From the Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118-2394
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ABSTRACT |
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The mannan chains of
Kluyveromyces lactis mannoproteins are similar to those of
Saccharomyces cerevisiae except that they lack mannose
phosphate and have terminal (1
2)-linked
N-acetylglucosamine. Previously, Smith et al.
(Smith, W. L. Nakajima, T., and Ballou, C. E. (1975)
J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal
N-acetylglucosamine in their mannoproteins. The former
mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in
the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked.
We have now cloned the gene encoding the K. lactis Golgi
membrane N-acetylglucosaminyltransferase by complementation
of the mnn2-1 mutation and named it GNT1. The mnn2-1
mutant was transformed with a 9.5-kilobase (kb) genomic fragment
previously shown to contain the gene encoding the UDP-GlcNAc
transporter; transformants were isolated, and phenotypic correction was
monitored after cell surface labeling with fluorescein
isothiocyanate-conjugated Griffonia simplicifolia II
lectin, which binds terminal N-acetylglucosamine, and a
fluorescence-activated cell sorter. The above 9.5-kb DNA fragment
restored the wild-type lectin binding phenotype of the transferase
mutant; further subcloning of this fragment yielded a smaller one
containing an opening reading frame of 1,383 bases encoding a protein
of 460 amino acids with an estimated molecular mass of 53 kDa, which
also restored the wild-type phenotype. Transformants had also regained
the ability to transfer N-acetylglucosamine to
3-0- Kluyveromyces lactis mannoproteins have mannan chains
that are similar to those of Saccharomyces cerevisiae except
that they have terminal We hypothesized based on the above genetic results and our previous
observation that a 9.5-kb1
K. lactis genomic clone contained near one end the open
reading frame of the UDP-GlcNAc transporter gene (see Fig. 2 in Ref. 5) that this clone might also contain the open reading frame for the
N-acetylglucosaminyltransferase. We therefore transformed the N-acetylglucosaminyltransferase mutant (mnn2-1) with a
plasmid containing the above 9.5-kb genomic clone and determined
whether the wild-type phenotype had been restored. This was measured by the ability of transformants to bind GSII-FITC, a
fluorescent-conjugated lectin that detects terminal We found that the above 9.5-kb genomic clone did, indeed, restore the
wild-type phenotype of the N-acetylglucosaminyltransferase mutant and that this phenotype was the result of the mutants having regained their ability to transfer N-acetylglucosamine to
mannose acceptors. After subcloning of the above 9.5-kb genomic
fragment, a smaller one containing an ORF of 1,383 kb encoding a
protein of 460 amino acids with an estimated molecular mass of 53 kDa also restored the wild-type phenotype. Together, these studies demonstrate that this latter protein is the K. lactis
[ DNA ligations were performed with T4 DNA ligase (Life Technologies,
Inc.). Escherichia coli plasmid DNA was isolated with either
Wizard Plus Miniprep kit (Promega) or Nucleobond AX (the Nest Group,
Southport, MA). All transformations were performed by electroporation
as described before (5). Polymerase chain reactions were made using
Pfu DNA polymerase (Stratagene) in a PTC-100 thermal
controller (MJ Research, Watertown, MA).
Strains, Media, and Growth Conditions--
K. lactis
strain MW103-1C (Mat
K. lactis cells were grown at 26 °C either in yeast
extract/peptone/dextrose media or SD-U synthetic uracyl-free media
(0.67% yeast nitrogen base without amino acids, 2% glucose, and 2%
SCM-URA (synthetic mix of amino acids and inositol) (8) (Bufferad, Inc.)
Plasmids--
Two additional plasmids were derived from the
initial genomic clone in pCA66 to narrow the position of the
N-acetylglucosaminyltransferase. After digestion of pCA66
with NheI and SalI, the resulting 4.3-kb Nhe-Nhe fragment was subcloned into the
Nhe site of the K. lactis expression vector Kep 6 (9) generating pCE1. The 2.3-kb Nhe-Sal fragment
was subcloned into Kep 6 linearized with Nhe and
Sal, generating pCE2. Plasmid pCA73 was described previously
(5).
Labeling of K. lactis with GSII-FITC and Fluorescence-activated
Cell Sorting--
K. lactis cells, grown either in yeast
extract/peptone/dextrose media or uracyl-free media, were labeled with
GSII-FITC and run through a FACScan (Becton Dickinson) as described
previously (10).
Subcellular Fractionation and Assay for Sequencing and Data Analysis--
The 4.3-kb NheI
fragment derived from pCA66 and containing the complete ORF for the
N-acetylglucosaminyltransferase was subcloned into
pBluescript II SK (+) (Stratagene) linearized with XbaI. Sequencing was performed in an Applied Biosystems DNA sequencer by
using the dideoxy chain termination method (Molecular Genetics Facility, University of Georgia, Athens, GA). Sequences were assembled and analyzed using DNAstar programs (DNAstar, Madison, WI), and data
base searches were made through the NCBI Blast server (National Center
for Biotechnology Information, Bethesda, MD)
Northern Blot Analysis--
Total RNA was obtained from Y-43
Probes were obtained by polymerase chain reaction using pCA66 as
template and the primers 7N0740 (5'-CAGAGTTCATCCCAGATTTGAC-3') and
7H2017 (5'-TATGGTACTACCTTTTACGCACGG-3') for
the-N-acetylglucosaminyltransferase and 3P1234
(5'-AGCTCATTTCTGGTACTATTTCG-3') and 7H0286
(5'-ATTTACGCACTAGAAGCACGACAG-3') for the UDP
N-acetylglucosamine transporter. In both cases the product
included most of the ORF.
Polymerase chain reaction reaction products were separated on a 1.2%
agarose gel, and the band of interest was cut and eluted by freeze-thaw
and centrifugation in Spin-X centrifuge tube filters (Costar Inc.,
Corning, NY). DNA was purified by phenol/chloroform extraction and
ethanol-precipitated before being used in the labeling reaction.
The 9.5-kb Genomic Clone Correcting the K. lactis Golgi UDP-GlcNAc
Transporter Defective Mutant (mnn2-2) Also Corrects the
N-Acetylglucosaminyltransferase-defective Mutant (mnn2-1)--
Based
on the previous observation by Ballou and co-workers (2) that crossings
between what is now known to be the UDP-GlcNAc transporter mutant and
the N-acetylglucosaminyltransferase mutant indicated that
both genes were in close proximity, and the existence of sufficient DNA
at either side of the transporter gene in the genomic clone to contain
the transferase ORF led us to hypothesize that the 9.5-kb genomic clone
might also contain the transferase gene. Our strategy to isolate the
genomic DNA encoding the N-acetylglucosaminyltransferase relied on the isolation of those K. lactis mnn2-1
transformants that had acquired wild-type binding to GSII-FITC lectin
after transformation with the genomic insert-containing plasmid
encoding also the K. lactis Golgi UDP-GlcNAc transporter
(5).
K. lactis KL11, an
N-acetylglucosaminyltransferase-deficient strain, was
transformed with the above pCA66 plasmid containing the 9.5-kb genomic
DNA fragment. Cells were plated in uracyl-free media, individual
colonies were isolated and replated, and cells were labeled with
GSII-FITC lectin. Fig. 1 shows that the
transformed mutant K. lactis cells, growing in uracyl-free
media, had acquired fluorescence in the wild-type range. This suggests
a restoration of N-acetylglucosamine to cell wall mannans
and that the corresponding transferase activity was now functional (see
below).
The N-Acetylglucosaminyltransferase and the UDP-GlcNAc Transporter
Genes Are in Different Regions of the pCA66 Plasmid--
To further
determine the region of the genomic 9.5-kb fragment encoding the
N-acetylglucosaminyltransferase, the DNA was digested with
restriction enzymes so that an Nhe-Nhe fragment
of 4.3-kb (pCE1) and one Nhe-SalI of 2.3 Kb
(pCE2) were obtained (Fig.
2A). The 4.3-kb
insert-containing plasmid was still able to restore fully the GSII-FITC
binding phenotype as wild-type cells, whereas the one containing the
2.3 kb was not, as did pCA73 containing the entire ORF for the
transporter (Fig. 2B).
To determine directly whether this wild-type phenotype recovery was
indeed the result of restoration of
N-acetylglucosaminyltransferase activity, Golgi-enriched
membranes were prepared from KL11 transformed with pCE1, and enzymatic
activity was measured in vitro using 3-0-
Sequencing the genomic DNA of pCE1 revealed an ORF of 1383 bp encoding
a protein of 460 amino acids (Fig. 4).
The predicted molecular mass of the polypeptide is 53 kDa with a pI of
4.98, in excellent agreement with the pI of 4.9 previously determined during the enzyme purification (3). The reported molecular mass of the
partially purified native protein, obtained by size-exclusion chromatography of detergent-solubilized enzyme in detergent-free buffer, is 300 kDa (3), raising the possibly that the enzyme is
multimeric or tightly associated with other proteins. The ORF is
located upstream from the gene encoding the
UDP-N-acetylglucosamine transporter and separated by an
intergenic region of 948 bp. Both genes are transcribed in the same
direction. Analysis of the primary sequence reveals a putative type II
protein; toward the amino terminus there is a likely noncleavable
signal peptide determining a transmembrane-anchoring segment separating
10 amino acids, probably a cytoplasmic tail, from a 431-luminal domain
presumptively containing the substrate recognition and catalytic site.
There are four consensus N-glycosylation sites through the
protein (Fig. 5, panel A);
most likely some of them are utilized, as Douglas and Ballou (3) reported binding of enzymatic activity to concanavalin A (3).
UDP-N-Acetylglucosaminyltransferase and UDP N-Acetylglucosamine
Transporter Are Translated into Different Messengers--
Northern
blots of total RNA from wild-type strain Y-43
Although the 2.4-kb transcript has the size to contain the ORFs of the
transporter and the transferase, when the same membrane is probed for
the transporter, only a band of 1.5 kb could be seen (Fig. 6).
Furthermore, the intensity of the transcript for the transporter was
similar both in the wild type and in the mutant for the transferase;
therefore, it can be concluded that the
UDP-N-acetylglucosamine transporter and the
N-acetylglucosamine transferase are transcribed into
independent messenger RNAs.
Besides the N-acetylglucosaminyltransferase (Kl-GNT1)
reported in this paper, the only other known eukaryotic
UDP-N-acetylglucosamine:oligosaccharide An interesting possibility is that both YOR320p and YEL004p (the
S. cerevisiae sequence homolog of the K. lactis
UDP N-acetylglucosamine transporter) operate on the same,
yet unknown, pathway in a similar manner as their K. lactis
homologs do. In this case, probably solving the problem of the
biochemical role of one of the above proteins will answer the function
of the other as well.
Surprisingly, a domain with 23% identity and 48% similarity spanning
amino acids 97 through 210 in the putative Golgi luminal portion of
K1-GNT1p was found to be conserved with glycogenins of diverse origin
(Fig. 5B). This domain is also highly conserved among a
group of glycogenin-like proteins from nematodes, plants, fungi, and
bacteria (17). Interestingly, this domain also contains the sequence
IVYFDSDSI, comprising amino acids 193 to 201 (Fig. 5B). This
is a highly conserved motif found among a very broad group of
glycosyltransferases in the form of a consensus sequence hhhhDXDXh (where h is a hydrophobic amino acid)
(18). Aspartates in the motif DXD were found to be essential
for the activity of the S. cerevisiae Mnn1p
It is very interesting to find two genes of related functions being
contiguous in the genome of a eukaryote such K. lactis. Although such occurrence is common in prokaryotes, with their widespread occurrence of operons, its incidence in eukaryotes is much
rarer. The norm in eukaryotes is that genes of related functions are
transcribed individually and are scattered throughout the genome.
However, there are several examples of clustered genes of related
functions. Some even are cotranscribed from a single promoter into a
polycistronic messenger as in bacterial operons. Examples are found in
trypanosomatides, nematodes, mammals, and plants (reviewed in Ref.
20.)
Of particular appeal is the case of the MAL loci in S. cerevisiae. Each locus comprises at least three genes: a
trans-acting activator, a maltose permease, and maltase (21). They
determine a unit comprising a regulatory gene adjacent to the genes of
related functions it controls, translated as independent transcripts
(22). It is very tempting to speculate that this may very well be the case for the genes for the UDP-GlcNAc transporter and transferase in
K. lactis, and studies are currently under way in our
laboratory to address this question.
It has been proposed (23) that S. cerevisiae ancestors arose
from the fusion of two diploid cells to form a tetraploid, thus leading
to genome duplication, followed by extensive gene deletions and
reciprocal translocation between chromosomes leading to gene
rearrangement. This event seemingly happened after the common ancestor
of K. lactis and S. cerevisiae had diverged. This hypothesis may explain why the homologue genes of the transferase and
the transporter of UDP-N-acetylglucosamine, although
contiguous in the genome of K. lactis, are in a single copy
on different chromosomes in S. cerevisiae.
It remains to be seen if the contiguity in the genome of the
UDP-N-acetylglucosamine transporter gene and K1-GNT1 has any physiological significance. Nothing is known about the possible regulation of the expression of either protein depending upon particular physiological conditions of the organism. If such a variation exists, it will be interesting to see if the levels of
mRNA and/or activity for these two proteins are related.
-D-mannopyranosyl-D-mannopyranoside.
The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene
encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed
independently by their own promoter. To our knowledge this is the first
demonstration of two Golgi apparatus functionally related genes being
contiguous in a genome.
INTRODUCTION
Top
Abstract
Introduction
References
(1
2)-linked
N-acetylglucosamine and lack mannose phosphate (1). Ballou
and co-workers (2) previously isolated and characterized two mutants of
K. lactis lacking terminal N-acetylglucosamine in
their mannoproteins. One mutant, mnn2-1, lacks the terminal N-acetylglucosaminyltransferase activity, whereas the other
mutant, mnn2-2, has wild-type levels of this enzyme (3). Extensive crossings between these two mutants showed parental ditype segregation (Table IV in Ref. 2) leading Ballou and co-workers (2) to hypothesize
that the mutations were either allelic or tightly linked. More
recently, Abeijon et al. (4) discovered that mutant mnn2-2
lacked the Golgi apparatus membrane UDP-N-acetylglucosamine transporter activity and thereafter cloned the gene that corrected the
mutant phenotype (5).
- or
-linked
N-acetylglucosamine; this approach had been previously used
to detect wild-type phenotypic restoration of this sugar in the Golgi
transporter-deficient mutant mnn2-2 (5).
(1
2) N-acetylglucosaminyltransferase and that the
genes for this enzyme as well as the UDP-GlcNAc transporter, each with
their own promoter, are contiguous in the K. lactis genome.
MATERIALS AND METHODS
-32P]dCTP (3000 Ci/mmol) and
UDP[3H]GlcNAc (20 Ci/mmol) were purchased from NEN Life
Science Products. Griffonia simplicifolia II lectin labeled
with fluorescein isothiocyanate (GSII-FITC) was purchased from EY
Laboratories. Restriction enzymes were purchased from New England
Biolabs.
3-O-
-D-mannopyranosyl-D-mannopyranose was purchased from Sigma.
, uraA, lysA, K+,
pKD+) (6) was a gift from Dr. H. Fukuhara (Institute Curie,
France). Y-43
(Mat a, his3) and Y-43
(3-55) (Mat a, mnn2-1,
his3) (2) were obtained from Dr. C. Ballou (UCLA). KL11 (Mat a,
mnn2-1, uraA, lysA, K+, pKD+) was obtained by
crossing MW103-1C with Y-43
(3-55) as described in Ref. 7 followed
by sporulation and tetrad dissection.
(1
2)
N-acetylglucosaminyltransferase--
Golgi-enriched membranes from
K. lactis were obtained as described (4). Enzyme assays were
performed as described previously (2) using 50 µg of membrane protein
and 2 µM UDP-[3H]GlcNAc (240 cpm/pmol) and
0.5 mM
3-0-
-D-mannopyranosyl-D-mannopyranoside in 20 mM imidazole-HCl, 20 mM
MgCl2, 0.2% Triton X-100, pH 6.5. Incubations were at
30 °C for 20 min.
(3-55) and Y-43
by the acidic phenol method (11), and 15 µg/lane were separated on a 1% formaldehyde-agarose gel
and capillary-transferred to a nylon Hybond-N+ membrane (Amersham
Pharmacia Biotech) (12). Prehybridization was performed at high
stringency conditions by including the membrane in 50% formamide, 10×
Denhardt's, 6× saline/sodium phosphate/EDTA, 0.5% SDS, and 0.2 mg/ml
salmon sperm single-stranded DNA at 42 °C for 4 h.
Hybridization was done overnight at 42 °C with double-stranded DNA
probes labeled with an oligolabeling kit (Amersham Pharmacia Biotech)
and [
-32P]dCTP as indicated by the manufactures.
Membranes were washed at 42 °C in 2× SSC (1× SSC= 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS
two times, and then two times in 1× SSC, 0.1% SDS. The final wash was
done twice in 0.5× SSC, 0.1% SDS.
RESULTS
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Fig. 1.
The plasmid pCA66 containing the ORF for the
UDP N-acetylglucosamine transporter also includes the
ORF for the N-acetylglucosamine transferase.
K. lactis strains were surface-labeled with GSII-FITC lectin
and separated by fluorescence-activated cell sorter. Cells of wild-type
phenotype (MW103-1C) show higher fluorescence than the mutant mnn2-1
lacking N-acetylglucosamine transferase activity (KL-11).
When the latter is transformed with the plasmid pCA66, also containing
the ORF for the UDP N-acetylglucosamine transporter, the
fluorescence is restored to values similar to those of the wild
type.
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Fig. 2.
Panel A, positions of pCE1, pCE2, and
pCA73 on the K. lactis genomic clone pCA66. Panel
B, K. lactis mnn2-1 mutant for
N-acetylglucosamine transferase activity (KL11), was
transformed with pCA66-derived plasmids pCE1, pCE2, and pCA73. Cells
were labeled with GSII-FITC lectin and separated by
fluorescence-activated cell sorter. Only the plasmid pCE1 comprising a
4.3-kb fragment of pCA66, including the complete ORF for K1-GNT1,
restores the wild-type phenotype in the mutant.
-D-mannopyranosyl-D-mannopyranoside
as exogenous acceptor. As can be seen in Fig.
3, pCE1 was able to restore the
transferase activity to levels comparable or even slightly higher than
wild-type cells.
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Fig. 3.
In vitro N-acetylglucosaminyl
transferase activity. Golgi-enriched membranes were obtained as
described under "Materials and Methods" from wild-type
(wt) K. lactis MW103-1C, K. lactis
N-acetylglucosamine transferase mutant KL11, or the former
transformed with the plasmid pCE66, which restores the in
vivo phenotype. When indicated,
3-0- -D-mannopyranosyl-D-mannopyranose
was used as acceptor. Valves are the mean of three to six
determinations of two independent membrane preparations.
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Fig. 4.
Genomic K. lactis sequence
containing the UDP N-acetylglucosamine transferase
gene. The sequence shown is located directly upstream of GenBankTM
U48413 containing the coding region for the UDP
N-acetylglucosamine transporter (5). The one-letter amino
acid code is used on the translation of the coding region. The putative
transmembrane domain is underlined. The combined sequences
can be accessed from GenBankTM under accession number AF106080.
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Fig. 5.
A, hydrophilicity plot of the K. lactis N-acetylglucosaminyl transferase as determined
with the Kyte-Doolittle algorithm (24) and a window size of 17 amino
acids. Dots are asparagines that are part of
N-glycosylation consensus sequences. The horizontal
bar underlines the sequence shown in panel B.
B, alignment of a domain highly conserved between Kl-GNT1p
and glycogenins using the Jotum-Hein algorithm (25). The consensus
sequence hhhhDXDXh (18) is
boxed.
show two transcripts
of 2.4 and 1.5 kb using as probe the UDP-GlcNAc transferase gene (Fig.
6). The same two transcripts are in the mnn2-1 transferase mutant but with lesser intensity (Fig. 6). Preliminary results from our
laboratory2 indicate that
there is a nonsense mutation in the ORF of the transferase gene. This
type of mutation is known to induce message instability, and the
results of Fig. 5 appear to suggest that this indeed has occurred.
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Fig. 6.
Northern blot of total RNA for K. lactis wild type (wt;
Y-43 ) or mutant (mut.) for
the a N-acetylglucosaminyl transferase
Y-43
(3-55) using as probe the transferase
and UDP-GluNAc transporter genes.
DISCUSSION
-N-acetylglucosaminyltransferases are
-GlcNAc TI and
TII, which participate in the synthesis of heparin/heparan sulfate,
transferring
-N-acetylglucosamine to glucuronic acid in
the core or in the elongating polymer, respectively (13). Recently Lind
et al. (14) reported that the
-N-acetylglucosaminyltransferase and glucuronyl
transferase activities necessary to elongate glycans in heparan
sulfate-type proteoglycans were contained in a single polypeptide of 70 kDa. This polypeptide shows no significant overall homology with
Kl-GNT1p (15% identity and 23% similarity). Sequence analysis of the
glycosyltransferases cloned so far detects little if any sequence
homology between members of catalytically distinct families of enzymes
(15, 16). This is true even in most of the cases where they have in
common either the sugar donor or the acceptor. We found extensive
homology only with YOR320p of S. cerevisiae. (36%
identical, 55% similar at amino acid level throughout the entire ORF).
This is a 491-amino acid protein with a putative noncleavable
endoplasmic reticulum translocation signal determining a 10 amino acid
cytoplasmic tail and a large lumenal domain whose function is still
undetermined. It doesn't seem likely that YOR320p has a similar
enzymatic activity than its K. lactis homolog, and no
oligosaccharide or glycolipid modified with
N-acetylglucosamine has been described so far in S. cerevisiae, although the possibility that such modification is
transiently present at some point during the cell cycle or under
certain growth conditions cannot be ruled out.
-1,3-mannosyltransferase, with folding or oligomerization not being
affected (18). Furthermore, mutations of the aspartic acids of the same
motif in the lethal toxin of Clostridium sordelli abolishes
its glucosyltransferase activity and toxicity (19). Affinity-labeling
with azido UDP-glucose is also suppressed. It has been hypothesized
(18) that this domain may play a role in the coordination of the
interactions between the divalent cation required for these enzymes to
be active and the pyrophosphate in the sugar nucleotides they use as
donors of the glycosidic moiety. Thus, it could be the case that this homologous domain in K1-GNT1p may play a similar role, interacting with
the UDP-GlcNAc, although the significance of the more extended homology
with glycogenins is unclear.
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ACKNOWLEDGEMENT |
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We thank Glenn Paradis, Michael Jennings, and Michael Connelly (MIT flow/cytometry Core Facility) for help with cell sorting and Dr. Hiroshi Fukuhara, Institute Curie, Orsai, France, for K. lactis strains and plasmids.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 30365 (NIGMS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, Boston University Goldman School of Dental Medicine, 700 Albany St., W200, Boston, MA 02118-2594. Tel.: 617-414-1040; Fax:
617-414-1041; E-mail: chirschb{at}bu.edu.
2 L. Puglielli, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase(s); ORF, open reading frame; FITC, fluorescein isothiocyanate; GSII-FITC, G. simplicifolia II lectin conjugated to FITC.
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REFERENCES |
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