# Patent

## Title
UDP-galactose: β-N-acetyl-glucosamine β-1,4-galactosyltransferase, β4Gal-T2

## Abstract

A novel gene defining a novel enzyme in the UDP-D-galactose: b-N-acetyl-glucosamine β-1,4-galactosyltransferase family, termed β4Gal-T2, with unique enzymatic properties is disclosed. The enzymatic activity of β4Gal-T2 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding β4Gal-T2 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting β4Gal-T2 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing β4Gal-T2. The enzyme β4Gal-T2 and β4Gal-T2-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of β4Gal-T2. Further, the invention discloses methods of obtaining β-1,4-galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active β4Gal-T2 protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active β4Gal-T2 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the β4Gal-T2 gene by isolating DNA from a patient, amplifying β4Gal-T2-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

## Background
This is a continuation of application Ser. No. 10/132,652, filed Apr. 24, 2002, now U.S. Pat. No. 6,916,649, which is a continuation of application Ser. No. 09/118,464, filed Jul. 17, 1998, now U.S. Pat. No. 6,558,934. Each of these prior applications is hereby incorporated by reference, in its entirety.


TECHNICAL FIELD
The present invention relates generally to the biosynthesis of glycans found as free oligosaccharides or covalently bound to proteins and glycosphingolipids. This invention is more particularly related to a family of nucleic acids encoding UDP-D-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferases (β4Gal-transferases), which add galactose to the hydroxy group at carbon 4 of 2-acetamido-2-deoxy-D-glucose (GlcNAc). This invention is more particularly related to a gene encoding the second member of the family of β4Gal-transferases, termed β4Gal-T2, probes to the DNA encoding β4Gal-T2, DNA constructs comprising DNA encoding β4Gal-T2, recombinant plasmids and recombinant methods for producing β4Gal-T2, recombinant methods for stably transfecting cells for expression of β4Gal-T2, and methods for identification of DNA polymorphism in patients.
BACKGROUND OF THE INVENTION
The UDP-galactose: β-N-acetyl-glucosamine β-1,4-galactosyltransferase (β4Gal-T1) was the first animal glycosyltransferase to be isolated and cloned (Narimatsu et al., 1986; Shaper et al., 1986; Nakazawa et al., 1988; Shaper et al., 1988; D'Agostaro et al., 1989), and early searches for homologous genes by low stringency Southern hybridisation suggested that this gene was unique. Characterisation of β4Gal-transferase activities from different sources, however, indicate that distinct activities exist (Sheares and Carlson, 1984; Furukawa et al., 1990). Emerging evidence now reveal that several β4galactosyltransferase genes may exist. Shaper and colleagues (Shaper et al., 1995) have identified two different chick cDNA sequences, which have 65% and 48% sequence similarity to human β4Gal-T1. Both chick cDNAs were shown to encode catalytically active b4Gal-transferases (Shaper et al., 1997). Two independent groups have analysed β4Gal-transferase activities in mice homozygously deficient for β4Gal-T1 (Asano et al., 1997; Lu et al., 1997). Both studies showed residual β4Gal-transferase activity, providing clear evidence for the existence of additional β4Gal-transferases. Thus, the β4Gal-T1 gene is likely to be part of a homologous gene family with recognisable sequence motifs, and this is supported by a large number of human ESTs with sequence similarities to β4Gal-T1 in EST databases (National Center for Biotechnology Information).
β-1,4-Galactosyltransferase activities add galactose to different acceptor substrates including free oligosaccharides, N- and O-linked glycoproteins, and glycosphingolipids (Kobata, 1992). In addition, β4Gal-T1 is modulated by a-lactalbumin to function as lactose synthase and hence has a major role in lactation (Brew et al., 1968). Given the diverse functions of β-1,4-galactosyltransferase activities and the evidence that multiple b4Gal-transferases exist, it is likely that these enzymes may have different kinetic properties. Furukawa et al. (Furukawa et al., 1990) showed that liver β4Gal-transferase activity was near 20-fold higher with asialo-agalacto-transferrin compared to asialo-agalacto-IgG, whereas the activity found in T and B cells only showed a 4 to 5-fold difference with the two substrates. The β4Gal-transferase activity in B cells of rheumatoid arthritis patients appear to be similar to B cells from healthy controls with several substrates including asialo-agalacto-transferrin (Furukawa et al., 1990) and βGlcNAc-pITC-BSA (Keusch et al., 1995), but different with asialo-agalacto-IgG (Furukawa et al., 1990). Furthermore, the Km for UDP-Gal of β4Gal-transferase activity from B cells of rheumatoid arthritis patients were 2-fold higher (35.6 mM) than normal B cells (17.6 mM) (Furukawa et al., 1990). Finally, the activity in B cells for asialo-agalacto-transferrin was more sensitive to a-lactalbumin inhibition than the activity with asialo-agalacto-IgG. A number of studies have concluded that there was no change in β4Gal-transferase activity in B cells of rheumatoid arthritis patients (Wilson et al., 1993; Axford et al., 1994). However, if multiple β4Gal-transferases exist, it is possible that the contradictory findings of Furukawa et al. (Furukawa et al., 1990) can be explained by a model with two β4Gal-transferases with different kinetic parameters expressed in normal B cells, and a selective down regulation of one in B cells of rheumatoid arthritis patients.
Access to additional existing β4Gal-transferase genes encoding β4Gal-transferases with better kinetic properties than β4Gal-T1 would allow production of more efficient enzymes for use in galactosylation of oligosaccharides, glycoproteins, and glycosphingolipids. Such enzymes could be used, for example, in pharmaceutical or other commercial applications that require synthetic galactosylation of these or other substrates that are not or poorly acted upon by β4Gal-T1, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for additional UDP-galactose: β-N-acetyl-glucosamine β-1,4-galactosyltransferases and the primary structure of the genes encoding these enzymes. The present invention meets this need, and further presents other related advantages.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acids encoding human UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase (β4Gal-T2), including cDNA and genomic DNA. β4Gal-T2 has better kinetic parameters than β4Gal-T1, as exemplified by its lower Km for UDP-Gal and its better activity with saccharide derivatives, glycoprotein substrates, and βGlcNAc-glycopeptides. The complete nucleotide sequence of β4Gal-T2, SEQ ID NO:1, is set forth in FIG. 2.
In one aspect, the invention encompasses isolated nucleic acids comprising the nucleotide sequence of nucleotides 1-1116 as set forth in SEQ ID NO:1 or sequence-conservative or function-conservative variants thereof. Also provided are isolated nucleic acids hybridizable with nucleic acids having the sequence of SEQ ID NO:1 or fragments thereof or sequence-conservative or function-conservative variants thereof, preferably, the nucleic acids are hybridizable with β4Gal-T2 sequences under conditions of intermediate stringency, and, most preferably, under conditions of high stringency. In one embodiment, the DNA sequence encodes the amino acid sequence, SEQ ID NO:2, also shown in FIG. 2, from methionine (amino acid no. 1) to glycine (amino acid no. 372). In another embodiment, the DNA sequence encodes an amino acid sequence comprising a sequence from tyrosine (no. 31) to glycine (no. 372) of SEQ ID NO:2.
In a related aspect, the invention provides nucleic acid vectors comprising β4Gal-T2 DNA sequences, including but not limited to those vectors in which the β4Gal-T2 DNA sequence is operably linked to a transcriptional regulatory element, with or without a polyadenylation sequence. Cells comprising these vectors are also provided, including without limitation transiently and stably expressing cells. Viruses, including bacteriophages, comprising β4Gal-T2-derived DNA sequences are also provided. The invention also encompasses methods for producing β4Gal-T2 polypeptides. Cell-based methods include without limitation those comprising: introducing into a host cell an isolated DNA molecule encoding β4Gal-T2, or a DNA construct comprising a DNA sequence encoding β4Gal-T2; growing the host cell under conditions suitable for β4Gal-T2 expression; and isolating β4Gal-T2 produced by the host cell. A method for generating a host cell with de novo stable expression of β4Gal-T2 comprises: introducing into a host cell an isolated DNA molecule encoding β4Gal-T2 or an enzymatically active fragment thereof (such as, for example, a polypeptide comprising amino acids 31-372 of SEQ ID NO:2), or a DNA construct comprising a DNA sequence encoding β4Gal-T2 or an enzymatically active fragment thereof; selecting and growing host cells in an appropriate medium; and identifying stably transfected cells expressing β4Gal-T2. The stably transfected cells may be used for the production of β4Gal-T2 enzyme for use as a catalyst and for recombinant production of peptides or proteins with appropriate galactosylation. For example, eukaryotic cells, whether normal or diseased cells, having their glycosylation pattern modified by stable transfection as above, or components of such cells, may be used to deliver specific glycoforms of glycopeptides and glycoproteins, such as, for example, as immunogens for vaccination.
In yet another aspect, the invention provides isolated β4Gal-T2 polypeptides, including without limitation polypeptides having the sequence set forth in SEQ ID NO:2, polypeptides having the sequence of amino acids 31-372 as set forth in SEQ ID NO:2, and a fusion polypeptide consisting of at least amino acids 31-372 as set forth in SEQ ID NO:2 fused in frame to a second sequence, which may be any sequence that is compatible with retention of β4Gal-T2 enzymatic activity in the fusion polypeptide. Suitable second sequences include without limitation those comprising an affinity ligand or a reactive group.
In another aspect of the present invention, methods are disclosed for screening for mutations in the coding region (exons I-VII) of the β4Gal-T2 gene using genomic DNA isolated from, e.g., blood cells of patients. In one embodiment, the method comprises: isolation of DNA from a patient; PCR amplification of coding exons I-VII; DNA sequencing of amplified exon DNA fragments and establishing therefrom potential structural defects of the β4Gal-T2gene associated with disease.
These and other aspects of the present invention will become evident upon reference to the following detailed description and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the strategy for identification and cloning of β4Gal-T2. Identified ESTs are indicated by their GenBank accession numbers with available sequence lengths in parenthesis. Vertical stippled lines labelled with numbers indicate 5′ positions of EST clones compared to the coding sequence of the gene.
FIG. 2 depicts the DNA sequence of the β4Gal-T2 (accession # Y12509) gene (SEQ ID NO: 1) and the predicted amino acid sequence of β4Gal-T2 (SEQ ID NO: 2). The amino acid sequence is shown in single letter code (SEQ ID NO: 2). The hydrophobic segment representing the putative transmembrane domain is double underlined, and adjacent charged amino acids are single-stipple underlined. Potential N-linked glycosylation sites are indicated by an asterisk. The locations of primers used for RT-PCR preparation of the expression construct are indicated by single underlining.
FIGS. 3A and 3B are an illustration of a sequence comparison between human β4Gal-T1 (SEQ ID NO: 4) (GenBank accession # M22921), human β4Gal-T2 (SEQ ID NO: 2), human β4Gal-T3 (SEQ ID NO: 5) (GenBank accession # Y12510), chick gene one (SEQ ID NO: 6) (GenBank accession # U19890), chick gene two (SEQ ID NO: 7) (GenBank accession # U19889), and a snail β4GlcNAc-transferase (SEQ ID NO: 8).
FIGS. 4A and 4B depict α-lactalbumin modulation of β4galactosyltransferase activities. 4A: Activities with glucose in the presence of increasing amounts of α-lactalbumin. The results are presented relative to the activities obtained with 40 mM glucose. 4B: Activities with GlcNAc in the presence of increasing amounts of α-lactalbumin. The results are presented relative to the activities obtained with 2 mM (for bovine milk enzyme and β4Gal-T3) or 0.25 mM βGlcNAc-benzyl (for β4Gal-T2). Purified bovine milk enzyme or media from Sf9 cells expressing secreted forms of either β4Gal-T2 or -T3 were used as enzyme sources. Designations: ▴ Bovine milk Gal-transferase mainly representing β4Gal-T1; ▪ β4Gal-T2; ● β4Gal-T3.
FIGS. 5A and 5B depict differential inhibition of β4Gal-transferase activities by high acceptor substrate concentrations. 5A: βGlcNAc-benzyl. 5B: GlcNAc. Designations as in FIGS. 4A and 4B.
FIG. 6 is a photographic illustration of Northern blot analysis of the expression patterns of β4Gal-T2 in different tissues. MTN signifies Multiple Tissue Northern blots (Clontech).
FIG. 7 is a schematic representation of the genomic structure of the coding region of the human β4Gal-T2 gene. The six identified introns are indicated with the nucleotide positions of the 3′ exon boundaries. The coding region is placed in 6 exons designated I-VI.
FIG. 8 is a schematic representation of forward and reverse PCR primers that can be used to amplify different regions of the β4Gal-T2.
FIG. 9 show sequences of the primers that were used for amplification of all exons.



DETAILED DESCRIPTION OF THE INVENTION
All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In the case of conflict, the present description, including definitions, is intended to control.
Definitions:
1. “Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases (see below).
2. “Complementary DNA or cDNA” as used herein refers to a DNA molecule or sequence that has been enzymatically synthesized from the sequences present in an mRNA template, or a clone of such a DNA molecule. A “DNA Construct” is a DNA molecule or a clone of such a molecule, either single- or double-stranded, which has been modified to contain segments of DNA that are combined and juxtaposed in a manner that would not otherwise exist in nature. By way of non-limiting example, a cDNA or DNA which has no introns is inserted adjacent to, or within, exogenous DNA sequences.
3. A plasmid or, more generally, a vector, is a DNA construct containing genetic information that may provide for its replication when inserted into a host cell. A plasmid generally contains at least one gene sequence to be expressed in the host cell, as well as sequences that facilitate such gene expression, including promoters and transcription initiation sites. It may be a linear or closed circular molecule.
4. Nucleic acids are “hybridizable” to each other when at least one strand of one nucleic acid can anneal to another nucleic acid under defined stringency conditions. Stringency of hybridization is determined, e.g., by a) the temperature at which hybridization and/or washing is performed, and b) the ionic strength and polarity (e.g., formamide) of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two nucleic acids contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5×SSC, at 65° C.) requires that the sequences exhibit some high degree of complementarity over their entire sequence. Conditions of intermediate stringency (such as, for example, an aqueous solution of 2×SSC at 65° C.) and low stringency (such as, for example, an aqueous solution of 2×SSC at 55° C.), require correspondingly less overall complementarily between the hybridizing sequences. (1×SSC is 0.15 M NaCl, 0.015 M Na citrate.)
5. An “isolated” nucleic acid or polypeptide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated.
6. A “probe” refers to a nucleic acid that forms a hybrid structure with a sequence in a target region due to complementarily of at least one sequence in the probe with a sequence in the target region.
7. A nucleic acid that is “derived from” a designated sequence refers to a nucleic acid sequence that corresponds to a region of the designated sequence. This encompasses sequences that are homologous or complementary to the sequence, as well as “sequence-conservative variants” and “function-conservative variants”. Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Function-conservative variants of β4Gal-T2 are those in which a given amino acid residue in the polypeptide has been changed without altering the overall conformation and enzymatic activity (including substrate specificity) of the native polypeptide; these changes include, but are not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like).
8. A “donor substrate” is a molecule recognized by, e.g., a galactosyltransferase and that contributes a galactosyl moiety for the transferase reaction. For β4Gal-T2, a donor substrate is UDP-galactose. An “acceptor substrate” is a molecule, preferably a saccharide or oligosaccharide, that is recognized by, e.g., a galatosyltransferase and that is the target for the modification catalyzed by the transferase, i.e., receives the galatosyl moiety. For β4Gal-T2, acceptor substrates include without limitation oligosaccharides, glycoproteins, O-linked GlcNAc-glycopeptides, and glycosphingolipids containing the sequences GlcNAcβ1-3Gal, GlcNAcβ1-6Gal, GlcNAcβ1-6GalNAc, GlcNAcβ1-3GalNAc, GlcNAcβ1-2Man, GlcNAcβ1-4Man, GlcNAcβ1-6Man, GlcNAcβ1-3Man, Glcβ1-ceramide.
The present invention provides the isolated DNA molecules, including genomic DNA and cDNA, encoding the UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase (β4Gal-T2).
β4Gal-T2 was identified by analysis of EST database sequence information, and cloned based on EST and 5′RACE cDNA clones. The cloning strategy may be briefly summarized as follows: 1) synthesis of oligonucleotides derived from EST sequence information, designated EBER102 (SEQ ID NO: 30) and EBER 104 (SEQ ID NO: 31); 2) successive 5′-rapid amplification of cDNA ends (5′RACE) using commercial Marathon-Ready cDNA; 3) cloning and sequencing of 5′RACE cDNA; 4) identification of a novel cDNA sequence corresponding to βGal-T2; 5) construction of expression constructs by reverse-transcription-polymerase chain reaction (RT-PCR) using Colo205 human cell line mRNA; 6) expression of the cDNA encoding β4Gal-T2 in Sf9 (Spodoptera frugiperda) cells. More specifically, the isolation of a representative DNA molecule encoding a novel second member of the mammalian UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase family involved the following procedures described below.
Identification of DNA Homologous to β4Gal-T1.
Novel human DNA sequences with apparent homology to the human β4Gal-T1 gene (Masri et al., 1988) were identified by sequence similarity searches of the dbEST database at The National Center for Biotechnology Information, USA, using the BLASTn and tBLASTn algorithms. Composites for identified novel genes were compiled and analysed for sequence similarity to human β4Gal-T1. EST cDNA clones with the longest inserts (FIG. 1) were obtained from Genome Systems Inc, USA.
Cloning of Human β4Gal-T2.
Two partly overlapping ESTs with predicted sequence similarity to β4Gal-T1 were identified (FIG. 1). Sequencing of the inserts revealed an open reading frame which potentially encoded a sequence similar to β4Gal-T1, but the 5′ sequence was shorter and without an initiation codon. Further 5′ sequence was obtained by 5′ RACE using human fetal brain Marathon-Ready cDNA (Clontech) in combination with anti-sense primers EBER102 and EBER104. The 5′RACE products were cloned and multiple clones were sequenced. The entire sequence was confirmed by sequencing genomic P1 clones. The composite sequence contained an open reading frame of 1116 bp (FIG. 2), with an overall sequence identity of approximately 63% to β4Gal-T1. The predicted open reading frame has one potential initiation codon in agreement with Kozak's rule (Kozak, 1992). The predicted coding sequence depicts a type II transmembrane glycoprotein with a 11 amino acid residue N-terminal cytoplasmic domain, a transmembrane segment of 21 residues, and a stem region and catalytic domain of 340 residues, with three potential N-linked glycosylation sites (FIG. 2). Multiple alignment analysis (ClustalW) of human β4Gal-T1 (accession # M22921), human β4Gal-T2, and human β4Gal-T3 (accession # Y12510) presented in FIGS. 3A and 3B demonstrated sequence significant similarities especially in the central and C-terminal region and conservation of cysteine residues. The N-terminal regions show no sequence similarity. A 3′ untranslated region without polyadenylation signals was included in the oligo-dT primed EST cDNA clones sequenced. The 3′ ESTs (STsG4681) were linked to chromosome 1 between D1S2861 and D1S211 microsatellite markers at 73-75 cM (NCBI).
Expression of β4Gal-T2.
An expression construct designed to encode amino acid residues 31-372 of β4Gal-T2 was prepared by RT-PCR with mRNA from Colo205 cell line, using the primer pair EBER100FOR (SEQ ID NO: 9) and EBER114 (FIG. 2; SEQ ID NO: 10). Expression of a soluble construct of β4Gal-T2 in Sf9 cells (Pharmingen) resulted in marked increase in galactosyltransferase activity using the βGlcNAc-benzyl acceptor substrate compared to uninfected cells or cells infected with control constructs for polypeptide GalNAc-transferases or histo-blood group A and O genes (Bennett et al., 1996; Gentzsch and Tanner, 1996) (Table 1).







TABLE I







Substrate specificity of β4Gal-transferases









β4Gal-T2a(nmol/min/ml)










Substrate Concentration
1 mM
3 mM
9 mM













D-GlcNAc
1.4
3.2
4.8


Bz-β-D-GlcNAc
6.8
3.6
1.5


Bz-α-D-GlcNAc
0.4
1.1
1.7


o-Nph-α-D-GlcNAc
0.4
0.8
1.5


p-Nph-β-D-GlcNAc
3.0
2.3
0.9


p-Nph-1-thio-β-D-GlcNAc
1.2
1.6
0.2


4-Me-lumb-β-D-GlcNAc
0.8
0.6
0.4


β-D-GlcNAc-(1-3)-β-D-Gal-1-OMe
5.8
7.7
 NDb


β-D-CLcNAc-(1-6)-α-D-Man-1-OMe
8.5
11.3
ND


Bz-2-(2-β-D-GlcNAc)-α-D-GlcNAc
9.9
2.6
1.3


4-Me-lumb-β-D-GalNAc
ND
0.0
ND


o-Nph-β-D-GalNAc
ND
0.0
ND


Bz-*-D-GalNAc
ND
0.0
ND


4-Me-lumb-β-D-Gal
ND
0.0
ND


o-Nph-β-D-Gal
ND
0.0
ND





aEnzyme sources were media of infected Sf9 cells. Background values obtained with uninfected cells or cells infected with an irrelevant construct were subtracted. The background rates were not higher than 0.5 nmol/min/ml.


bND, not determined






Analysis of the substrate specificity of the soluble β4Gal-T2 activity showed that only βGlcNAc-benzyl and not βGlcNAc-benzyl or βGalNAc-benzyl was an acceptor substrate. Free glucose was not an acceptor, but in the presence of increasing concentrations of α-lactalbumin incorporation rates similar to bovine milk β4Gal-transferase was observed (FIG. 4A). Differences in the concentration of α-lactalbumin to achieve maximum activity with Glc were observed with 0.4 mg/ml required for β4Gal-T2 and only 0.1 mg/ml for the bovine milk enzyme. The activities of both β4Gal-T2 and the bovine milk enzyme with GlcNAc were inhibited by α-lactalbumin, but β4Gal-T1 (bovine milk transferase preparation) was overall more sensitive to inhibition (FIG. 4B). The apparent Km for benzyl-βGlcNAc was 0.16 mM, and the Km for UDP-Gal using benzyl-βGlcNAc was 0.011 mM. The bovine milk β4-galactosyltransferase showed higher Km for UDP-Gal in agreement with previous studies (Fujita-Yamaguchi and Yoshida, 1981; Paquet and Moscarello, 1984; Furukawa et al., 1990; Nakazawa et al., 1991; Malissard et al., 1996), and the measured Km for GlcNAc was similar to that determined in some studies (Powell and Brew, 1974; Moscarello et al., 1985), but 5-10 fold higher than compared to other studies (Fujita-Yamaguchi and Yoshida, 1981; Paquet and Moscarello, 1984; Nakazawa et al., 1991; Malissard et al., 1996). As shown in FIGS. 5A and 5B β4Gal-T2 was inhibited at high concentrations of both benzyl-βGlcNAc and free N-acetylglucosamine to higher degree than bovine milk β4Gal-transferase and β4Gal-T3 (Shur, 1982). β4Gal-T2 showed strict donor substrate specificity for UDP-Gal and did not utilise UDP-GalNAc or UDP-GlcNAc with the acceptor substrates tested. β4Gal-T2 utilised the Lc3Cer glycosphingolipid substrates, and the product formed with this substrate was confirmed by 1H-NMR to be nLc3Cer similar to what was found for the activity of β4Gal-T3 (Almeida et al., 1997). β4Gal-T2 exhibited the overall best activities with the glycoprotein acceptors ovalbumin, asialo-agalacto-fetuin, and asialo-agalacto-transferrin (Table II).







TABLE II







Substrate specificity of β4-galactosyltransferases with glycopeptide and


glycoprotein acceptors









Acceptor substratea











β4Gal-T2
β4Gal-T3
Bovine milk β4Gal-T



nmol/min/ml
nmol/min/ml
nmol/min/μg














β-D-GlcNAC-1-Bz
3.5
3.9
3.4


β-D-GlcNAC-1-
1.3
0.9


(FAPGSYPAL)


*-D-GalNAc-1-
0.0
0.0
0.0


(FAPSNYPAL)


Hen egg albumin
2.0
1.0
0.7


Asialo-agalacto-
2.8
0.7
0.8


Fetuin


Asialo-Fetuin
0.2
0.0
0.1





aβ-D-GlcNAc-1-Bz was used at 0.25 mM with β4Gal-T2, 0.625 mM with bovine milk β4Gal-T, 2 mM with β4Gal-T3, and 20 mM with β4Gal-T5; glycopeptides were used at 0.1 mM; glycoproteins were used at 10 mg/ml.






The activities of the b4Gal-transferases were analysed relative to benzyl-β-GlcNAc, and β4Gal-T2 showed 2-3 fold higher activity than other β4Gal-transferases tested. β4Gal-T2 also showed the best activity with a synthetic O-linked βGlcNAc-glycopeptide (Table II), suggesting that this enzyme will show higher sensitivity in labeling O-linked βGlcNAc-glycoproteins as well.

Northern Blot Analysis of Human Organs

Northern analysis with mRNA from 16 human adult organs showed a single transcript of both genes of approximately 2.2 kb (FIG. 6). β4Gal-T2 was expressed weakly in several adult organs with highest expression in prostate, testis, ovary, intestine, and muscle.
Genomic Organization of β4Gal-T2 Gene.
The present invention also provides isolated genomic DNA molecules encoding β4Gal-T2. A human P1 library (DuPont Merck Pharmaceutical Company Human Foreskin Fibroblast P1 Library) was screened using primer pairs EBER100 (SEQ ID NO: 29) and EBER102 (SEQ ID NO: 30). Three clones; DPMC-HFF#10638:515:G9, DPMC-HFF#10639:516:G4, and DPMC-HFF#10640:924:A11, were obtained from Genome Systems. Southern blot analysis with various oligonucleotides covering the 3′ and 5′ coding sequence of the existing full length β4Gal-T2 cDNA indicated that the entire coding sequence was included in the P1 clone. A comparative Southern blot analysis between cloned P1 DNA and total human genomic DNA using a full length cDNA as probe gave similar patterns, validating the use of cloned P1 DNA as a model. The coding region of β4Gal-T2 were found in six exons (FIG. 7; The nucleic acid sequences for these exons, numbered as exons I, II, III, IV, V and VI, are depicted as SEQ ID NOS: 11-16 respectively). Human and mouse β4Gal-T1 is encoded in six exons (Hollis et al., 1989; Mengle-Gaw et al., 1991). Comparison of the intron/exon boundaries of β4Gal-T1, -T2, and -T3, revealed that the five introns in the coding regions of the three genes are placed identically. FIGS. 8 and 9 depict a PCR strategy and primer sequences for amplification of all coding exons in β4Gal-T2 using genomic DNA. (The primer sequences for cloning each exon are depicted in FIG. 9 and defined as EBER151 and EBER143 for Exon I, SEQ ID NOS: 17 and 18, respectively; EBER142 and EBER144 for Exon II, SEQ ID NOS: 19 and 20, respectively; EBER145 and EBER146 for Exon III, SEQ ID NOS: 21 and 22, respectively; EBER147 and EBER148 for Exon IV, SEQ ID NOS: 23 and 23, respectively; EBER149 and EBER150 for Exon V, SEQ ID NOS: 25 and 26, respectively; and EBER132 and 1003pri2 for Exon VI, SEQ ID NOS: 27 and 28, respectively.)
DNA, Vectors, and Host Cells
In practicing the present invention, many conventional techniques in molecular biology, microbiology, recombinant DNA, and immunology, are used. Such techniques are well known and are explained fully in, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively); Immunochemical Methods in Cell and Molecular Biology, 1987 (Mayer and Waler, eds; Academic Press, London); Scopes, 1987, Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.) and Handbook of Experimental Immunology, 1986, Volumes I-IV (Weir and Blackwell eds.).
The invention encompasses isolated nucleic acid fragments comprising all or part of the nucleic acid sequence disclosed herein as SEQ ID NO:1. The fragments are at least about 8 nucleotides in length, preferably at least about 12 nucleotides in length, and most preferably at least about 15-20 nucleotides in length. The invention further encompasses isolated nucleic acids comprising sequences that are hybridizable under stringency conditions of 2×SSC, 55° C., to SEQ ID NO:1; preferably, the nucleic acids are hybridizable at 2×SSC, 65° C.; and most preferably, are hybridizable at 0.5×SSC, 65° C.
The nucleic acids may be isolated directly from cells. Alternatively, the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incorporation into a given vector for recombinant expression.
The nucleic acids of the present invention may be flanked by natural human regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3′-noncoding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
According to the present invention, useful probes comprise a probe sequence at least eight nucleotides in length that consists of all or part of the sequence from among the sequences designated SEQ ID NO:1 or sequence-conservative or function-conservative variants thereof, or a complement thereof, and that has been labelled as described above.
The invention also provides nucleic acid vectors comprising the disclosed sequence or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple cloning or protein expression.
Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. The inserted coding sequences may be synthesized by standard methods, isolated from natural sources, or prepared as hybrids, etc. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences may be achieved by known methods. Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl2 mediated DNA uptake, fungal infection, microinjection, microprojectile, or other established methods.
Appropriate host cells included bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are Saccharomyces cerevisiae, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, and the like. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced β4Gal-T2 derived peptides and polypeptides.
Advantageously, vectors may also include a transcription regulatory element (i.e., a promoter) operably linked to the β4Gal-T2-coding portion. The promoter may optionally contain operator portions and/or ribosome binding sites. Non-limiting examples of bacterial promoters compatible with E. coli include: â-lactamase (penicillinase) promoter; lactose promoter; tryptophan (trp) promoter; arabinose BAD operon promoter; lambda-derived P1 promoter and N gene ribosome binding site; and the hybrid tac promoter derived from sequences of the trp and lac UV5 promoters. Non-limiting examples of yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter, galactokinase (GALI) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Suitable promoters for mammalian cells include without limitation viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly A addition sequences and enhancer sequences which increase expression may also be included; sequences which cause amplification of the gene may also be desirable. Furthermore, sequences that facilitate secretion of the recombinant product from cells, including, but not limited to, bacteria, yeast, and animal cells, such as secretory signal sequences and/or prohormone pro region sequences, may also be included. These sequences are known in the art.
Nucleic acids encoding wild-type or variant polypeptides may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell, and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
The nucleic acids of the present invention find use, for example, as probes for the detection of β4Gal-T2 in other species and as templates for the recombinant production of peptides or polypeptides. These and other embodiments of the present invention are described in more detail below.
Polypeptides and Antibodies
The present invention encompasses isolated peptides and polypeptides encoded by the disclosed genomic sequence. Peptides are preferably at least five residues in length.
Nucleic acids comprising protein-coding sequences can be used to direct the recombinant expression of polypeptides in intact cells or in cell-free translation systems. The known genetic code, tailored if desired for more efficient expression in a given host organism, can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The phosphoramidite solid support method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem. 764:17078, or other well known methods can be used for such synthesis. The resulting oligonucleotides can be inserted into an appropriate vector and expressed in a compatible host organism.
The polypeptides of the present invention, including function-conservative variants of the disclosed sequence, may be isolated from native or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which a protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins.
Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel elctrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against a protein or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.
The present invention also encompasses derivatives and homologues of polypeptides. For some purposes, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e., function-conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids.
The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.
The present invention encompasses antibodies that specifically recognize immunogenic components derived from β4Gal-T2. Such antibodies can be used as reagents for detection and purification of β4Gal-T2.
β4Gal-T2 specific antibodies according to the present invention include polyclonal and monoclonal antibodies. The antibodies may be elicited in an animal host by immunization with β4Gal-T2 components or may be formed by in vitro immunization of immune cells. The immunogenic components used to elicit the antibodies may be isolated from human cells or produced in recombinant systems. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA. Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains. The antibodies include hybrid antibodies (i.e., containing two sets of heavy chain/light chain combinations, each of which recognizes a different antigen), chimeric antibodies (i.e., in which either the heavy chains, light chains, or both, are fusion proteins), and univalent antibodies (i.e., comprised of a heavy chain/light chain complex bound to the constant region of a second heavy chain). Also included are Fab fragments, including Fab′ and F(ab)2 fragments of antibodies. Methods for the production of all of the above types of antibodies and derivatives are well-known in the art. For example, techniques for producing and processing polyclonal antisera are disclosed in Mayer and Walker, 1987, Immunochemical Methods in Cell and Molecular Biology, (Academic Press, London).
The antibodies of this invention can be purified by standard methods, including but not limited to preparative disc-gel elctrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. Purification methods for antibodies are disclosed, e.g., in The Art of Antibody Purification, 1989, Amicon Division, W.R. Grace & Co. General protein purification methods are described in Protein Purification: Principles and Practice, R. K. Scopes, Ed., 1987, Springer-Verlag, New York, N.Y.
Anti-β4Gal-T2 antibodies, whether unlabeled or labeled by standard methods, can be used as the basis for immunoassays. The particular label used will depend upon the type of immunoassay used. Examples of labels that can be used include, but are not limited to, radiolabels such as 32P, 125I, 3H and 14C; fluorescent labels such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone; chemiluminescers such as luciferia and 2,3-dihydrophthal-azinediones; and enzymes such as horseradish peroxidase, alkaline phosphatase, lysozyme and glucose-6-phosphate dehydrogenase.
The antibodies can be tagged with such labels by known methods. For example, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bisdiazotized benzadine and the like may be used to tag the antibodies with fluorescent, chemiluminescent or enzyme labels. The general methods involved are well known in the art and are described in, e.g., Chan (Ed.), 1987, Immunoassay: A Practical Guide, Academic Press, Inc., Orlando, Fla.
The following examples are intended to further illustrate the invention without limiting its scope.
EXAMPLE 1
A: Identification of cDNA Homologous to b4Gal-T1 by Analysis of EST Database Sequence Information.
Database searches were performed with the coding sequence of the human β4Gal-T1 sequence (Masri et al., 1988) using the BLASTn and tBLASTn algorithms against the dbEST database at The National Center for Biotechnology Information, USA. The BLASTn algorithm was used to identify ESTs representing the query gene (identities of ≦95%), whereas tBLASTn was used to identify non-identical, but similar EST sequences. ESTs with 50-90% nucleotide sequence identity were regarded as different from the query sequence. The results of tBLASTn searches were evaluated by visual inspection after elimination of ESTs regarded as identical to the query sequence (<95% nucleotide sequence identity). ESTs with several apparent short sequence motifs and cysteine residues arranged with similar spacing were selected for further sequence analysis. Initially, the identified ESTs (5′ sequence) were used in BLASTn searches of the dbEST database to search for overlapping ESTs (95-100% identity in at least 30 bp) (FIG. 1). If new ESTs were identified, the procedure was repeated and sequences merged. In addition, all identified ESTs were analysed in the Unigene database in order to confirm that they were from the same gene transcript, and to select cDNA clones with the longest inserts as well as information for each set of ESTs were compiled and analysed for sequence similarity to human β4Gal-T 1.
B: Cloning and Sequencing of β4Gal-T2.
Two partly overlapping ESTs were identified (FIG. 1). Sequencing of the inserts revealed an open reading frame which potentially encoded a sequence similar to β4Gal-T1, but the 5′ sequence was shorter and without an initiation codon. Further 5′ sequence was obtained by 5′ RACE using human fetal brain Marathon-Ready cDNA (Clontech) in combination with anti-sense primers EBER102 (5′-GAAACTGAGCCTTACTCAGGC; SEQ ID NO: 30) and EBER104 (5′-TCCACATCGCTGAAGATGAAGC; SEQ ID NO: 31) for 35 cycles at 95° C., 45 sec; 55° C., 15 sec; 68° C., 3 min, using the Expand kit enzyme (Boehringer Mannheim). The RACE products were cloned into the BamHI site of pT7T3UI9 and multiple clones were sequenced. The entire sequence was confirmed by sequencing genomic P1 clones.
EXAMPLE 2
A. Expression of βGal-T2 in Sf9 Cells.
An expression construct designed to encode amino acid residues 31-372 of β4Gal-T2 was prepared by RT-PCR with mRNA from Colo205 cell line, using the primer pair EBER100FOR (5′-TACTTTGACGTCTACGCCCAG; SEQ ID NO: 9) and EBER114 (5′-GAAAACAGAGCCCAGCTCAG; SEQ ID NO: 10) with BamH1 restriction sites (FIG. 2). The PCR product were cloned into the BamHI site of pAcGP67 (Pharmingen), and the construct sequenced to verify correct insertion and sequence. The plasmid pAcGP67-β4Gal-T2-sol was co-transfected with Baculo-Goldä DNA (Pharmingen) as described previously (Bennett et al., 1996). Recombinant Baculo-virus were obtained after two successive amplifications in Sf9 cells grown in serum-containing medium, and titres of virus were estimated by titration in 24-well plates with monitoring of enzyme activities. Controls included pAcGP67-β4Gal-T3-sol (Almeida et al., 1997) and pAcGP67-GalNAc-T3-sol (Bennett et al., 1996).
B. Analysis of βGal-T2 Activity.
Standard assays were performed in 50 ml total reaction mixtures containing 25 mM Tris (pH 7.5), 10 mM MnCl2, 0.25% Triton X-100, 100 mM UDP-[14C]-Gal (2,300 cpm/nmol) (Amersham), and varying concentration of acceptor substrates (Sigma) (see Table I for structures). The soluble constructs were assayed with 5-20 ml of culture supernatant from infected cells, whereas the full length construct was assayed with 1% Triton X-100 homogenates of washed cells. Bovine milk P1,4Gal-transferase (Sigma) was used as control. Assays used for determination of Km of acceptor substrates were modified to include 200 mM UDP-[14C]-Gal, and assays for donor substrate Km were performed with 2 mM (for β4Gal-T3 and bovine milk Gal-T) or 0.25 mM OGlcNAc-benzyl.
Reaction products were quantified by Dowex-1 chromatography. Assays with hen egg Ovalbumin (Sigma), asialo-fetuin (Sigma), and asialo-agalacto-fetuin (Sigma, treated with bgalactosidase) were performed with the standard reaction mixture modified to contain 200 mM UDP-Gal, 54 mM NaCl, and 0.5 mg Ovalbumin. The transfer of Gal was evaluated after precipitation by filtration through Whatman GF/C glass fiber filters.
C: Stable Expression of Full Coding Sequence of βGal-T2 in CHO Cells.
A cDNA sequence encoding the full coding sequence of the putative β4Gal-T2 gene was derived by RT-PCR using primers EBER 120 (5′-AGCGGATCCATGAGCAGACTGCTGGGG-3′; SEQ ID NO: 32) and EBER 114 with BamHI restriction sites introduced. The PCR product was designed to yield a β4Gal-T2 protein with a hydrophobic transmembrane retention signal in order to have the enzyme expressed and positioned in the appropriate Golgi compartment of the transfected cell. The PCR product was inserted into the BamHI site of a mammalian expression vector pCDNA3 (Invitrogen), and the construct, pCDNA3-β4Gal-T2-mem, was transfected into CHO and stable transfectants were selected.
D: Stable Expression of the Soluble Form of bGal-T2 in CHO Cells.
cDNA pAcGP67-b4Gal-T2-sol containing the coding sequence of the putative soluble b4Gal-T2 enzyme was cloned into the BamHI site of a modified mammalian expression vector pCDNA3 (Invitrogen). pcDNA3 had been modified by insertion of an interferon signal peptide sequence into the KpnI/BamHI site of ensuring secretion of the expressed product when cloned into the vector. The pcDNA3γINF-β4Gal-T2-sol construct was transfected into CHO and stable transfectants were selected.
EXAMPLE 3
Restricted Organ Expression Pattern of βGal-T2
Human Multiple Tissue northern blots were obtained from Clontech. The soluble expression construct of β4Gal-T2 was used as probe. The probe was random primed labelled using αP32dCTP (Amersham) and an oligo labelling kit (Pharmacia). The blots were probed 18 hours at 42° C. as previously described (Bennett et al., 1996), and washed 2×10 min at RT with 2×SSC, 1% Na4P202; 2×20 min at 65° C. with 0.2×SSC, 1% SDS, 1% Na4P2O2; and once 10 min with 0.2×SSC at RT.
EXAMPLE 4
Genomic Structure of the Coding Region of β4Gal-T2
A human foreskin genomic P1 library (DuPont Merck Pharmaceutical Company Human Foreskin Fibroblast P1 Library) was screened using primer pair EBER100 (5′-TGAAGGAGGATGCCGCCTATGAC; SEQ ID NO: 29)/EBER102 (5′-GAAACTGAGCCTTACTCAGGC; SEQ ID NO: 30). P1 clones were obtained from Genome Systems Inc, and DNA from P1 phages prepared as recommended by Genome Systems Inc. The entire coding sequence of each gene was sequenced in full using automated sequencing (AB1377, Perkin Elmer) with dye terminator chemistry. Intron/exon boundaries were determined by comparison with the cDNA sequences optimising for the gt/ag rule (Breathnach and Chambon, 1981).
EXAMPLE 5
Analysis of DNA Polymorphism of β4Gal-T2 Gene
Primer pairs as described in FIGS. 8 and 9 have been used for PCR amplification of individual coding sequence of the 6 exons (SEQ ID NOS: 11-16). Each PCR product was subcloned and the sequence of 10 clones containing the appropriate insert was determined assuring that both alleles of each individual are characterized.
From the foregoing it will be evident that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
REFERENCES


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## Claims

1. An isolated UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase polypeptide, encoded by a nucleic acid that hybridizes to the nucleic acid of SEQ ID NO:1 at 0.5×SSC, 65° C.

2. The isolated UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase polypeptide of claim 1, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO: 2.

3. The isolated UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase polypeptide of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 3.

4. The isolated UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase polypeptide of claim 1, wherein said polypeptide has a lower Km for UDP-galactose as a donor substrate than bovine β4Gal-T1.

5. The isolated UDP-galactose: β-N-acetylglucosamine β-1,4-galactosyltransferase polypeptide of claim 3, wherein said polypeptide has a lower Km for UDP-galactose as a donor substrate than bovine βGal-T1.