezequel-Cuer, M., Daliz, A.-M., Flejou, J.-F., and Durand, G. (1992) Liver 12,140-146 [Medline] [Order article via Infotrieve]
  13. Candelier, J. J., Mollicone, R., Mennesson, B., Bergemer, A. M., Henry, S., Couillin, P., and Oriol, R. (1993) Lab. Invest. 69,449-459 [Medline] [Order article via Infotrieve]
  14. Sakamoto, J., Furukawa, K., Cordon-Cardo, C., Yin, B. W. T., Rettig, W. J., Oettgen, H. F., Old, L. J., and Lloyd, K. O. (1986) Cancer Res. 46,1553-1561 [Abstract]
  15. Itzkowitz, S., Yuan, M., Fukushi, Y., Palekar, A., Phelps, P., Shamsuddin, A., Trump, B., Hakamori, S., and Kim, Y. S. (1986) Cancer Res. 46,2627-2632 [Abstract]
  16. Macher, B. A., Holmes, E. H., Swiedler, S. J., Stults, C. L. M., and Srnka, C. A. (1991) Glycobiology 6,577-584
  17. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes & Dev. 4,1288-1303
  18. Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63,1349-1356 [Medline] [Order article via Infotrieve]
  19. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R., and Ernst, L. K. (1991) J. Biol. Chem. 266,17467-17477 [Abstract/Free Full Text]
  20. Kumar, R., Potvin, B., Muller, W. A., and Stanley, P. (1991) J. Biol. Chem. 266,21777-21783 [Abstract/Free Full Text]
  21. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992) J. Biol. Chem. 267,4152-4160 [Abstract/Free Full Text]
  22. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem. 267,24575-24584 [Abstract/Free Full Text]
  23. Koszdin, K. L., and Bowen, B. R. (1992) Biochem Biophys. Res. Commun. 187,152-157 [Medline] [Order article via Infotrieve]
  24. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269,14730-14737 [Abstract/Free Full Text]
  25. Natsuka, S., Gersten, K., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269,16789-16794 [Abstract/Free Full Text]
  26. McCurley, R. S., Recinos, A., Olsen, A., Gingrich, J., Szczepaniak, D. A., Cameron, H. S., Krauss, R., and Weston, B. W. (1995) Genomics 26,142-146 [CrossRef][Medline] [Order article via Infotrieve]
  27. Mollicone, R., Candelier, J., Reguigne, I., Couillin, P., Fletcher, A., and Oriol, R. (1994) Transfus. Clin. Biol. 2,91-97
  28. Yago, K., Zenita, K., Ginya, H., Sawada, M., Ohmori, K., Minoru, O., Kannagi, R., and Lowe, J. (1993) Cancer Res. 53,5559-5565 [Abstract]
  29. Messner, H. A., Izaguirre, C. A., and Jamal, N. (1981) Blood 58,402-405 [Abstract]
  30. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology , J. Wiley and Sons, New York
  31. Paulson, J. C., Rearick, J. I., and Hill, R. L. (1977) J. Biol. Chem. 252,2363-2371 [Abstract]
  32. Birnstiel, M. L., Busslinger, M., and Strub, K. (1985) Cell 41,349-359 [Medline] [Order article via Infotrieve]
  33. Wickens, M., and Stephenson, P. (1984) Science 226,1045-1051 [Medline] [Order article via Infotrieve]
  34. Peterson, M. L., and Perry, R. P. (1989) Mol. Cell. Biol. 9,726-738 [Medline] [Order article via Infotrieve]
  35. Leff, S. E., Evans, R. M., and Rosenfeld, M. G. (1987) Cell 48,517-524 [Medline] [Order article via Infotrieve]
  36. Kan, J. L. C., and Moran, R. G. (1995) J. Biol. Chem. 270,1823-1832 [Abstract/Free Full Text]
  37. Fleming, S., and Brown, G. (1986) Histochem. J. 18,61-66 [Medline] [Order article via Infotrieve]
  38. Jezequel-Cuer, M., N'Guyen-Cong, H., Biou, D., and Durand, G. (1993) Biochim. Biophys. Acta 1157,252-258 [Medline] [Order article via Infotrieve]
  39. Sasaki, M., Kono, N., and Nakanuma, Y. (1994) Hepatology 19,138-144 [Medline] [Order article via Infotrieve]
  40. Okada, Y., Jin-no, K., Ikeda, H., Sakai, N., Sotozono, M., Yonei, T., Nakanishi, S., Moriwaki, S., and Tsuji, T. (1994) Cancer 73,1811-1816 [Medline] [Order article via Infotrieve]
  41. Dohi, T., Haskigunchi, M., Yamamoto, S., Morita, H., and Oshima, M. (1994) Cancer 73,1552-1561 [Medline] [Order article via Infotrieve]
  42. Johnson, P. H., Yates, A. D., and Watkins, W. M. (1981) Biochem. Biophys. Res. Commun. 100,1611-1618 [Medline] [Order article via Infotrieve]
  43. Mollicone, R., Reguigne, I., Kelly, R. J., Fletcher, A., Watt, J., Chatfield, S., Aziz, A., Cameron, H. S., Weston, B. W., Lowe, J. B., and Oriol, R. (1994) J. Biol. Chem. 269,20987-20994 [Abstract/Free Full Text]
  44. Tamagawa, H., Iwakura, K., Amano, A., Shizukuishi, S., and Tsunemitsu, A. (1987) J. Dental Res. 66,756-760 [Abstract]
  45. Tamagawa, H., Inoshita, E., Takeshita, T., Takagaki, M., Shizukuishi, S., and Tsunemitsu, A. (1987) J. Dental Res. 66,72-77 [Abstract]
  46. Mandel, U., Orntoft, T., Holmes, E., Sorensen, H., Clausen, H., Hakamori, S., and Dabelsteen, E. (1991) Vox Sang. 61,205-214 [Medline] [Order article via Infotrieve]
  47. Langkilde, N. C., Wolf, H., and Orntoft, T. (1991) Br. J. Haematol. 79,493-499 [Medline] [Order article via Infotrieve]
  48. Orntoft, T. F., Holmes, E. H., Johnson, P., Hakomori, S., and Clausen, H. (1991) Blood 77,1389-1396 [Abstract]
  49. Boularand, S., Darmon, M., and Mallet, J. (1995) J. Biol. Chem. 270,3748-3756 [Abstract/Free Full Text]
  50. Green, M. R. (1991) Annu. Rev. Cell Biol. 91,559-595 [CrossRef]
  51. McKeown, M. (1992) Annu. Rev. Cell Biol. 8,133-155 [CrossRef]
  52. Dominski, Z., and Kole, R. (1994) J. Biol. Chem. 269,23590-23596 [Abstract/Free Full Text]
  53. Burch, J. B. E., and Davis, D. L. (1994) Nucleic Acids Res. 22,4733-4741 [Abstract]
  54. Sussenbach, J. S. (1989) Prog. Growth Factor Res. 1,33-48 [Medline] [Order article via Infotrieve]
  55. Ball, G. E., O'Neill, R., Schultz, J. E., Lowe, J. B., Weston, B. W., Nagy, J. O., Brown, E. G., Hobbs, C., and Bednarski, M. D. (1992) J. Am. Chem. Soc. 114,5449-5451
  56. Karlsson, S., and Nienhuis, A. W. (1985) Annu. Rev. Biochem. 54,107-118
  57. Mollicone, R., Reguigne, I., Fletcher, A., Aziz, A., Rustam, M., Weston, B. W., Kelly, R. J., Lowe, J. B., and Oriol, R. (1994) J. Biol. Chem. 269,12662-12671 [Abstract/Free Full Text]
  58. Bingham, P. M., Chou, T., Mims, I., and Zachar, Z. (1988) Trends Genet. 4,134-138 [Medline] [Order article via Infotrieve]
  59. Peng, S. B., Crider, B. P., Xie, X. S., and Stone, D. K. (1994) J. Biol. Chem. 269,17262-17266 [Abstract/Free Full Text]
  60. Nah, H. D., Niu, Z., and Adams, S. L. (1994) J. Biol. Chem. 269,16443-16448 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.