Complete cDNA Cloning, Genomic Organization, Chromosomal
Assignment, Functional Characterization of the Promoter, and Expression
of the Murine Bamacan Gene*
Giancarlo
Ghiselli
,
Linda D.
Siracusa§, and
Renato V.
Iozzo
§¶
From the
Department of Pathology, Anatomy, and Cell
Biology and the § Kimmel Cancer Center, Jefferson
Medical College, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
 |
ABSTRACT |
Bamacan is a chondroitin sulfate proteoglycan
that abounds in basement membranes. To gain insights into the bamacan
gene regulation and transcriptional control, we examined the genomic
organization and identified the promoter region of the mouse bamacan
gene. Secondary structure analysis of the protein reveals a sequential organization of three globular regions interconnected by two
-helix coiled-coils. The N- and the C-terminal ends carry a P-loop and a DA
box motif that can act cooperatively to bind ATP. These features as
well as the high sequence homology with members of the SMC (structural maintenance of
chromosome) protein family led us to conclude that bamacan
is a member of this protein family. The gene comprises 31 exons and is
driven by a promoter that is highly enriched in GC sequences and lacks
TATA and CAAT boxes. The promoter is highly functional in transient
cell transfection assays, and step-wise 5' deletions identify a strong
enhancer element between
659 and
481 base pairs that includes
Jun/Fos proto-oncogene-binding elements. Using backcrossing
experiments we mapped the Bam gene to distal chromosome 19, a locus syntenic to human chromosome 10q25. Bamacan is differentially
expressed in mouse tissues with the highest levels in testes and brain.
Notably, bamacan mRNA levels are low in normal cells and markedly
reduced during quiescence but are highly increased when cells resume
growth upon serum stimulation. In contrast, in all transformed cells
tested, bamacan is constitutively overexpressed, and its levels do not
change with cell cycle progression. These results suggest that bamacan
is involved in the control of cell growth and transformation.
 |
INTRODUCTION |
Proteoglycans are specialized glycoproteins found in all
connective tissues and on the surfaces of cells (1). The heterogeneity of proteoglycan structure is a reflection not only of the variation in
amino acid sequence of the protein core but also variation in the type
and size of their glycosaminoglycan chains (2, 3). There is mounting
evidence that the proteoglycan constituents of the extracellular matrix
play a crucial role in modulating cell phenotype and growth. This has
been particularly well established with regard to the neoplastic
process inasmuch as remodeling of the extracellular matrix represents
an important hallmark of the disease (4). A large body of literature
exists documenting the presence of increased levels of proteoglycans
with altered composition in human tumors, primarily those of epithelial
origin, such as breast, colon, and lung (5). Furthermore there is
convincing evidence that tumor formation is associated with aberrant
expression of proteoglycans (6). For instance, altered proteoglycan
biosynthesis is a phenotypic trait of neoplastic cells that is
maintained at the metastatic site (7, 8) and changes in proteoglycan
expression either precede (9) or are induced by malignant
transformation (3).
Bamacan is a chondroitin sulfate proteoglycan originally isolated from
organ cultures of embryonic parietal yolk sac (Reichert's membrane)
(10, 11). Bamacan has been recently identified as a component of the
basement membrane in the Engelbreth-Holm-Swarm tumor matrix (12), the
renal mesangial matrix (13) and possibly of the basement membrane
of other tissues (14). Its amino acid sequence, deduced from the cloned
rat cDNA (15), reveals unique features among proteoglycans such as
the presence of two coiled-coil domains and protein motifs potentially
implicated in cell adhesion. The attachment of glycosaminoglycan chains
is possible at five potential glycanation sites located at both the N-
and the C-terminal ends. The functional role of bamacan can be only
speculated at present, although developmental studies suggest that
bamacan expression is tightly regulated during mammalian embryonic
development (16, 17).
In this study, we report the complete characterization and sequencing
of the murine bamacan (Bam) cDNA, the genomic
organization, and the structural-functional characterization of the
promoter. In addition, we mapped the Bam gene to distal
chromosome 19. The high conservation of the deduced protein core in
comparison with rat, human, and bovine bamacan proteins supports the
concept that the protein may play crucial roles in cell biology. The
strong sequence homology with the SMC (structural
maintenance of chromosome) family of proteins
and in particular the similarity to the SMC3 subclass predicts that
this proteoglycan may be implicated in chromosome dynamics including
chromosome condensation, duplication, and X chromosome dosage (18, 19).
The architecture of the bamacan promoter reveals several
cis-acting elements capable of recognizing nuclear factor
proto-oncogenes and factors involved in cell cycle regulation, whereas
the remarkable complexity of the genomic organization raises the
possibility that bamacan splicing variants might be expressed. Notably,
bamacan is differentially expressed in murine tissues, and its levels
are dramatically enhanced in transformed cells. In normal cells, its
expression varies as a function of their growth status. Collectively,
these results suggest that bamacan may play a role in cell growth and transformation.
 |
EXPERIMENTAL PROCEDURES |
Materials and Cells--
All reagents were of molecular biology
grade. [
-32P]dATP and [
-32P]dATP
(~3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. Cells
were cultured in Dulbecco's modified Eagle medium supplemented with
10% fetal bovine serum. The cell lines utilized were: 3T3 Swiss albino
mouse fibroblasts that display contact inhibition and serum dependence
and a 3T3-L1 clone obtained in our laboratory that has lost sensitivity
to contact inhibition; mouse mammary C3 epithelial cells and their
malignant counterpart C7 (a gift of J. E. Knepper, Villanova
University) derived from normal and neoplastic mammary tissue of
MMTV/v-Ha-ras transgenic mice, respectively; a highly
metastatic variant of the Lewis lung carcinoma cells (HM-LLC); the
immortalized MLE-10 hepatocytes (20); BAM1, a Engelbreth-Holm-Swarm
tumor-derived cell line producing basement membrane components (21);
J774-A1 murine macrophages; YAC-1, a lymphoma cell line growing in
suspension; M2 melanoma; CMT-93 colon carcinoma; and COMMA-D, a mammary
tumor cell line.
Identification and Characterization of the cDNA and Genomic
Clones--
Based on the published sequence for rat bamacan cDNA
(15) and BLAST search of the dbEST Data Base, we procured
five mouse expressed sequence tag
(EST)1 clones (AA144255,
AA164123, AA119690, AA240205, and AA209705) from the IMAGE consortium
through the Genome Systems service. DNA sequencing was performed by an
automated sequencing system (Applied Biosystems) using oligonucleotides
(18-21 bp) external to the polylinker sequences of the cloning
vectors. The obtained base sequences were aligned and analyzed using a
Omiga 1.1 software for Windows 95. For genomic analysis, we screened a
129SvJ mouse liver genomic library (Stratagene) in Lambda FIX II
vector. The library was screened by Southern blotting using PCR-generated probes corresponding to selected internal regions of EST
clones AA144255, AA164123, and AA240205 (henceforth identified as
probes P144, P164, and P240, respectively). The probes were
32P-labeled using the Prime-it II random-primed labeling
kit (Stratagene) according to the manufacturer's instructions. DNA
hybridization was performed under stringent conditions by washing the
hybridized filters at 65 °C in solutions of progressively low salt
concentration in the presence of 1% SDS. Approximately 106
recombinant clones were screened. Positive colonies were identified through four rounds of colony lifting. Phage DNA was isolated by a
modification of the polyethylenglycol precipitation method in the
presence of RNase followed by DNA precipitation with ethanol. To
achieve purification at level suitable for direct sequencing, phage DNA
was subjected to agarose electrophoresis, and the DNA was recovered by
absorption onto glass beads using the Geneclean II kit (Bio101).
Automatic sequencing was performed from either purified phage DNA
preparations or suitable fragments subcloned into pBluescript plasmid.
Identification and Functional Analysis of the Promoter--
A
segment of DNA 5' of exon 1 or step-wise deletions were subcloned into
a pGL3 luciferase reporter vector (Promega). Specifically, a
DraI-BsrGI insert was excised from the genomic
EcoRI subclone containing this sequence and ligated at the
SmaI-NcoI sites of the reporter vector. To
achieve directional insertion and at the same time convert the
BsrGI and NcoI ends to compatible 5' overhang ends, the promoter insert and the linearized vector were incubated with
T4 DNA polymerase in the presence of dNTPs mixture to achieve partial
filling. Further manipulation of the promoter sequence was carried out
by taking advantage of the SacI, ApaI, and
BalI restriction sites located within the promoter sequence.
All the constructs were analyzed by restriction digestion, and the
relevant ligation sites were fully sequenced. Transient transfection
was performed by a modified calcium phosphate method. Briefly, various constructs were mixed with a solution containing calcium -phosphate Maximizer (Stratagene) in 2 M CaCl2 and added
dropwise to a 2× Hanks phosphate butter. After careful
mixing, the solution was added to the cells, and the incubation was
continued for 8 h. The transfection mixture was then removed, and
the cells were fed with regular medium. After 24 h the luciferase
activity was assayed using the dual luciferase reporter assay system
(Promega). As an internal control we used an empty firefly luciferase
vector and a SV40-pGL3 vector in which the firefly luciferase
expression is driven by the early SV40 promoter. The cytomegalovirus
Renilla luciferase vector was used as internal control to
normalize for the efficiency of the transient transfection. All the
samples were analyzed in quadruplicate, and two sets of experiments
were carried out.
Primer Extension, Northern Blotting, and Chromosomal
Mapping--
Total RNA was isolated using the Tri-Reagent solution
from subconfluent cells and stored in 70% ethanol at
20 °C until
used. Synthetic oligonucleotides (21 bp) antisense and complementary to
bamacan cDNA spanning a 21-bp region 3' to the ATG site, were end-labeled with [32P]ATP using 10 units of T4
polynucleotide kinase (Promega). Following incubation at 37 °C for
10 min, the enzyme was heat inactivated at 90 °C for 15 min, and the
radiolabeled primers were purified by chromatography on a prepacked
RNase-free Sephadex G-25 column. Specific activity of the labeled probe
was >1016 cpm/mmol. For primer extension, ~2 ng of the
labeled probe was incubated with 10 µg of total RNA and heated at
85 °C for 5 min, and 32P-labeled DNA-RNA annealing was
allowed to proceed at 50 °C for 1 h. Primer extension was
completed by addition of reverse transcriptase (Promega) in the
presence of dNTPs at 42 °C for 30 min and stopped by the addition of
70% ethanol. The precipitated DNA was dissolved in 30% formamide and
loaded onto a 6% polyacrylamide sequencing gel. Northern blotting with
probes spanning various regions of the bamacan cDNA was performed
as described before (22). Additional details are provided in the text
and legends to figures. For chromosomal mapping, we used an
interspecific backcross of (AEJ/Gn-a bpH/a bpH × Mus spretus) × AEJ/Gn-a
bpH/a bpH mice (23). Segregation patterns for the loci
were determined from random subsets of 195 N2 progeny. The
Bam locus was identified using two distinct probes, P144 and
P164, from the murine bamacan cDNA. Primer pairs identifying the
D19Mit1 and D19Mit75 loci were purchased from
Research Genetics, Inc. (Huntsville, AL). PCR conditions were 94 °C
for 4 min, followed by 40 cycles at 94 °C for 30 s, 55 °C
for 45 s, and 72 °C for 45 s, ending with a single cycle
at 72 °C for 7 min. PCR products were analyzed on 2.5% agarose gels.
 |
RESULTS AND DISCUSSION |
cDNA and Genomic Cloning of Mouse Bamacan--
Based on the
published sequence for rat bamacan cDNA (15), a computer search of
the EST Data Base for homologous mouse and human sequences was
performed. This search led to the identification of 20 potential clones
for mouse bamacan originating from 6 mouse cDNA libraries and 16 potential clones for human bamacan from 3 human cDNA libraries.
Seven mouse clones were fully sequenced providing more than 95% of the
mouse bamacan cDNA (Fig.
1A). The leading sequence of
the 5'-untranslated region and an internal segment (bp 1469-1526 of
the cDNA) were not present in the clones considered; these
sequences were determined by analyzing genomic clones. To this end, a
129/SvJ mouse genomic library was screened using
32P-labeled cDNA probes P144, P164, and P240, covering
respectively 1404 bp of the 5' region, 857 bp of the middle region, and
843 bp of the 3' end. In addition, a PCR-generated probe spanning 330 bp at the 5' end of cDNA clone AA144255 was also used. With this
strategy we isolated a series of overlapping genomic clones
encompassing the entire coding sequence and further extending ~7 kb
upstream of the putative transcription start site. Clone Xba-6.2, derived from phage 3/164, encompassed the sequence
that was missing from the EST clones, thereby allowing the
determination of the complete mouse cDNA. The strategy utilized for
the identification of this clone is illustrated in Fig. 1B.
To identify the size of the bamacan transcript, murine RNA was
hybridized with 32P-labeled probes spanning two separate
areas of the bamacan cDNA. The results revealed a single bamacan
transcript of ~4.2 kb (Fig. 1C) using the two cDNA
probes.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
Determination of mouse bamacan cDNA
sequence. A, alignment of the EST clones utilized for
the determination of the cDNA sequence of mouse bamacan. The
location of the Mmip1 cDNA (24) matching part of the bamacan
cDNA sequence is also shown. The full cDNA sequence of the
proteins is illustrated with a thin line. The coding region
is represented by the shaded bar. B, Southern
blot hybridization of restriction fragments of the genomic phage 3. The
strategy for the identification of the coding region of the bamacan
gene not present in the EST clones is illustrated. Aliquots of phage 3 DNA were digested with the indicated enzymes, and the reaction products
were subjected to Southern blot hybridization with two nonoverlapping
cDNA probes (P144 and P164) obtained by PCR using EST clones
AA144255 and AA164123 as templates, respectively. A 6.2-kb fragment
generated by digestion of the genomic clone with XbaI
(indicated by an arrow) hybridized with both probes
indicating the presence of exonic sequences spanning the sequence
comprised between the two probes. This was later confirmed by direct
sequencing. C, Northern blot hybridization of RNA extracted
from mouse testes and probed with nonoverlapping bamacan cDNA
probes. Total RNA was extracted from freshly excised tissue and
analyzed by Northern blot hybridization first using mouse P144 probe.
After autoradiography, the filter was washed in the presence of 1% SDS
at 98 °C, checked for removal of the P144 probe by overnight
exposure, and hybridized with probe P164. Both autoradiographs show a
single band of ~4.2 kb. The migration of the band relative to that of
ribosomal RNA is illustrated.
|
|
While this work was underway, Gupta et al. (24) reported the
cloning of a mouse protein denoted Mmip1 with cDNA sequence identical to that of mouse bamacan between bp 1523 and 4138 (Fig. 1A). The size of the Mmip1 transcript corresponded to that
of mouse and rat bamacan (i.e. ~4 kb). Mmip1 cDNA
coded for a protein of 485 amino acids whose sequence matches that of
mouse bamacan between residue Met-537 and residue Met-1018. This short
form of bamacan cDNA was generated by the presence of additional G and A intercalating the cDNA at bp 1655 and 3150, respectively, thereby generating two stop codons that truncated the cDNA. These presumed insertions occur within exon 18 and exon 27 (see below) of the
bamacan gene. Presently, there is no evidence of gene transcription alteration by base insertion; mRNA editing by base substitution has
been reported as an extremely rare event in eukaryotic organisms and
involves an enzymatic conversion of one base into another (25). Several
lines of evidence, however, lead to the conclusion that the bamacan
gene is encoding a single protein. First, a single major transcript of
~4.2 kb can be identified using either probe P164, which is common to
both bamacan and Mmip1 cDNA, or probe P144, which should recognize
only bamacan because it is located 5' to Mmip1 (Fig. 1C).
Second, when the same probes were used for the chromosomal mapping, the
results were consistent with the two probes mapping to the same locus
(see below and see Fig. 6). Third, sequencing of both the mouse EST and
genomic clones provided the same coding sequence, thereby corroborating
the correctness of our sequencing. Fourth, both the cDNA and
deduced amino acid sequences of mouse bamacan matched those of rat
bamacan with a high degree of homology. Fifth, the open reading frames
are of identical size. Finally, the mouse and the rat bamacan sequences are highly homologous (~98%) to HCAP, a human protein that is associated with chromosomes (henceforth identified as human bamacan) (26). Based on this compelling evidence, we conclude that Mmip1 is
either the product of an as yet unidentified mRNA editing process or, more likely, a truncated cDNA with scrambled ends.
The Murine Bamacan Protein--
Compared with the published
sequence of rat bamacan (15), the mouse protein (Fig.
2) extends for an additional 32 amino acids at the C terminus. The early termination of the rat bamacan coding sequence is due to the presence of a stop codon located at bp
3668. In contrast, mouse and human bamacan cDNAs are of the same
length and code for proteins of identical amino acid number. Bamacan,
unlike other proteoglycan core proteins, displays a high degree of
sequence conservation in the three species thus far examined. Protein
conservation is at the ~98% level in human, rat, and mouse. In
particular, mouse bamacan differs from the rat protein at residue 517 (Asp to Glu), 735 (Ile to Thr), 786 (Leu to Pro), 810 (Glu to Lys), 865 (Glu to Gln), and 1051 (Gly to Ala). The difference between mouse and
human bamacan is even more contained, consisting of a conservative Glu
to Asp substitution at residue 517. Primary structure analysis fails to
reveal a signal peptide sequence. Potential glycanation sites (Ser-Gly)
are present at residues 36, 249, 1073, 1081, and 1116. The sequences
ETSGE and GEGSGE beginning at residues 247 and 1071, respectively, are also present in the cell surface heparan sulfate proteoglycan syndecan-1 (27) where they act as the attachment sites for chondroitin sulfate. The latter motif is also found in the chondroitin sulfate proteoglycan versican (28). The ability of some of these sequences to
act as functional glycanation sites has been directly demonstrated by
expression in COS-7 cells of a fusion protein composed of bamacan amino
acid 1029-1128 sequence fused to the IgG-binding portion of protein A
(15). Metabolic labeling of the transfected cells gave rise to a
product that migrated as a broad smear on SDS-polyacrylamide gel
electrophoresis in untreated or heparinase III-treated samples but as a
discrete product after chondroitinase ABC digestion, consistent with
the fusion protein being secreted as a chondroitin sulfate proteoglycan
(15).

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2.
Deduced amino acid sequence of mouse bamacan
from cloned cDNA. The potential glycanation sites
(SG) are boxed and are contained within ETSGE and
GEGSGE sequences at residues 247 and 1070, respectively. The LRE and
the VTXG motifs, both potentially mediating cell adhesion,
are double underlined. The leucine zipper domain extending
from residues 800 to 821 (underlined) is harbored in an
amphipathic -helical region. The sequences NGSGKSN and
LSGGQX24DEX4LD beginning
at residues 32 and 1114, respectively, are also shown in
bold and are highly conserved in proteins involved in the
SMC protein family. The putative nuclear localization signal motif
KKLEK located at residue 395 is shown in bold and
underlined. Finally, a 33-amino acid sequence at the
C-terminal end of the protein (bold italics) represents the
additional sequence of mouse bamacan not detected in the rat
cDNA.
|
|
Other potentially relevant motifs are found at position 552 (VTxG),
which has been implicated in mediating cell adhesion and an LRE motif
at residue 850. Further, a leucine zipper DNA-binding domain can be
identified extending from residue 800 to 821 and is harbored in an
amphipathic
-helical region (24). Finally, the sequences NGSGKSN
(P-loop motif) and LSGGQ
X24DEX4LD (DA box motif)
starting at residues 32 and 1114, respectively, are highly conserved in
SMC proteins and are potential sites for ATP binding (19, 29).
Organization of the Bamacan Gene vis à vis the Protein
Modules--
Sequencing of the genomic clones spanning the
Bam locus identified 31 exons (Table
I) contained within ~45 kb of genomic DNA (Fig. 3). Exons range between 39 bp
(exon 3) and 440 bp (exon 31) in size. Exons 14-21 are located within
large intronic sequences. The rat bamacan protein has been postulated
to be organized into five structural domains (15). The information we
have assembled on the genomic organization allowed the matching of
these domains to specific groups of exons of the bamacan gene. Domain I
is predicted to assume a globular conformation and is coded by exons
1-8. The highly conserved NGSGKSN P-loop motif is encoded by a single
exon (exon 3). Exons 9-17 code for a protein domain that can assume
-helix coiled-coil conformation. Exon 10 contains the ETSGE
consensus sequence for glycanation. The third structural domain of the
protein is coded by exons 18-20. This short connecting domain can
assume a globular conformation. This domain is followed by a fourth
domain that can assume an
-helix coiled-coil conformation similar to that of domain II. This domain is encoded by exons 21-27 and harbors the leucine zipper DNA-binding domain (exons 23 and 24). Domain V may
assume a globular conformation and is coded by the exons 28-31. This
domain contains the GEGSGE consensus sequence for glycanation, which is
coded by exon 28. The DA box motif is coded by exon 29.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Genomic organization of mouse bamacan.
The position and size of the five genomic clones encompassing the 31 exons of the bamacan gene are shown in the top panel, and
the intron-exon organization is shown in the lower left
panel. The bamacan gene is annotated to indicate the
correspondence between the protein structural domains and the bamacan
exons (lower right panel). The model of bamacan is modified
from Wu and Couchman (15). For additional information see Table
I.
|
|
Domains I, III, and IV share a high degree of sequence homology with
members of the SMC protein family, whereas domains II and IV are
homologous to the myosin heavy chain. The sequential structural
organization and protein homology as well as the presence of a P-loop
and DA box motifs, respectively, at the N- and the C-terminal ends of
the protein identify bamacan as a member of the SMC protein family.
Recently the protein sequence of bovine SMC3 has been made available
(GenBankTM accession number AF072713). The protein has the
same degree of homology to mouse bamacan as the rat and the human
proteins. We therefore assign bamacan to the SMC3 subfamily. The other
members of this family are located intracellularly and are implicated in the condensation of chromatin and in gene dosage mechanism (30).
These proteins can directly interact with DNA. The P-loop and the DA
box are believed to act cooperatively to bind ATP. When this occurs it
triggers the structural alteration of the molecule leading to its
contraction and the condensation of the bound DNA to form chromatin.
Extracellular versus Intracellular Species--
Bamacan, unlike
the other members of the SMC protein family, has been detected both
extracellularly and intracellularly within the nucleus. The lack of a
signal peptide is at odds with the observation that all the
extracellular proteoglycans thus far identified have a well conserved
signal peptide. On the other hand, the structural motif analysis of the
bamacan amino acid sequence has further revealed that the protein
harbors a "bipartite" nuclear localization motif. This motif
(KKELK), located at residue 395 in exon 15, has been shown to signal
transport to the nucleus in other proteins (31). Although these results
do not exclude the possibility that bamacan can be secreted, they do
corroborate the observation that this protein may have primarily a
nuclear localization. An intriguing possibility is that the glycanation of bamacan may mediate its extracellular transfer. Perhaps part of the
newly translated protein may escape its nuclear fate and be
post-translationally modified to become a proteoglycan. Notably, other
chromatin-associated proteins have been identified extracellularly despite their lacking a signal peptide. The most remarkable case is
that of histone H1, which can act as cell surface receptor for
thyroglobulin and has been shown to be secreted and act as a potent
growth factor (32). Proteoglycans can also play an important functional
role within the nucleus. For example glypican and biglycan, a cell
surface proteoglycan and a extracellular matrix proteoglycan,
respectively, have been immunologically detected in the nuclei of
neuronal cells and are thought to play a specific function during the
cell cycle (33). Both display chromosomal binding, but in addition
glypican exhibits dynamic properties moving from the cytoplasmic
compartment to the nucleus during mitosis. The core proteins of both
proteoglycans harbor a nuclear localization signal that appears to be
functional inasmuch as its mutation leads to ablation of their ability
to move to the nucleus during mitosis.
Structural and Functional Characterization of the Bamacan
Promoter--
Sequence analysis of the 5'-flanking region of the
Bam gene (Fig. 4A)
revealed the presence of several cis-acting factor-binding motifs involved in growth control and cytokine stimulation such as:
IRF1, a primary target of signal transduction induced by interferon-
and interleukin-6; NF- I, which binds a nuclear factor induced by
TGF-
; two GR motifs elements recognizing the glucocorticoid receptor; and a E2F-1 recognizing a factor involved in cell cycle regulation (34). The presence of E2F-1 cis-acting elements
is noteworthy insofar as it has not yet been identified in the promoter region of any other proteoglycan thus far investigated (35). Members of
the E2F transcription factors regulate the expression of a number of
genes important in cell proliferation, particularly those involved in
progression through the G1 phase and into the S phase of
the cell cycle. The activity of E2F factors is regulated through
association with the retinoblastoma tumor suppressor protein. The
presence of cis-acting elements for proto-oncogene acting as
nuclear transcription factors is noteworthy in relation to the possible
involvement of bamacan in cell transformation. In particular, a
v-jun/c-fos binding motif is located ~0.6 kb
upstream of the major transcription start site (see below).

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 4.
Structural and functional analysis of the
bamacan promoter. A, putative regulatory motifs
(bold and underlined) are labeled above the
coding strand. Sp1, ubiquitous zinc finger transcriptional
factor. GCN4, ubiquitous activator of genes involved in
protein and purine biosynthesis. UCRBP and TGT3,
positive transcriptional elements. UCRF-L, negative
transcriptional factor. TFIID, a general transcriptional
factor required for transcriptional initiation by RNA polymerase II.
GR, a factor that mediates gene induction/repression by
glucocorticoids, member of the steroid hormone receptor superfamily.
AP-1, ubiquitous factor that binds homodimers of
Jun or heterodimers of Jun/Fos or
Jun/ATF family members. NF1 and CTF,
ubiquitous factors induced by TGF- that interact with DNA polymerase
to enhance DNA replication. C/EBP , a factor that
regulates the balance between cell growth and differentiation, binds
Myc/Max heterodimers, and is down-regulated by TNF- .
IRF1, positive regulator for interferon- , inducible by
interleukin-6 immediate-early gene product of differentiated cells.
E2F-1, involved in the regulation of cell cycle,
overexpression of this factor may cause neoplastic transformation and
autoregulate its own promoter in a cell cycle-dependent
manner; its expression is lost in senescent cells. USF, a
factor involved in development. The analysis of the transcription
factor-binding motifs was performed on line using the TESS program
accessible at the server of the Department of Human Genetics of the
University of Pennsylvania. B, 5' extension using total RNA
from various mouse cells. Total RNA was extracted from subconfluent
3T3-L1 fibroblasts, HM-LLC Lewis lung carcinomas cells, and M2 melanoma
cells. 5' extension of labeled primers was initiated by annealing a
32P-labeled 21 bp primer complementary to the cDNA
sequence 3' to the transcriptional start codon of the bamacan gene by
incubating the reagents for 30 min at 55 °C. Transcription of the
bamacan RNA was initiated by the addition of MV-reverse transcriptase
and was allowed to proceed for 30 min at 40 °C. The sample was
subjected to electrophoresis on a 6% polyacrylamide sequencing gel.
After drying, the polyacrylamide gel was subjected to autoradiography.
32P-labeled Mr standards were used
to establish the size of the primer extension products. The
arrow denotes the major transcription start site common to
the three cell lines. The two asterisks point to other major
extension products. C, summary of luciferase expression
assays of bamacan gene promoter construct transfected in a variety of
transformed and normal mouse cells. The cells were grown to 50%
confluence and were transiently transfected with the 659-bp bamacan
promoter linked to the luciferase reporter gene. Normal cells were
harvested 36 h later when they had reached 90-95% confluence.
Transformed cells were also harvested at this time in a stage of
overconfluence and growth. Firefly luciferase values are expressed as a
percentage relative to the values recorded in C7 cells. In these
experiments, normalization of the values was based on the cell protein
content alone. The values are the means of quadruplicate
determinations ± S.D.
|
|
To establish the transcription start site(s), primer extension was
performed using total RNA from 3T3-L1 fibroblasts, HM-LLC lung
carcinoma cells, and M2 melanoma cells. The extension products were
analyzed on a 6% denaturating polyacrylamide sequencing gel using a
ladder of labeled DNA standards. Multiple transcription start sites
were identified using various mRNAs (Fig. 4B). 3T3-L1 RNA generated several bands that predict the location of the putative transcriptional start sites to be located between
285 and
59 bp
upstream of the ATG. RNA from HM-LLC and M2 cells generated fewer
extension products. The larger of these product was the same for both
cells and would be generated by a transcription start site located 95 bp upstream of the ATG site. Because the same fragment was also
detected as the major extension product in 3T3-L1, we use this as the
primary transcription start site. Multiple transcription start sites
are frequently observed in genes that lack TATA and CAAT boxes and are
GC-rich, as in the bamacan gene.
Next, we sought to determine whether this region could act as a
functional promoter in transient cell transfection assays. For this
purpose, a 752-bp genomic fragment flanking the 5' region of the
Bam gene was cloned upstream of a firefly luciferase
reporter gene in a pGL3 vector. This construct was transfected into six different cells with diverse histogenetic backgrounds along with a
Renilla luciferase reporter gene driven by a cytomegalovirus promoter to normalize for transfection efficiency. In all the transformed cell lines, the bamacan promoter displayed high activity when compared with the activity detected in normal, nontransformed cells (Fig. 4C). In particular the highest promoter activity
was detected in C7 mammary carcinoma cells. Expression in hepatoma MLE-10 cells and in melanoma M2 cells was approximately one-fourth that
detected in C7 cells. By comparison the promoter activity in normal
mammary cells (C3) and fibroblasts (3T3) was only small percentage of
the value detected in C7 cells. The bamacan promoter activity in CMT-93
rectal carcinoma cells was similarly low, suggesting that bamacan
expression may be regulated differentially in neoplastic cells.
Further experiments using the most responsive C7 mammary carcinoma
cells were carried out to investigate the functional role of the
cis-acting elements. For this purpose, step-wise deletions of the bamacan promoter were generated (Fig.
5A). Deletion of the distal
178 bp of the bamacan promoter, which harbor two GR and one AP-1 motif,
caused a sharp decrease in functional promoter activity (Fig.
5B). Recently an interaction between AP-1 jun/fos proto-oncogenes and the glucocorticoid receptor has been established and may be responsible for the coordination of the gene responses initiated by glucocorticoids (36). The relative proximity of these two
cis-acting elements suggests that these region may act as a
functional domain. Shortening of the upstream sequence from
489 to
70 did not result in a major change in the promoter activity in C7
cells. In contrast, ablation of the bamacan promoter activity was
observed after the deletion of a short 33-bp sequence harboring a UCRBP
binding motif, a transcription factor that binds the regulatory regions
of many viral and cellular genes.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 5.
Deletion construct of the bamacan gene
promoter and summary of the luciferase activity. A,
schematic representation of the 5' step-wise deletion constructs used
to test the functional activity of the bamacan promoter in transient
transfection assays and relative luciferase activity of each construct
are shown. Numbers to the left of each construct
indicate the 5' end of the promoter fragment relative to the major
transcription start site (+1) of the bamacan gene. B,
summary of luciferase expression assays of bamacan gene promoter and
various 5' deletion constructs. C7 mammary carcinoma cells were
co-transfected with the various bamacan promoter-firefly luciferase
constructs and pRL-CMV plasmid carrying the Renilla
luciferase gene. Firefly luciferase activity was assayed as described
in the text and is expressed as a percentage relative to the maximum
firefly luciferase activity produced by the 659-bp bamacan construct.
Value were normalized for transfection efficiency based on the
Renilla luciferase value recorded in the same cells. The
values represent the normalized means ± S.D. of four different
experiments.
|
|
Mapping of the Mouse Bamacan Gene to Distal Chromosome 19--
To
identify restriction fragment length polymorphisms useful for mapping
the Bam locus, genomic DNA from the progenitor strains of
the interspecific backcross, AEJ/Gn and M. spretus, were
digested with 14 restriction endonucleases, and individual digests were analyzed by Southern blot hybridization using the 5' and 3' probes for
the Bam gene independently. The restriction fragment length polymorphisms used to detect the Bam locus with the P144
probe and the sizes of the EcoRV restriction fragments that
distinguished the AEJ/Gn and M. spretus alleles are 10 and
5.9 kb versus >23.1 and 20 kb, respectively. The
restriction fragment length polymorphisms used to detect the
Bam locus with the P164 probe and the sizes of the
PvuII restriction fragments that distinguished the AEJ/Gn and M. spretus alleles are 2.4 and 1.8 kb versus
2.3 and 2.0 kb, respectively. No differences in the segregation
patterns for the P144 and P164 probes were detected among 127 N2
progeny (Fig. 6), consistent with the
possibility that these two probes represent the 5' and 3' ends of a
single gene. The segregation patterns obtained for the restriction
fragments were compared with markers that scanned the mouse genome, and
linkage was found to mouse chromosome 19. The order of the loci and the
ratio of the number of recombinants to the total number of N2 offspring
is shown in Fig. 6A. The genetic distance between
D19Mit75 and P144, P164 and between P144,
P164 and D19Mit1 is 0.8 ± 0.8 centimorgans (Fig. 6B). This location places the Bam gene in close
proximity to the Mxi1 gene, a helix-loop-helix leucine
zipper that forms heterodimers with Mmip1 and is implicated
in the transcriptional regulation of c-Myc (37). These
linkage data suggest that the human homolog of the Bam gene
most likely resides on human chromosome 10q25.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
Mapping of the bamacan (Bam)
gene to mouse chromosome 19. A, haplotype analysis of
the N2 progeny from the interspecific backcross. Loci mapped in the
backcross are listed to the left. Each column represents the
chromosome identified in N2 offspring that was inherited from the
(AEJ/Gn × M. spretus) F1 parent.
Black squares represent the AEJ/Gn allele. White
squares represent the M. spretus allele. The
asterisk indicates the one mouse that was genotyped with the
P144 probe but not with the P164 probe. The number of N2 progeny
carrying each type of chromosome is listed at the bottom.
B, genetic linkage map showing the chromosomal location of
the Bam locus. The chromosome to the left shows
the loci typed in the interspecific backcross (described in the text)
with the distance between loci given in centimorgans. The chromosome on
the right shows a partial version of the consensus linkage
map of mouse chromosome 19 (41). The dotted lines between
chromosomes indicate that the D19Mit75 and
D19Mit1 loci were used to align the maps. Our data establish
the order of the loci as centromere - D19Mit75 - Bam - D19Mit1 - telomere based on mapping with
respect to other D19Mit markers that were known to reside on
the proximal and distal sides of D19Mit75 and
D19Mit1 (A. M. Buchberg and L. D. Siracusa,
unpublished data). Loci mapped in humans are underlined;
locations of genes in the human genome are shown between the
chromosomes.
|
|
Bamacan Is Differentially Expressed in Mouse Tissues--
To
assess the levels of bamacan expression in different organs, bamacan
mRNA steady state levels were determined by Northern blotting. A
single band migrating slightly faster than the ribosomal 28 S band was
detected in all the tissues with an estimated size of ~4.2 kb (Fig.
7A). When normalized on total
RNA, the highest steady state levels were detected in the testes and
brain (Fig. 7B). Lower levels ranging between 32% and 62%
of those detected in the testes were found in muscle, heart, lung,
kidney, colon, and thymus. Much lower levels were detectable in
parenchymal organs such as spleen, kidney, and liver. In the latter,
the bamacan mRNA level was less than 5% that of testes. The same
results were obtained using probes spanning bp 60-1464 (P144) or bp
2802-3645 (P240) (data not shown), thus confirming that bamacan is
encoded by a single major transcript.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 7.
Northern blot analysis of bamacan mRNA in
various mouse tissues. A, autoradiograph of the
Northern blots of bamacan mRNA. The tissues were rapidly excised
from anesthetized animals and snap frozen in liquid nitrogen, and the
RNA was extracted with TRI reagent. The samples (~30 µg) were then
electrophoresed on an agarose gel (1%) containing 6% formaldehyde,
transferred onto a nitrocellulose membrane, and hybridized with the
P164 probe under stringent conditions. In the bottom panel
the ethydium bromide staining of ribosomal RNA (rRNA) prior
to transfer is presented for loading comparison. For the quantitative
assessment of the results, the autoradiograph was scanned and the
readings were normalized on the intensity of the ethydium bromide
stained RNA measured by reflective densitometry of the agarose gel
photograph. B, relative expression of bamacan in different
mouse adult tissues. Normalized results of the bamacan mRNA signal
in the various tissues were made relative to the value in the testes
and plotted. The bars represent the average value from the
results from two independent sets of tissues.
|
|
Bamacan Is Abnormally Expressed in Transformed Cells and Tumor
Tissues--
First, we investigated two sets of cell lines, each
composed of the normal and the corresponding transformed counterpart: (i) 3T3 Swiss albino mouse fibroblasts that maintain contact inhibition and are serum-dependent for growth and a 3T3-L1-derived
clone that has lost contact inhibition and (ii) mouse mammary C3
epithelial cells and the malignant counterpart C7 cell lines, obtained
from normal and neoplastic mammary tissue of MMTV/v-Ha-ras
transgenic mice, respectively. A significantly higher (2-4-fold)
expression of bamacan mRNA was consistently found in the
transformed cell lines (Fig.
8A). To compare the amount of
bamacan transcripts synthesized by other tumorigenic cell lines with
that of the two transformed cell lines examined above, a panel of mouse
transformed cell lines of different origins was investigated under the
same standardized condition of growth (Fig. 8B). Several
interesting observations were made: (i) a single transcript of ~4.2
kb was detected in all the samples, (ii) the levels of bamacan
expression were variable among the cell lines examined, (iii) the
highest expression of bamacan was detected in 3T3-L1 transformed cells, in a highly metastatic variant of Lewis Lung Carcinoma cells, and in
the mammary carcinoma C7 cells, (iv) lymphoma cells YAC-1 had the
lowest bamacan level, and (v) the immortalized hepatic cells MLE-10
(20) expressed significant amounts of bamacan in contrast to the very
low level displayed by hepatocytes.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 8.
Abnormal expression of bamacan in transformed
cells and neoplastic tissues. A, hybridization
autoradiograph of RNA from normal (3T3) and transformed (3T3-L1) mouse
fibroblasts, and normal (C3) and malignant (C7) mammary cells. Cells
were grown in the presence of 10% serum and harvested upon reaching
confluence. Total RNA was extracted with TRI reagent, and RNA was
separated by electrophoresis, transferred to nitrocellulose membrane,
and hybridized under stringent conditions using bamacan probe P144.
B, hybridization autoradiograph of a series of murine
tumorigenic cells as indicated. C, autoradiograph of the
Northern blot of mouse RNA derived from a normal thymus (lane
1) and three T-cell lymphomas (lanes 2-4)
spontaneously arising from p53/decorin double knockout mice (38). Total
RNA was extracted from freshly excised tissue and analyzed by Northern
blot hybridization using mouse bamacan cDNA probe P144. For loading
comparison, the ethydium bromide staining of ribosomal RNA is presented
in the matching panels. D, Northern blot of four sets of
normal (N) and tumor (T) samples from subjects
with malignant fibrous histiocytoma (lanes 1 and
2) or colon carcinoma (lanes 3-8). For loading
comparison, the ethydium bromide staining of ribosomal RNA is presented
in the matching panels. The human probe P554, a PCR-generated probe of
the human EST AA554698 spanning bp 567-1324, was used.
|
|
To further assess whether bamacan expression was altered in neoplastic
tissues, samples from tumor and unaffected areas of the same organ were
analyzed by Northern blot hybridization. The results from spontaneously
arising thymic lymphoma cells from p53
/
,
decorin
/
double knockout animals (38), and a thymus
from a control wild type animal are illustrated in Fig. 8C.
In all the samples, the hybridization signal of bamacan mRNA in
tumor tissues, when normalized on total RNA, was consistently higher
(2-5-fold) than in normal tissues.
To corroborate the observation of increased bamacan expression in
neoplastic tissue, we analyzed bamacan expression in normal human
tissues and neoplastic specimens from four cancer patients with
malignant fibrous histiocytoma or colon carcinoma. In all the sets of
samples, neoplastic tissue consistently displayed higher bamacan RNA
levels (Fig. 8D). Thus, bamacan expression is increased in
both transformed cell lines and tumor tissues, suggesting a role for
bamacan in tumorigenesis.
Dynamics of Bamacan Expression--
Because of the link between
bamacan expression and transformation we sought to investigate the
dynamics of bamacan expression under stimulation by serum. To this end,
we synchronized 3T3 and 3T3-L1 fibroblasts by incubating them for 3 days in serum-free medium to attain quiescence. The medium was then
supplemented with 10% serum, and the bamacan mRNA levels were
quantified at different time points thereafter. Striking differences
were observed between the normal and the corresponding transformed cell
lines (Fig. 9). Within 30 min after the
addition of serum, bamacan expression in the untransformed cells begun
to increase, and at 60 min it reached a level approximately two times
higher than that of the cells kept in serum-free medium. Subsequently,
bamacan mRNA level began to decrease, reaching base-line values by
2 h, and then began to increase again (not shown). This clearly
biphasic behavior in bamacan mRNA level in response to growth
stimulation was not observed in neoplastic cells. In this case, the
addition of serum did not cause major alteration in bamacan mRNA
levels, which remained essentially unchanged for up to 12 h. It
should be noted, however, that the absolute levels of bamacan mRNA
was 2-3-fold higher in the transformed 3T3-L1 fibroblasts. Thus, it
appears that bamacan is constitutively overexpressed in transformed
cells. Virtually similar results were obtained when normal and
SV40-transformed human skin fibroblasts cells were investigated (not
shown). Collectively, these data suggest that bamacan may be part of a
set of early responsive genes whose expression is activated upon entry
of the cells into the cell cycle. A similar phenomenon has been
reported for the transient expression of the proto-oncogenes
c-myc and jun-fos (39) and is regarded to have
functional significance in initiating a signal cascade leading to
activation of genes involved in cell growth.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Dynamics of bamacan expression: effect of
serum addition on the level of bamacan mRNA in normal and
transformed cells. Normal (3T3) and transformed (3T3-L1) mouse
fibroblasts were grown in 10% serum until they had reached 60%
confluence. The cells were then placed in 0.4% serum and incubated for
2 additional days. On the 3rd day, the cells were supplemented with
10% serum and harvested at different time intervals as indicated in
the figure. RNA was extracted, and bamacan mRNA was quantified by
Northern hybridization with probe P144. Autoradiographic results were
analyzed by scanning densitometry, and the results were normalized on
total RNA loaded and expressed as a percentage of the change over
serum-starved (synchronized) cells at time 0 (set arbitrarily as
100%).
|
|
Conclusions--
In this investigation we have cloned and
sequenced the entire mouse bamacan gene and investigated its promoter
activity, its expression in cells and tissues, and its aberrant levels
in transformed cells. The fact that bamacan belongs to the SMC3 family
of genes (30), which are implicated in gene dosage and DNA repair,
suggests that this proteoglycan is destined primarily to the nucleus.
More research needs to be done to establish with certainty whether this
gene product can be secreted under special circumstances because the
high conservation across species puts into doubt the specificity of the
polyclonal rabbit antisera that have been used to map bamacan to the
extracellular (15) and intracellular (24, 40) compartments. The study
of the genomic organization reveals that bamacan is a modular protein
in which the salient structural motifs are encoded by separate exons.
The presence in the promoter of cis-acting protein-binding
motifs for oncogenes as well as the constitutive elevation of bamacan
mRNA levels in transformed cells and tumor tissues suggest that the
expression of this gene may play a role in transformation. Because of
the virtual complete conservation of the protein among all the mammal
species thus far investigated, it will be of interest to examine
whether mutations or deletions of this gene may contribute to the
transformed phenotype.
 |
ACKNOWLEDGEMENTS |
We thank I. Eichstetter and D. L. Green
for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants RO1 CA39481 and RO1 CA47282 (to R. V. I.) and PO1
CA21124 (to L. D. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF141294.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Anatomy, and Cell Biology, Rm. 249, JAH, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail:
iozzo{at}lac.jci.tju.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
EST, expressed
sequence tag;
kb, kilobase pair(s);
bp, base pair(s), PCR, polymerase
chain reaction.
 |
REFERENCES |
-
Iozzo, R. V.
(1998)
Annu. Rev. Biochem.
67,
609-652[CrossRef][Medline]
[Order article via Infotrieve]
-
Kjellén, L.,
and Lindahl, U.
(1991)
Annu. Rev. Biochem.
60,
443-475[CrossRef][Medline]
[Order article via Infotrieve]
-
Jackson, R. L.,
Busch, S. J.,
and Cardin, A. D.
(1991)
Physiol. Rev.
71,
481-539[Free Full Text]
-
Iozzo, R. V.,
and Cohen, I.
(1993)
Experientia
49,
447-455[Medline]
[Order article via Infotrieve]
-
Iozzo, R. V.
(1995)
Lab. Invest.
73,
157-160[Medline]
[Order article via Infotrieve]
-
Esko, J. D.,
Rostand, K. S.,
and Weinke, J. L.
(1988)
Science
241,
1092-1096[Medline]
[Order article via Infotrieve]
-
Nakanishi, H.,
Takenaga, K.,
Oguri, K.,
Yoshida, A.,
and Okayama, M.
(1992)
Virchows Archiv. A. Pathol. Anat.
420,
163-170
-
Iozzo, R. V.,
Cohen, I. R.,
Grässel, S.,
and Murdoch, A. D.
(1994)
Biochem. J.
302,
625-639[Medline]
[Order article via Infotrieve]
-
Adany, R.,
Heimer, R.,
Caterson, B.,
Sorrell, J. M.,
and Iozzo, R. V.
(1990)
J. Biol. Chem.
265,
11389-11396[Abstract/Free Full Text]
-
Iozzo, R. V.,
and Clark, C. C.
(1986)
J. Biol. Chem.
261,
6658-6669[Abstract/Free Full Text]
-
Iozzo, R. V.,
and Clark, C. C.
(1987)
Histochemistry
88,
23-29[Medline]
[Order article via Infotrieve]
-
Couchman, J. R.,
Kapoor, R.,
Sthanam, M.,
and Wu, R.-R.
(1996)
J. Biol. Chem.
271,
9595-9602[Abstract/Free Full Text]
-
McCarthy, K. J.,
Abrahamson, D. R.,
Bynum, K. R.,
St. John, P. L.,
and Couchman, J. R.
(1994)
J. Histochem. Cytochem.
42,
473-484[Abstract/Free Full Text]
-
McCarthy, K. J.,
and Couchman, J. R.
(1990)
J. Histochem. Cytochem.
38,
1479-1486[Abstract]
-
Wu, R.-R.,
and Couchman, J. R.
(1997)
J. Cell Biol.
136,
433-444[Abstract/Free Full Text]
-
McCarthy, K. J.,
Bynum, K. R.,
St. John, P. L.,
Abrahamson, D. R.,
and Couchman, J. R.
(1993)
J. Histochem. Cytochem.
41,
401-414[Abstract/Free Full Text]
-
Couchman, J. R.,
Abrahamson, D. R.,
and McCarthy, K. J.
(1993)
Kidney Int.
43,
79-84[Medline]
[Order article via Infotrieve]
-
Hirano, T.,
Mitchison, T. J.,
and Swedlow, J. R.
(1995)
Curr. Opin. Cell Biol.
7,
329-336[CrossRef][Medline]
[Order article via Infotrieve]
-
Koshland, D.,
and Strunnikov, A.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
305-333[CrossRef][Medline]
[Order article via Infotrieve]
-
Kanda, H.,
Tajima, H.,
Lee, G. H.,
Nomura, K.,
Ohtake, K.,
Matsumoto, K.,
Nakamura, T.,
and Kitagawa, T.
(1993)
Oncogene
8,
3047-3053[Medline]
[Order article via Infotrieve]
-
Danielson, K. G.,
Martinez-Hernandez, A.,
Hassell, J. R.,
and Iozzo, R. V.
(1992)
Matrix
11,
22-35
-
Santra, M.,
Skorski, T.,
Calabretta, B.,
Lattime, E. C.,
and Iozzo, R. V.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7016-7020[Abstract]
-
Argeson, A. C.,
Druck, T.,
Veronese, M. L.,
Knopf, J. L.,
Buchberg, A. M.,
Huebner, K.,
and Siracusa, L. D.
(1995)
Genomics
25,
29-35[CrossRef][Medline]
[Order article via Infotrieve]
-
Gupta, K.,
Anand, G.,
Yin, X.,
Grove, L.,
and Prochownik, E. V.
(1998)
Oncogene
16,
1149-1159[CrossRef][Medline]
[Order article via Infotrieve]
-
Chang, B. H.,
and Chan, L.
(1998)
Methods
15,
41-50[CrossRef][Medline]
[Order article via Infotrieve]
-
Shimizu, K.,
Shirataki, H.,
Honda, T.,
Minami, S.,
and Takai, Y.
(1998)
J. Biol. Chem.
273,
6591-6594[Abstract/Free Full Text]
-
Bernfield, M.,
Kokenyesi, R.,
Kato, M.,
Hinkes, M. T.,
Spring, J.,
Gallo, R. L.,
and Lose, E. J.
(1992)
Annu. Rev. Cell Biol.
8,
365-393[CrossRef]
-
Zimmermann, D. R.,
and Ruoslahti, E.
(1989)
EMBO J.
8,
2975-2981[Abstract]
-
Hirano, T.
(1998)
Curr. Opin. Cell Biol.
10,
317-322[CrossRef][Medline]
[Order article via Infotrieve]
-
Jessberger, R.,
Frei, C.,
and Gasser, S. M.
(1998)
Curr. Opin. Genet. Dev.
8,
254-259[CrossRef][Medline]
[Order article via Infotrieve]
-
Boulikas, T.
(1994)
J. Cell. Biochem.
55,
32-58[Medline]
[Order article via Infotrieve]
-
Brix, K.,
Summa, W.,
Lottspeich, F.,
and Herzog, V.
(1998)
J. Clin. Invest.
102,
283-293[Abstract/Free Full Text]
-
Liang, Y.,
Roughley, P. J.,
Margolis, R. K.,
and Margolis, R. U.
(1997)
J. Cell Biol.
139,
851-864[Abstract/Free Full Text]
-
Johnson, D. G.,
and Schneider-Broussard, R.
(1998)
Front. Biosci.
3,
447-448
-
Iozzo, R. V.,
and Danielson, K. G.
(1999)
Prog. Nucleic Acid Res. Mol. Biol.
62,
19-53[Medline]
[Order article via Infotrieve]
-
Zhang, X. K.,
Dong, J. M.,
and Chiu, J. F.
(1991)
J. Biol. Chem.
266,
8248-8254[Abstract/Free Full Text]
-
Edelhoff, S.,
Ayer, D. E.,
Zervos, A. S.,
Steingrímsson, E.,
Jenkins, N. A.,
Copeland, N. G.,
Eisenman, R. N.,
Brent, R.,
and Disteche, C. M.
(1994)
Oncogene
9,
665-668[Medline]
[Order article via Infotrieve]
-
Iozzo, R. V.,
Chakrani, F.,
Perrotti, D.,
McQuillan, D. J.,
Skorski, T.,
Calabretta, B.,
and Eichstetter, I.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3092-3097[Abstract/Free Full Text]
-
Greenberg, M. E.,
and Ziff, E. B.
(1984)
Nature
311,
433-438[Medline]
[Order article via Infotrieve]
-
Shimizu, K.,
Kawabe, H.,
Minami, S.,
Honda, T.,
Takaish, K.,
Shirataki, H.,
and Takai, Y.
(1996)
J. Biol. Chem.
271,
27013-27017[Abstract/Free Full Text]
-
Poirier, C.,
and Guerret, S.
(1998)
Mamm. Genome
8,
S356-S360
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.