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Published ahead of print on October 30, 2003, doi:10.1165/rcmb.2003-0202OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 710-719, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0202OC

Mucin Biosynthesis

Bovine C2GnT-M Gene, Tissue-Specific Expression, and Herpes Virus-4 Homologue

Kyung H. Choi, Fernando A. Osorio and Pi-Wan Cheng

Department of Biochemistry and Molecular Biology, College of Medicine, and Eppley Institute for Research in Cancer and Allied Diseases, Nebraska Medical Center, Omaha; and Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska

Address correspondence to: Pi-Wan Cheng, Ph.D., Department of Biochemistry and Molecular Biology, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. E-mail: pcheng{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mucin glycans are the major determinant of mucin functions. Mucin glycan branch structures, which increase structural heterogeneity and thus functional potential, are extended from ß6 N-acetylglucosaminides formed by ß6 N-acetylglucosaminyltransferases (ß6GnT). Core 2 ß6GnT-M (C2GnT-M) is the only branching enzyme that can synthesize all known mucin ß6 N-acetylglucosaminides. We report the cloning of four different bovine (b) C2GnT-M transcripts that are different only at 5'-untranslated regions. Two bC2GnT-M transcripts are found exclusively in tracheal epithelium and testis, whereas the other two are found in all other mucus-secreting tissues. The bC2GnT-M gene contains four exons spanning 5.3 kb, and the entire open reading frame is in one exon. The bC2GnT-M ORF has 95, 83, and 75% sequence identity to those of bovine herpes virus type 4 (BHV-4), human, and rat C2GnT-Ms, respectively. The homology between bovine and BHV-4 C2GnT-M genes is in the region between 170 nucleotides upstream from ATG start codon and 114 nucleotides downstream from TGA stop codon of the viral gene. Localized at the nonconserved region of the viral genome, the BHV-4 C2GnT-M gene is the only known viral C2GnT-M gene. The results suggest that BHV-4 acquired its C2GnT-M gene from the bovine gene. The mechanism of the viral acquisition of bC2GnT-M gene and the roles of the C2GnT-M gene in the survival and pathogenesis of this virus remain to be elucidated.

Abbreviations: ß6-N-acetylglucosaminyltransferases, ß6GnT • bovine herpes virus type 4, BHV-4 • bovine serum albumin, BSA • core 2 ß6 N-acetylglucosaminyltransferase, C2GnT • C2GnT thymus type, C2GnT-3 • C2GnT leukocyte type, C2GnT-L • C2GnT mucus-type, C2GnT-M • galactose, Gal • N-acetylgalactosamine, GalNAc • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • N-acetylglucosamine, GlcNAc • blood group I ß6 N-acetylglucosaminyltransferase, IGnT • blood group i ß3 N-acetylglucosaminyltransferase, iGnT • lipopolysaccharide, LPS • long unique genome region, LUR • mannoside ßN-acetylglucosaminyltransferases, MGAT • open reading frame, ORF • reverse transcription-polymerase chain reaction, RT-PCR • sodium dodecyl sulfate, SDS • short interspersed element, SINE • standard saline citrate, SSC • Tris-buffered saline, TBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There are two types of mucins, secreted and membrane-bound (1). The secreted mucins are found in the mucus that covers the epithelial tissues, such as airways, gastrointestinal tract, cervix, etc. The primary function of these mucins, such as MUC2, MUC5AC, and MUC5B, is to maintain the viscoelastic property of the mucus, and in the case of airway mucins also serve as a trap for airborne pathogens because many bacteria contain lectins, which can bind to mucin carbohydrates (2, 3). To perform this important function, the peptide backbone of the secreted mucins is decorated with oligosaccharides of heterogeneous structures. For example, more than 100 different oligosaccharides were detected in the tracheobronchial mucins of a single individual (3, 4). The membrane-bound mucins also can serve as ligands for many cell adhesion molecules involved in cell–cell interactions, which play important roles in immune functions (5). For example, upon activation of immune cells, the glycans on cell surface mucins shift from simple sialylated oligosaccharides to core 2–based complex carbohydrates (6) on which sLeX, an essential component of the ligands for P-, E-, and L-selectins, is formed. Interactions between the glycans that contain core 2–associated sLeX and these cell adhesion molecules on the cell surface of opposing participants, such as leukocytes and endothelial cells, play critical roles during the initial phase of inflammatory response (5, 7). Core 2 O-glycan–deficient neutrophils show a significant decrease in rolling activity on P-, L-, and E-selectins, resulting in defective inflammatory response (8). Overexpression of C2GnT activity also can lead to impaired humoral and cellular immune defects as described in C2GnT transgenic mice (9) and patients with Wiskott-Aldrich syndrome (10).

Following the synthesis of mucin protein at the endoplasmic reticulum, glycans are assembled in the Golgi apparatus as catalyzed by glycosyltransferases (6). These glycosyltransferases can be grouped into chain termination and chain elongation enzymes. Competition of these two groups of glycosyltransferases for common glycan substrates determines the eventual complexity of the glycan structures. Several mucin carbohydrate core structures can be generated after the addition of N-acetylgalactosamine (GalNAc) to serine or threonine of newly synthesized mucin protein (Figure 1). Addition of ß3-linked galactose to the GalNAc forms "core 1" structure. On the other hand, N-acetylglucosamine can be attached to the GalNAc via ß3-link to form "core 3." Core 1 and core 3 can be branched by ß6-linked GlcNAc to form "core 2" and "core 4," respectively, from which longer carbohydrate structures are produced. ß6 N-acetylglucosaminyltransferases (ß6GnTs) belong to the group of glycan chain elongation enzymes responsible for the synthesis of mucin glycan branch structures. These branch structures are synthesized by several different ß6GnT isozymes, including C2GnT-L (or 1) (11, 12), C2GnT-M (or 2) (13–15), C2GnT-3 (16), and blood group I ß6 N-acetylglucosaminyltransferases (IGnT) (17) (Figure 1).



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  		Figure 1. Synthesis of core 2, core 4, and blood group I branch structures of mucin glycans. Core 2 can be synthesized by C2GnT-L, C2GnT-M, and C2GnT-3; Core 4 by C2GnT-M; and blood group I by C2GnT-M and IGnT.

 
C2GnT-L cDNA was first cloned in a leukemia cell line, but was found to be ubiquitously distributed (11). C2GnT-M is expressed primarily in mucus-secreting tissues (13, 14) and C2GnT-3 is expressed in the thymus (16). IGnT is expressed in many tissues (17). Our lab previously purified a ß6GnT from bovine tracheal epithelium and showed that it could synthesize all known mucin glycan branch structures (18). A viral C2GnT-M gene from bovine herpes virus type 4 (BHV-4) was recently identified (19). This BHV-4 C2GnT-M gene is the only known ß6GnT in virus. It is localized in the region of BHV-4 genome not conserved among {gamma}herpes virus family (19). However, BHV-4 C2GnT-M is highly homologous with human C2GnT-M gene and encodes an enzyme (19) exhibiting similar substrate specificity as that of rat and human C2GnT-Ms (13).

In this communication, we report the cloning and characterization of bovine C2GnT-M transcripts (GenBank accession no. AY283763–6) and its genomic clone (GenBank accession no. AY283767). We found four different C2GnT-M transcripts including two that are expressed only in tracheal epithelium and testis. The open reading frame (ORF) of bovine C2GnT-M gene and its 5'- and 3'-immediate flanking regions are highly homologous to that of BHV-4 C2GnT-M gene, suggesting that BHV-4 acquires its C2GnT-M gene from the bovine host. The mechanism of the viral acquisition of a host gene and the significance of this C2GnT-M gene in the survival and pathogenesis of this virus in the host remain to be elucidated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Molecular biology reagents and DNA-modifying enzymes were purchased from New England Biolabs Inc. (Beverly, MA), Promega (Madison, WI), or Life Technologies, Inc. (Grand Island, NY), and used as recommended by the suppliers. SMART RACE cDNA Amplification Kit was purchased from Clontech (Palo Alto, CA). The cloning vectors (pcDNA6/Myc-His/bsd, and pCRII) and anti-Myc monoclonal antibody were from Invitrogen (Carlsbad, CA). Galß1,3GalNAc{alpha}-O-benzyl, GlcNAcß1,3GalNAc{alpha}-O-benzyl, and GlcNAcß1,3Galß-O-methyl were purchased from the Toronto Research Chemicals Inc. (Downsview, ON, Canada) and Sigma (St. Louis, MO). UDP-[3H]GlcNAc was from American Radiolabeled Chemicals (St. Louis, MO). Transferrin (iron-saturated) was from Collaborative Biomedical Products (Bedford, MA). Bond Elute C18 cartridges were from Varian (Sunny Vale, CA). The Panc1/Muc1 cell line, which is a pancreatic cancer cell line stably expressing MUC1 tandem repeat, was a gift from Dr. Michael A. Hollingsworth at the University of Nebraska Medical Center (Omaha, NE). Bovine tissues were obtained from a local slaughterhouse. Oligonucleotide primers were synthesized by the Molecular Biology Core Lab, the University of Nebraska Medical Center. Unless otherwise indicated, all other chemicals were purchased from Sigma.

Preparation of Bovine Genomic DNA
Bovine liver (100 mg) was cut into small pieces and ground in liquid nitrogen using a mortar and pestle. Then, the genomic DNA was purified with Wizard Genomic DNA Purification Kit (Promega).

Preparation of Polyadenylated RNA
Total RNA was isolated from various bovine tissues using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH). Polyadenylated RNA was isolated from the total RNA with PolyATract mRNA Isolation System III (Promega).

Cloning and Sequencing of Bovine C2GnT-M cDNA
Because the ORF of human C2GnT-M is encoded in a single exon, it was predicted that the ORF of bC2GnT-M gene also resides in one exon. The ORF of bC2GnT-M gene was cloned by PCR using bovine genomic DNA as the template and a pair of primers, 11,222 and 11,223 (5'-TGCGAATTCAAGTTCAGTCCCATAGATGG-3' and 5'-AAAGGATCCTGTTCTGTCATCCCATGAAG-3', 30 cycles at 94°C for 40 s, 55°C for 40 s, and 70°C for 1 min 30 s) that are specific to the 3'- and 5'-ends of the ORF of BHV-4 C2GnT-M gene, respectively. A DNA fragment of ~ 1.3 kb was amplified by two independent PCR reactions and cloned into pCRII vector. Three independent clones from each PCR reaction were completely sequenced to minimize aberrant mutations generated by PCR. The 1.3-kb DNA fragment was highly homologous to the comparable region of BHV C2GnT-M gene without any intervening sequence. To obtain the 5' upstream and 3' downstream sequences of the bC2GnT-M cDNA, 5'- and 3'-RACE were performed using the SMART RACE cDNA Amplification Kit (Clontech). The 5'- and 3'-RACE were performed with polyadenylated RNAs isolated from colon and tracheal epithelium. RACE was performed with first-strand cDNA synthesized according to the manufacturer's protocol. The gene specific primers 13,046 (5'-GTCCCTACAACGCTGACTTTGGAAG-3') and 13,047 (5'-ACCCTGCTCTGGAATCCATCAGCGG-3') were used for the 5'- and 3'-RACE, respectively. The 5'-RACE product was reamplified using Nested Universal Primer and the gene-specific primer BR (5'-ACTCCAGATCCAAGGAATCTACATC-3' 30 cycles at 94°C 30 s, 55°C 30 s, 72°C 40 s). The RACE products were cloned into the pCRII vector. Multiple clones from each RACE reaction were sequenced.

Analysis of C2GnT-M Gene Expression by Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed with polyadenylated RNA isolated from bovine colon, heart, liver, lung, small intestine, stomach, thymus, trachea, and testicle. PCR without the RT step performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers generated no DNA fragment, indicating no genomic DNA contamination in the polyA RNAs. The polyA RNAs were used for 5'- and 3'-RACE and RT-PCR studies. First strand cDNA was synthesized with Superscript II RNase H- Reverse Transcriptase (Life Technology Inc.) from 200 ng of the polyadenylated RNAs. The primer pairs, which were specific for the ORF region, type 1, type 2, types 1 and 3, and types 2 and 4 transcripts of bC2GnT-M and the PCR conditions using a 30-s elongation time, were OF/OR (5'-AGTTTAAGGCCCAAAGGAAG-3' and 5'-ACTCCAGATCCAAGGAATCTACATC-3', 30 cycles at 94°C 15 s, 55°C 30 s, and 72°C 40 s), AF/BR (5'-TGCACTGAATCCGGTTAACAGTAAC-3' and 5'-CCTATCTAGGGGAGATCTATTGCC-3' 30 cycles at 94°C, 30 s, 49°C 30 s, and 72°C 40 s), AF/ER (ER=5'-TGCATAAGATTGTATCCACTTGAGG-3', 30 cycles at 94°C 30 s, 49°C 30 s, and 72°C 40 s), BF/BR (BF=5'-GGAGAGGCTGAAAGGAGACCAG-3', 30 cycles at 94°C 30 s, 49°C 30 s, and 72°C 40 s), and CF/BR (CF=5'-TTTGCAGCATCAGGTTACAGATCTC-3', 30 cycles at 94°C 30 s, 50°C 30 s, and 72°C 40 s), respectively. GAPDH was employed as an internal standard of the RT-PCRs. The primer pair and the conditions for GAPDH were 5'-TCCACCACCCTGTTGCTGTA-3' and 5'-ACCACAGTCCATGCCATCAC-3' and 30 cycles at 94°C 15 s, 55°C 30 s, and 72°C 40 s. Buffers for the PCRs were optimized with MasterAmp PCR Optimization Kit (Epicentre, Madison, WI).

Northern Blot Analysis
Thirty-five micrograms of total RNA from colon, lung, small intestine, testis, and tracheal epithelium were fractionated by glyoxyal-1% agarose gel electrophoresis and transferred to a nitrocellulose membrane and ultraviolet crosslinked. The membrane was prehybridized in 50% formamide, 5x standard saline citrate (SSC), 5x Denhardt's solution, 5 mM EDTA, 0.1% SDS, and 100 µg /ml denatured salmon sperm DNA at 42°C for 4 h. Probe 1 was prepared by hot asymmetric PCR with a reverse primer (5'-CAGATGGTACCTGGGTCACT-3', 40 cycles at 95°C, 45 s, 50°C, 45 s, and 72°C, 45 s) to make the antisense strand alone. PCR was performed with the cDNA of type 1 transcript in 50 µl reaction mixture containing: 2.5 mM MgCl2, dNTPs (0.2 mM of dGTP, dATP, and dTTP, and 1.2 µM dCTP), [{alpha}-32P]dCTP (100 µCi), 10 pmole of the reverse primer, 2.5 U Taq DNA polymerase, and 1x Taq buffer (Promega). Probes 2, 3, and Bov-A2 were labeled with [{alpha}-32P]dCTP (ICN, Costa Mesa, CA) using Prime-It II Random Primer DNA Labeling Kit (Stratagene, Cedar Creek, TX). The membrane was hybridized with a probe in the prehybridzation solution supplemented with 10% dextran sulfate at 42°C for 14 h. The membrane was washed with 0.1% SDS in 2x SSC at room temperature for 15 min and twice with 0.1% SDS in 0.2x SSC at 58°C for 15 min and exposed to X-OMAT AR film (Kodak, Rochester, NY) with intensifying screen at –80°C for 48–72 h. The membrane was stripped and subsequently rehybridized with GAPDH probe to assess equivalent loading of RNA.

Transient Expression of Bovine and BHV-4 C2GnT-Ms in CHO Cells and Generation of bC2GnT-M Stable Clone in Panc1/MUC1 Cells
The 1.3-kb ORF of bovine and BHV-4 C2GnT-M genes was subcloned into a mammalian expression vector, pcDNA6/Myc-His/bsd, using EcoRI and BamHI restriction sites to construct pbC2GnT-M and pBHV-4 C2GnT-M. These two expression vectors were transfected into CHO cells using a protocol modified from the transferrin-facilitated lipofection protocol previously reported (20). Lipofectin was replaced with DMRIE-cholesterol (Invitrogen) in the modified protocol. After 48 h, the transfected CHO cells were harvested for measurement of C2GnT and C4GnT activities. The pbC2GnT-M stable clones were selected based on resistance to 10 µg/ml blasticidin after transfection of Panc1/MUC1 cells with pbC2GnT-M using the transfection protocol described above.

Western Blot Analysis
Cell lysates were analyzed by SDS-polyacrylamide gel (4% stacking and 6% separating gels) electrophoresis. Proteins were electroblotted to PVD membrane (Immobilon-P, 0.45 µ; Millipore, Bedford, MA) overnight at 100 mA, then blocked with 5% nonfat milk in Tris-buffered saline (TBS) (0.9% NaCl, 10 mM Tris, pH7.5) at room temperature for 1 h. The membranes were then incubated for 1 h at room temperature with anti-Myc antibody (1:2,000 dilution) in 5% nonfat milk. The membranes were washed with 5% nonfat milk in TBS for 15 min and then 5 min twice. The treated membranes were incubated with peroxidase-conjugated goat anti-mouse IgG secondary antibodies (1:2,000 dilution) in TBS containing 5% nonfat milk at room temperature for 1 h. The membranes were washed as described above and rinsed with TBS. ECL reagents (Pierce, Rockford, IL) were applied per the manufacturer's instruction, and the blots were exposed to ECL-sensitive film (Amersham Pharmacia Biotech, Uppsala, Sweden).

Assay of ß6 N-Acetylglucosaminyltransferase Activity
Enzyme assays were performed with crude cell homogenates of pbC2GnT-M stably-transfected clone. Confluent cells from a T25 flask were washed twice with cold phosphate-buffered saline, scraped off the flask in 250 µl of 0.25 M sucrose, and disrupted by passing the cells successively through 20- and 25-gauge needles. Twenty-nine microliters of the cell lysate was used in a 50-µl reaction mixture containing: 50 mM MOPS (pH 7.5), 5 mM MnCl2, 2% Tween-20, 1 mM ATP, 1 mg/ml bovine serum albumin (BSA), 2 mM UDP-[3H]GlcNAc (~ 1500 dpm/nmol), and 2–5 mM of appropriate acceptors. Galß1,3GalNAc{alpha}-O-Benzyl (2 mM), GlcNAcß1,3GalNAc{alpha}-O-Benzyl (2 mM), and GlcNAcß1,3Galß1-O-Me (5 mM) were used as acceptors for assaying C2GnT, C4GnT, and IGnT activities, respectively as previously described (18). The reaction mixtures were incubated at 37°C for 2 h and terminated with 0.3 ml of 10 mM ZnCl2. The products of C2GnT and C4GnT reactions were isolated by solid phase extraction on C18 cartridges with 2 ml of methanol. The purified products were dried by Automatic Environmental SpeedVac System (Savant, Holbrook, NY) and resuspended in 500 µl H2O. The IGnT reaction product was isolated on a DoweX-1 Cl- column. Radioactivity associated with the product was measured with a liquid scintillation counter (Packard, Meriden, CT). Protein concentration was measured using Commassie Plus Protein Assay Reagent (Pierce) with BSA as the standard. Enzyme activity was calculated by subtracting the endogenous activity measured without exogenous acceptor from total activity and was expressed as nmol of sugar donor transferred/h/mg protein.

Cloning and Characterization of Bovine C2GnT-M Genomic DNA
A bC2GnT-M genomic clone was obtained by screening a male bovine BAC library. High-density hybridization filters spotted with male bovine BAC library were purchased from BACPAC Resource Center at the Children's Hospital Oakland Research Institute (Oakland, CA) and screened with a 32P-labeled DNA probe derived from the coding region of bC2GnT-M cDNA. Three positive BAC clones were identified from the screening and then purchased from BACPAC Resource Center. A BAC DNA was purified from one of the clones (clone K18) by a Large-Construct Kit (Qiagen Inc., Valencia, CA) and used for characterization of the genomic structure of C2GnT-M and analysis of the putative promoters. The genomic structure of bC2GnT-M was characterized by PCR using several primer pairs followed by sequencing. The primer pairs include AF/AR, BF/BR, and DF/DR. The PCR protocol was 94°C for 4 min followed by 30 cycles of 94°C-30 s, 49°C-30 s, and 72°C-2 min, and completed with 72°C for 5 min.

Cloning of BHV-4 C2GnT-M Gene
Bovine lung cells prepared from the explants of a neonate calf lung tissue were infected with BHV-4 strains DN599, Movar 33/63, or 3,374 at an M.O.I. of 5 as previously described (21, 22). At 48 h after inoculation, the virus particles in the conditioned medium were semi-purified by centrifugation and pelleting through 40% sucrose cushion. The virus-containing fractions were treated with RNAse-free nuclease and incubated for 3 h at 37°C. After being boiled for 3 min, these virus-derived samples were cooled on ice and used as DNA template for PCR. PCR was performed with a pair of primers, 11,222/11223 (5'-TGCGAATTCAAGTTCAGTCCCATAGATGG-3' and 5'-AAAGGATCCTGTTCTGTCATCCCATGAAG-3', 30 cycles at 94°C for 40 s, 55°C for 40 s, and 70°C for 1 min 30 s) that are specific to the 3'- and the 5'-ends of the BHV-4 C2GnT-M ORF, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of Four Bovine C2GnT-M Transcripts
The 1.3-kb ORF region of bC2GnT-M was cloned from bovine genomic DNA with primers specific to the 5'- and 3'-ends of the BHV-4 C2GnT-M ORF. The same DNA fragment was amplified from the polyadenylated RNA of colon and tracheal epithelium by RT-PCR, suggesting that bovine C2GnT-M ORF is monoexonic. The 5'- and 3'-untranslated regions of C2GnT-M were cloned by 5'- and 3'-RACE from colon and tracheal epithelium. Four different C2GnT-M transcripts were cloned (Figure 2A). They have identical ORF and 3'-untranslated region, but different 5'-untranslated regions. These transcripts are designated as types 1, 2, 3, and 4. Type 1 and type 2 transcripts were found in tracheal epithelium, whereas type 3 and type 4 transcripts were identified in colon. Type 1 transcript has 2,111 nucleotides and is identical with type 3 transcript, but has an additional 128 nucleotides at the 5'-end. Type 2 and type 4 transcripts are identical except that type 2 transcript at its 5'-end has the same extra 128 nucleotides as those found in type 1 transcript. Type 2 transcript is 3,697 nucleotides long. In addition, as compared with type 1 transcript, type 2 transcript has 1,586 extra nucleotides near the 5'-end of the ORF. Similarly, type 4 transcript is 1,586 nucleotides longer than type 3 transcript.



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  		Figure 2. Cloning of four bC2GnT-M transcripts and characterization of their tissue-specific expression. (A) Four bC2GnT-M transcripts were cloned from poly A RNAs of colon and tracheal epithelium by RT-PCR and 5'- and 3'-RACEs. Identical regions among these transcripts are indicated with same shading. Arrows denote location and direction of the primers used for RT-PCR. (B) RT-PCRs were performed with polyA RNAs from various bovine tissues. Bovine C2GnT-M expresses mainly in mucus-secreting tissues. The primer pair, OF/OR, detected the common ORF region. The AF/BR primer pair, detected type 1 (480 bp product) but not type 2 transcript (2,055 bp). AF/ER primer pair detected only type 2 transcript. BF/BR primers detected only type 1 and type 3 transcripts (303 bp product) but not type 2 and type 4 transcripts (2,055 bp product), whereas CF/BR primers detected only type 2 and type 4 transcripts. The short (30 s) PCR elongation time coupled with greater abundance of type 1 and type 3 transcripts as compared with type 2 and type 4 transcripts (C) did not allow the generation of PCR products longer than 1 kb. GAPDH is the internal control. (C) Northern blot analyses of C2GnT-M transcripts were performed with three different probes which were specific to type 1 and type 2 transcripts (probe 1), type 2 and type 4 transcripts (probe 2), or all four transcripts (probe 3). The probe 3 detected a transcript in Panc1/bC2GnT-M cell which is a Panc1 cell line stably transfected with bovine C2GnT-M ORF region. The probes were stripped off and the membrane was hybridized with GAPDH probe.

 
Tissue-Specific Expression of Bovine C2GnT-M Transcripts
Tissue-specific expression of different bC2GnT-M transcripts was investigated by RT-PCR using sets of primers that can distinguish the four transcripts, Northern blot analysis using transcript-specific probes, and 5'-RACE. When ORF-specific primers OF/OR was used, C2GnT-M messages were detected in all mucus-secreting tissues such as colon, small intestine, stomach, testis, and tracheal epithelium (Figure 2B). Heart and thymus expressed low level of the C2GnT-M message, whereas liver did not have detectable C2GnT-M transcript. Although primers AF/BR can generate 480-bp and 2,055-bp PCR fragments from type 1 and type 2 transcripts, respectively, only a 480-bp fragment was amplified because the 30-s PCR elongation time was not long enough to amplify a fragment longer than 1 kb. In addition, type 1 transcript is more abundant than type 2 transcript (Figure 2C), which could further magnify the difference in PCR reaction between these two transcripts. Therefore, only type 1 and not type 2 transcript was detected in tracheal epithelium and testis under the PCR conditions employed. The presence of type 2 transcript in these two tissues was shown by the amplification of a 275-bp PCR product using type 2 transcript-specific primers AF/ER. Similarly, the primers BF/BR can amplify 303-bp and 1,888-bp PCR fragments from types 1 and 3 transcripts and types 2 and 4 transcripts, respectively. But, for the same reason as described above, the primers BF/BR amplified only a 303-bp fragment from all mucus-secretory tissues. The type 2 and type 4 transcript-specific primers CF/BR amplified a 361-bp PCR fragment from all mucus-secretory tissues. Because BF/BR primer pair could not distinguish type 1 from type 3 transcript and neither could CF/BR primer pair distinguish type 2 from type 4 transcript, the results shown above could not confirm or prove that transcript 3 and transcript 4 are not present in tracheal epithelium and testis. To solve this problem, additional experiments, such as Northern blot analysis using transcript-specific probes (Figure 2C) and 5'-RACE were performed.

Probe 1, which is a full-length antisense probe to the 5'-end sequences found only in type 1 and type 2 transcripts, detected two bands at ~ 2.1 and 3.7 kb only in tracheal epithelium and testis. They were the expected sizes for type 1 and type 2 transcripts, respectively. Probe 1 also detected a strong 0.8-kb band exclusively in the testis. This unknown transcript was at least 10-fold more abundant than that of type 1 and type 2 transcripts based on the Northern blot analysis. But probes derived from other regions of the four C2GnT-M transcripts did not recognize this transcript, suggesting that it is derived from another gene. Probe 2, which was derived from the sequence unique to type 2 and type 4 transcripts, detected only the 3.7-kb band and not the 2.0- to 2.1-kb band. The 3.7-kb band could be either type 2 or type 4 transcripts, because they differ in only 128 nucleotides and the RNA gel electrophoresis could not resolve their difference. 5'-RACE designed to detect the type 2 and type 4 transcripts only detected the type 2 transcript in tracheal epithelium and testis, which indicated that the type 4 transcript is not expressed in these tissues. Probe 3, which is specific to the C2GnT-M ORF, detected 2.1- and 3.7-kb bands in tracheal epithelium and testis, and 2- and 3.7-kb bands in small intestine, colon, and lung. The 2-kb band matches the expected size of 1,983 nucleotides of type 3 transcript, which is 128 nucleotides shorter than type 1 transcript. 5'-RACE did not detect type 3 transcript in testis and tracheal epithelium. Therefore, RT-PCR, Northern blot analyses, and 5'-RACE clearly showed tissue-specific expression of different C2GnT-M transcripts among C2GnT-M–expressing tissues. Expression of type 1 and type 2 transcripts is highly restricted to tracheal epithelium and testis, which do not express type 3 and type 4 transcripts. Type 3 and type 4 transcripts are found in other tissues that express C2GnT-M.

Characterization of the Recombinant bC2GnT-M
The complete cDNA sequence of type 1 transcript and the deduced amino acid sequence are shown in Figure 3. The ORF of bC2GnT-M gene is 1,323 bp long and encodes 440 amino acid residues with a calculated molecular weight of 50.8 kD. This enzyme is a type II transmembrane protein containing a short cytoplasmic tail (15 amino acid residues), followed by a transmembrane domain (19 amino acid residues) and then the catalytic domain. It has two potential N-glycosylation sites, N-72 and N-108, and 13 cysteines. N-72 and all 13 cysteines are conserved among all C2GnT-Ms (Figure 4). In addition, the nine cysteines located at the catalytic domain are conserved among all ß6GnTs (23).



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  		Figure 3. cDNA and deduced amino acid sequences of C2GnT-M type 1 transcript. The transmembrane region is underlined and putative N-glycosylation sites are indicated by bold characters. Polyadenylation signal is indicated by underlined bold characters. Nine cysteine residues that are located at the catalytic domain and conserved among all ß6GnTs are underlined and shaded.

 


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  		Figure 4. Alignment of the deduced amino acid sequences of bovine, BHV-4, human, and rat C2GnT-M ORF. Identical amino acid residues of C2GnT-Ms from these four sources are shaded in black, and residues that are identical in any three sequences are shaded in gray. Dashes denote gaps in the sequence. Bovine C2GnT-M has 95, 83, and 75% amino acid sequence identity with those of BHV-4, human, and rat homologs, respectively. BC2GnT-M is bC2GnT-M, VC2GnT-M is BHV-4 C2GnT-M, HC2GnT-M is human C2GnT-M, and RC2GnT-M is rat C2GnT-M.

 
Figure 4 shows the comparison of the deduced amino acid sequences of the C2GnT-M from bovine, BHV-4, human, and rat. The bovine C2GnT-M has 95, 83, and 75% amino acid sequence identity with those of BHV-4, human, and rat homologs, respectively.

The ORFs of bovine and BHV-4 C2GnT-M genes were cloned into a mammalian expression vector, pcDNA-6/Myc-His, to generate recombinant C2GnT-Ms fused with Myc epitope and His tag at the C-terminus. Assay of C2GnT-M activities in CHO cells transiently transfected with these constructs showed that the recombinant bovine and BHV-4 C2GnT-Ms had similar core 4 and I branching activity profiles, but recombinant bC2GnT-M had higher C2GnT activity than recombinant BHV-4 C2GnT-M (Figure 5). Stable cell lines expressing bC2GnT-M were generated in Panc1/MUC1 cells, a pancreatic cancer cell line expressing MUC1. The recombinant bC2GnT-M in these cell lines showed the same disaccharide acceptor specificity profile as shown above (data not shown). In addition, GalNAcß1–3Galß-BSA and Galß1–3Galß-methyl can serve as acceptors (data not shown), suggesting that the carbon-2 substituents of GalNAc and Gal are not critical for the disaccharide acceptor specificity of the enzyme. We also found that the enzyme activity was abolished after treatment with 5 mM N-ethylmaleimide (data not shown), suggesting that free cysteine(s) is required for catalysis. Western blot analysis of the recombinant C2GnT-M/Myc-His revealed a 65-kD band (data not shown), which is larger than the expected size (i.e., 55.7 kD), suggesting that the recombinant protein is glycosylated.



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  		Figure 5. Profile of C2GnT (open bars), C4GnT (solid bars), and IGnT (shaded bars) activities in the CHO cells transfected with pbC2GnT-M and pBHV-4 C2GnT-M (DN599). Transiently transfected CHO cells were assayed for C2GnT, C4GnT, and IGnT activities. The net C2GnT and IGnT activities in these transfected cells were obtained by subtracting the corresponding activities present in the nontransfected CHO cells and then normalized to C4GnT activity. The nontransfected CHO cells had very low C2GnT (0.13 nmol GlcNAc transferred/h/mg protein) and C4GnT (0.18 nmol GlcNAc transferred/h/mg protein) activity but significant IGnT activity (2.6 nmol GlcNAc transferred/h/mg protein). This activity was ~ 26% and 57% of the net IGnT activity in the CHO cells transfected with pbC2GnT-M or pBHV4 C2GnT-M (DN599), respectively. The C4GnT activities in the CHO cells transfected with either pbC2GnT-M or pBHV-4 C2GnT-M were 22.3 and 9.1 nmol GlcNAc transferred/h/mg protein, respectively.

 
Genomic Structure of Bovine C2GnT-M Gene
The genomic structure of bC2GnT-M gene was characterized by PCR using bovine genomic DNA as the template and primer sets that cover all regions of type 2 transcript. Type 2 transcript is the longest transcript, which contains all the sequences found in the four transcripts (Figure 6A). Primers AF and AR yielded a 1.7-kb PCR product, which is considerably larger than the expected fragment size (274 bp) based on the sequence of transcript 2, suggesting that there is a 1.4-kb intron between these two primers (Figure 6A). Primers BF and BR, which are located near the 5'-end of the primer AR binding site and in the ORF, respectively, generated a 1.9-kb PCR product. The size is equal to the distance between the two primers on type 2 transcript, suggesting that there is no intron between the primer BF and the ORF. However, type 1 and type 3 transcripts do not contain substantial portion (1.6-kb) of this region, implying alternative splicing of C2GnT-M transcripts with or without the usage of alternative transcription initiation sites. The ORF of bC2GnT-M is located in one exon because the entire ORF was obtained from genomic DNA by PCR without any intervening sequence. A 500-bp PCR product was amplified with the primer pair DF and DR, which are specific to the 3'-end of the ORF and the very end of the 3'-untranslated region, respectively (Figure 6A). Generation of the 500-bp PCR product indicates that there was no intron in the 3'-untranslated region. All the PCR products were cloned into the pCRII vector and sequenced. The data show that the bC2GnT-M gene has four exons spanning 5.3 kb, including 128 bp of exon 1, 1,427 bp of intron 1, 186 bp of exon 2, 1,771 bp of intron 2, and 1,798 bp of exon 3 (Figure 6B). In addition, bC2GnT-M gene has an alternative exon, i.e., 3,775 bp exon 2', which includes exon 2, intron 2, and exon 3. Exon 3 contains 51 bp of 5'-untranslated region, 1,323 bp of ORF, and 424 bp of 3'-untranslated region. The splice-junction signatures (GT-AG) were identified at each junction of the exons and introns. These results were confirmed later by cloning and sequencing of a BAC clone containing the bC2GnT-M gene.



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  		Figure 6. Characterization of the genomic structure of bC2GnT-M gene. PCR employing a 2-min elongation time at 72°C was performed using a BAC DNA as the template and with primer pairs specific to various regions of C2GnT-M type 2 transcript to identify introns. (A) Binding sites and orientation of primers are indicated as arrows in type 2 transcript, which is the longest transcript and contains all the sequences found in all four C2GnT-M transcripts. PCR with AF and AR primer pair generated a 1.7-kb DNA fragment, which is larger than the expected size of 274 bp assuming no intron between the two primers, suggesting a 1.4-kb intron. BF and BR primer pair produced a 1.9-kb DNA fragment from type 2 transcript, implying that there is no intron between the two primers. However, type 1 and type 3 transcripts do not have a substantial portion of the region, suggesting alternative splicing in different C2GnT-M transcripts. DF and DR primers amplified a 0.5-kb DNA fragment, indicating that there is no intron between the 3'-end of the ORF and the end of the 3'-UTR of the C2GnT-M transcripts. All PCR products were cloned and confirmed by sequencing. Various shadings represent type 2 transcript identical regions among these transcripts as shown in Figure 2. PCR products are depicted as solid lines with arrows indicating primers used. The PCR products are arranged based on the nucleotide sequence order found in type 2 transcript. Dotted line indicates the entire ORF of which PCR product is not shown in the gel. (B) Structure of bC2GnT-M gene and origins of the four transcripts. Bovine C2GnT-M gene is 5.3 kb long and has four exons. The four transcripts were derived from various combinations of the exons. Boxes indicate exons and lines without boxes represent introns. Exons are named by numbers. Exon 2' includes exon 2, intron 2, and exon 3. Arrows denote the direction of SINE sequences (Bov-A2). A full-length (~ 260 bp) Bov-A2 sequence is located in intron 1 and a partial (105 bp) 5' portion of Bov-A2 sequence in intron 2 and exon 2'.

 
Four different transcripts were derived from various combinations of the exons. Type 1 transcript consists of exons 1, 2, and 3, whereas type 2 has exons 1 and 2'. Type 3 transcript is derived from exons 2 and 3, whereas type 4 transcript is made of exon 2'.

A full-length (260-bp) copy of Bov-A2, a bovine short interspersed element (SINE; GenBank accession no. AF327250), was found in an inverted orientation in intron 1, and a partial length (105-bp) copy was detected in intron 2 (Figure 6B). Northern blot analysis using a probe specific to the Bov-A2 SINE sequence showed a strong smear extending from over 9 kb to 0.2 kb in all bovine tissues tested, but not in Panc1, a human pancreatic carcinoma cell line (data not shown). The results suggest that mRNAs containing Bov-A2 sequence are abundantly expressed, and that they are highly bovine-specific.

Sequence Comparison of Bovine and BHV-4 C2GnT-M Genes
The immediate flanking sequences of the ORFs of bovine and BHV-4 C2GnT-M genes are highly homologous. But, when compared with the bC2GnT-M gene, the BHV-4 C2GnT-M gene has a 13-nucleotide deletion at the 5'-flanking region and an 18-nucleotide deletion at the 3'-flanking region. If the sequences of these two deletions are excluded, the 5' flanking region (from the ATG start codon up to 182 nucleotide upstream) and the 3' flanking region (from the TGA stop codon to 133 nucleotides downstream) of the bC2GnT-M gene have 96% and 98% sequence identity with the corresponding regions of the BHV4 C2GnT-M gene (Figures 7A and 7B). The high degree of sequence identity between these two genes ends abruptly beyond these flanking regions, suggesting that BHV-4 acquired the bC2GnT-M gene from around 182 nucleotides upstream from the start codon to 133 nucleotide downstream of the stop codon of the ORF of the bC2GnT-M gene.



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  		Figure 7. Alignment of bovine and BHV-4 C2GnT-M genes. Alignment of the genomic DNA sequences of bovine and BHV-4 C2GnT-M genes. Homology between the two genes ends abruptly outside of the ORFs. The end points of the homology are indicated by asterisks. VC2GnT-M is BHV-4 C2GnT-M and BC2GnT-M is bC2GnT-M. (A) Alignment of the 5'-region upstream of C2GnT-M ORF. Minus number indicates residues upstream from the translation initiation codon, ATG. (B) Alignment of the 3'-region downstream of C2GnT-M ORF. Plus number indicates residues downstream from the translation termination codon, TGA. Identical residues between the two sequences are shaded. Dashes indicate gaps. Translation initiation (ATG) and termination (TGA) codons are boxed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our laboratory previously purified a ß6GnT from bovine tracheal epithelium and demonstrated a multifunctional capability of this enzyme (18). The current communication extends this work by cloning the cDNA encoding this enzyme. The very similar profile of C2GnT, C4GnT, and IGnT activities for the recombinant bC2GnT-M and the previously purified bovine ß6GnT (18) indicates that the recombinant enzyme is the same as the one we previously purified and characterized. It is of interest to note that bovine herpes virus type 4 contains a functional C2GnT-M gene (19). Although bovine and BHV-4 C2GnT-M genes have 95% sequence identity and both recombinant enzymes exhibit core 2, core 4, and IGnT activities, the C2GnT activity of BHV-4 C2GnT-M is only 50% of that exhibited by bC2GnT-M (Figure 5). Such a change in acceptor specificity may be resulted from a change of few amino acids in the catalytic domain of bC2GnT-M. These amino acids likely are involved in the binding of core 1 disaccharide acceptor during catalysis. Identification of these amino acids should help elucidate the catalytic mechanism of the C2GnT activity exhibited by C2GnT-M.

We have characterized four bC2GnT-M transcripts. These transcripts have identical ORF and 3'-UTR but different 5'-UTRs as demonstrated by 5'- and 3'-RACEs, and RT-PCR. These observations were confirmed by Northern blot analysis using probes specific to each of the four C2GnT-M transcripts. The 2.1- and 2.0-kb bands, which correspond to type 1 and type 3 transcripts, respectively, are the predominant messages in all tissues that expressed these and either type 2 or type 4 transcript. The 3.7-kb band, which is considerably less intense, contains either type 2 or type 4 transcript. Type 1 and type 2 transcripts were found only in tracheal epithelium and testis, but their expression in testis is far weaker than that in tracheal epithelium. In addition, the expression of type 1 transcript in tracheal epithelium is higher than the expression of other transcripts in all the tissues analyzed. Interestingly, type 1 and type 2 transcripts were not detected in the lung, further confirming tracheal epithelium-specific expression of C2GnT-M messages. On the other hand, type 3 and type 4 transcripts are ubiquitously distributed in many mucus-secreting tissues other than tracheal epithelium and testis.

Probe 1, which is a full-length antisense of exon 1, strongly hybridized to a band of ~ 0.8-kb exclusively in testis in addition to 2.1- and 3.7-kb bands. The signal intensity of the 0.8-kb band is at least 10-fold higher than that of type 1 or type 2 transcript, but other probes specific for the four C2GnT-M transcripts including the ORF-specific probe failed to detect the 0.8-kb band. This result indicates that the hybridizing band is highly testis-specific and does not contain ORF of C2GnT-M. Therefore, the 0.8-kb band is likely a transcript of another gene, which shares the exon 1 of C2GnT-M gene. It will be very interesting to characterize the gene and its relationship with C2GnT-M gene in the regulation of gene expression and other biological functions of their protein products.

Expression of multiple transcripts occurs not only for bC2GnT-M but also C2GnT-L and C2GnT-M genes from other animal species. For example, Northern blot analysis of C2GnT-M transcripts from human gastrointestine and trachea showed three bands at 2.4, 3.4, and 6 kb (13, 14). C2GnT-L transcripts from HL-60, a human promyelocytic cell line, displayed three bands at 2.1, 3.3, and 5.4 kb (11). Mouse C2GnT-L transcripts revealed two hybridizing bands at 2.2 and 5.4 kb (24). The multiple transcripts of mouse and human C2GnT-L and C2GnT-M were originally proposed to be the result of differential usage of polyadenylation signals (25). These authors predicted that these transcripts contained varying lengths of 3'-UTR, which has never been confirmed. Instead, two mouse C2GnT-L cDNAs that are ~ 2 kb long and differ only at the 5'-UTR have been characterized. One of the mouse C2GnT-L transcripts was detected exclusively in the kidney, whereas the other one was expressed in various other organs. Four transcripts of human C2GnT-L ranging from 2.2 to 2.8 kb were reported recently (25). Derived from 6 exons spanning over 60 kb of genomic DNA, the transcripts have identical ORF and 3'-UTR but different 5'-UTR (25). Based on these and our current findings, we propose that the various C2GnT transcripts are derived from alternative promoter usage and splicing in a tissue-specific fashion.

Expression of C2GnT-M in the airway epithelial cells can be regulated by different stimuli, including EGF (26), retinoic acid (27), Th2 cytokines (Beum and coworkers, unpublished observation), and lipopolysaccharide (28). EGF inhibits the expression of C2GnT-M but not C2GnT-L, resulting in a shift of carbohydrate structure from complex core 2–containing glycans to simple core 1–containing glycans (26). On the other hand, retinoic acid and Th2 cytokines, such as interleukin-4 and interleukin-13, enhance C2GnT-M expression. Recently, we observed that following a single intratracheal instillation of lipopolysaccharide, C2GnT-M expression was induced in the mouse airways and the induction coincided with mucus cell hyperplasia and hypertrophy (28). The sequence of the 1.2-kb fragment upstream of exon 1 and the entire sequence of intron 1 of bC2GnT-M gene analyzed by the PromoterInspector promoter analysis software (29) yielded no putative promoter. It is possible that the promoters reside in the region beyond the 1.2-kb sequence upstream of the transcription start site or novel promoters are present in this region and intron 1. Future characterization of the promoter(s) of this gene is needed to understand the mechanism of the regulation of C2GnT-M gene expression and the functions of core 2–associated carbohydrate in airway inflammation.

The bC2GnT-M gene contains four exons spanning over 5.3 kb, and the ORF is in exons 2' and 3 (Figure 6B). The entire coding sequences of C2GnT-L (11) and C2GnT-3 (16) have also been shown to localize in a single exon. However, analysis of the genomic structure of IGnT, which is a ß6GnT with a high sequence homology with other C2GnTs, revealed that its coding sequence is distributed over several exons (30). Phylogenetic analysis (31) (Figure 8) of human C2GnT-L (11), C2GnT-M (13, 14), C2GnT-3 (16), IGnT (17), iGnT (32), and five other human GnTs involved in N-linked glycan synthesis (33–37) showed that C2GnTs are closely related among themselves as compared with other GnTs. Furthermore, among the five ß6GnTs, only C2GnT-L, C2GnT-M, and C2GnT-3 are more closely related among themselves, suggesting that they are evolved from one single ancestral gene by duplication.



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  		Figure 8. Phylogenetic tree of human N-acetylglucosaminyltransferases involved in the synthesis of mucin-type and N-linked glycans. C2GnT-3 (16), C2GnT-L (11), C2GnT-M (13, 14), IGnT (17), and MGAT5 (37) are ß6 GnTs. MGAT1 (33) and MGAT2 (34) are ß2 GnT. iGnT (32) is a ß3 GnT. MGAT3 (35) and MGAT4 (36) are ß4 GnTs. C2GnT-3, C2GnT-L, and C2GnT-M, which have ORF residing in one exon, are more closely related to each other, suggesting that these three genes are derived from same ancestral gene through gene duplication.

 
We noticed that there are two inverted SINE sequences, Bov-A2 (38), in the bC2GnT-M gene, a full-length sequence (~ 260 bp) in intron 1 and a partial 5' portion (105 bp) of the sequence in exon 2'. SINEs are widely distributed in mammalian genomes and can be replicated by transcription, reverse transcription, and integration into other parts of the genomes (39). Northern blot analysis with a probe specific to Bov-A2 SINE sequence produced strong smears in all of the bovine tissues tested, but no signal was detected in a human pancreatic cancer cell line, Panc1/Muc1/bC2GnT-M. The data suggest that Bov-A2 SINE is bovine specific and that mRNA species of various lengths containing Bov-A2 SINE are highly expressed (data not shown). Because the Bov-A2 SINEs in bC2GnT-M are inverted in relation to the bC2GnT-M gene itself, their transcription will be opposite to that of bC2GnT-M gene. Transcription of SINEs can be induced by viral infection or stress (40). Also, it has been reported that active SINE can affect the transcription activity of its host genes. It will be interesting to study the effect of Bov-A2 SINE in the transcription of bC2GnT-M gene and possible role in the acquisition of the C2GnT-M gene by BHV-4.

BHV-4 is a linear, double-stranded DNA virus. Its genome consists of a 108-kb unique genome region (LUR) flanked by two stretches of tandem repeats designated as polyrepetitive DNA (25). The BHV-4 C2GnT-M gene is located at the right end of the LUR of BHV-4 genome localized at the nonconserved region of the genome for all members of the Gammaherpesvirinae family (19, 41). Sequence alignment of the bovine and BHV-4 C2GnT-M genes showed that the ORF and its 5'- and 3'-immediate flanking regions of BHV-4 C2GnT-M gene are essentially identical with those of bovine C2GnT-M (Figure 7), with the exception of 13-bp and 19-bp deletions at the 5'- and 3'-regions, respectively. The results strongly implicate BHV-4's acquisition of its C2GnT-M gene from the bovine host. However, the mechanism of such an acquisition is not clear. It is important to note that the C2GnT-M gene retained in BHV-4 genome is functional (19). Given the important roles mucin core 2–associated glycans play in immune functions (9), we believe the retention of the ORF region of bC2GnT-M gene in BHV-4 genome confers on this virus a survival advantage. The possible functions of this gene include generation of core 2 and possibly core 4 and blood group I branch structures on cell surface glycoproteins of infected cells and/or viral coat protein to evade host immune surveillance during acute infection phase and subsequent latent period. The BHV-4 can persist for a long time, possibly for life, in several lymphoreticular organs in cattle (21) and rabbits (22) following experimental inoculation. The possible pathogenic role of BHV-4 is not yet clear, although its function in important diseases such as chronic reproductive failure has long been suspected (42). Understanding the role of C2GnT-M in the pathophysiology of BHV-4 should help us develop means to prevent or manage BHV-4 viral infection in cattle.


   Acknowledgments
 
The authors acknowledge the research supports from NIH R01 HL48282, state of Nebraska LB506 and NRI-Center for Glycobiotechnology (P.-W.C.), and a graduate fellowship from the University of Nebraska Medical Center (K.H.C.). They thank Dr. Stanley Cox for critical review of the manuscript and Dr. Guillermo Orti for assistance in phylogenetic analysis.

Received in original form May 27, 2003

Received in final form September 29, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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