From the Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Suppressin (SPN) is an inhibitor of cell
proliferation that was originally identified and purified to
homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns,
J. N., Bost, K. L., and Blalock, J. E. (1990)
J. Biol. Chem. 265, 158-165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides
of 5- and 3
-untranslated sequences, respectively. Northern gel
analysis of rat pituitary mRNA showed a single hybridizing species
at ~2 kilobases. Sequence analyses showed that the nucleotide and
deduced amino acid sequences of SPN are novel and unrelated to any
known vertebrate inhibitors of proliferation. However, the deduced
amino acid sequence of SPN contains two domains that have extensive
sequence identity with a recently cloned transcription activator in
Drosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J. 15, 1961-1970) suggesting that SPN represents a vertebrate
cognate of deformed epidermal autoregulatory factor-1. Reverse
transcriptase-polymerase chain reaction and immunohistochemical
analyses showed that the SPN mRNA and the SPN protein
are expressed in every tissue examined including testis, spleen,
skeletal muscle, liver, kidney, heart, and brain suggesting that SPN
may be involved in the control of proliferation in a variety of cell
types.
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INTRODUCTION |
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Suppressin (SPN)1 is a 63-kDa monomeric protein identified and purified to homogeneity from the bovine pituitary based on its ability to inhibit mitogen-stimulated murine splenocyte proliferation (1). Initial immunologic and species-specificity studies indicated that SPN was a structurally conserved molecule. Specifically, (i) anti-SPN antibodies prepared against purified bovine SPN cross-reacted with human, mouse, and rat SPN (1-4), and (ii) purified bovine SPN was active on mouse, rat, and human cells. Other cross-species activities could also be shown for SPN (e.g. human SPN was active on rat cells). In the rat pituitary, SPN production is restricted to five hormone-secreting cell phenotypes (somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs) in the anterior pituitary (2). The primary biological activity, inhibition of cell proliferation, has been most extensively studied in vitro in murine and human lymphocytes (1, 3). The inhibition of cell proliferation by SPN does not occur by either a cytotoxic mechanism or by increasing the rate of apoptosis (1). The results of cell cycle analyses on SPN-treated lymphocytes have shown that SPN arrests cells in G0 or early G1 stages of the cell cycle (3). Suppressin also inhibits the proliferation of tumor cells. The addition of exogenous SPN to cultures of leukemia, lymphoma, and thymoma cells and tumor cells from brain, adrenal, breast, and pituitary resulted in markedly reduced proliferation (4). The results of metabolic labeling have shown that SPN is synthesized and secreted as an active molecule by human and mouse lymphocytes and GH3 cells (1, 3, 5). Moreover, neutralization of secreted SPN in culture supernatants by anti-SPN antibodies (Ab) increases proliferation in the absence of exogenous growth factors (6) showing that SPN acts as an autocrine/paracrine inhibitor of entry into the cell cycle. Collectively, the results of our studies show that SPN is a fundamental component of a regulatory circuit that functions to maintain cells in a nondividing state.
To understand the structure, function, and regulation of SPN, we
cloned, sequenced and characterized the full-length SPN
cDNA from the rat pituitary. The molecular cloning of SPN was
accomplished by immunoscreening a pituitary cDNA library with an
anti-SPN Ab, by DNA hybridization screening of cDNA libraries with
a partial SPN cDNA, and by polymerase chain reaction (PCR) and
5-rapid amplification of cDNA ends (RACE) using rat pituitary
mRNA. The results of sequence analyses and comparisons showed that
SPN is a novel vertebrate regulatory molecule. However, SPN is highly homologous to DEAF-1, a recently cloned molecule from
Drosophila (7). In addition, we provide results from studies
on the tissue distribution of SPN expression, on structural
characteristics of SPN, and on the expression of recombinant SPN.
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EXPERIMENTAL PROCEDURES |
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RNA Analysis--
Whole pituitaries were surgically removed from
male Sprague-Dawley rats (Harlan) as described previously (2), and
total RNA was isolated by the guanidinium thiocyanate/cesium chloride method (8). Poly(A)+ mRNA was obtained by two rounds of
chromatography over oligo(dT)-cellulose type 7 (Pharmacia Biotech Inc.)
as described previously (9). Total RNA (10-20 µg) or
poly(A+) mRNA (1-5 µg) was denatured in 50% (v/v)
formamide and 2.2 M formaldehyde and resolved by
agarose-formaldehyde gel electrophoresis, and the gel was transferred
to Biotrans (ICN, Irvine, CA) nylon membranes by capillary diffusion in
10 × SSC using standard procedures (9). Membranes were baked for
2 h at 80 °C in a vacuum oven and prehybridized and hybridized
in the following buffer (0.5 M NaHPO4, pH 7.2, 1 mM EDTA, 7% SDS, and 0.1% (w/v) bovine serum albumin).
32P-Labeled SPN cDNA probes were prepared from
gel-purified cDNAs using a Nick Translation System (Promega,
Madison, WI) and [-32P]dCTP (3000 Ci/mmol, DuPont).
For hybridization, radiolabeled SPN cDNA probes were added at
2 × 106 cpm/ml. Pre-hybridization (4-6 h) and
hybridization (10-12 h) were done at 42 °C in 50% formamide in
5 × SSC, and membranes were washed in 2 × SSC, 0.1% SDS at
50 °C followed by two washes in 0.2 × SSC, 0.1% SDS at
50 °C. The membrane was then exposed to Kodak XAR film at
70 °C
for 10-20 h. Slot blot analyses were performed with total rat RNA, and
the membranes were prehybridized, hybridized, and washed as described
above except the temperature for these procedures was 45 °C. Two
oligodeoxynucleotides were used as probes as follows: one that was
complementary to the cDNA sequence containing the largest open
reading frame (5
-TGATGGCTTCTCAGTAGAG-3
), and the complement to this
oligodeoxynucleotide that is homologous to the cDNA sequence
containing the open reading frame. Both oligodeoxynucleotides were
labeled with 32P using T4 polynucleotide kinase by standard
procedures (9).
cDNA Library Construction and Screening--
An oligo(dT)
adaptor-primed unidirectional rat pituitary cDNA library was
constructed from rat pituitary poly(A)+ mRNA using a
Uni-Zap XR/Gigapack Cloning system (Stratagene, La Jolla, CA). This
cDNA library was immunoscreened (1 × 106
individual plaque-forming units) using a previously characterized (1)
monospecific polyclonal anti-SPN Ab and Ab-positive clonal plaques
detected using a BioStain Super ABC alkaline-phosphatase immunodetection kit (Biomeda, Foster City, CA). After tertiary replating, the Ab-positive clonal plaques were analyzed for
clonality and cDNA insert size by PCR with vector-specific primers
that flank the XhoI/EcoRI cloning site in the
Uni-Zap XR vector as described previously (10). The largest cDNA
insert (691 bp) was direct-sequenced and then used as a probe to
rescreen the rat pituitary Uni-Zap XR cDNA library. After tertiary
replating, one larger clone was obtained by nucleic acid hybridization
screening, with an insert of 924 bp corresponding exactly to the SPN
691-bp cDNA sequence but extended at its 5 end. The SPN 924-bp
cDNA was subcloned in M13mp18 and mp19 by standard methods (9), and
the sequence of this cDNA was determined in both orientations. DNA
sequencing was performed using the Sanger dideoxy sequencing method
with Sequenase (U. S. Biochemical Corp.). Sequence analyses were
performed with the Genepro program (Riverside Scientific, Bainbridge
Island, WA). Homology searches and protein structure analyses were
performed using the GCG programs (Genetics Computer Group,
Madison, WI) against all available public sequence data bases.
5-RACE and Elucidation of the Full-length cDNA Sequence of
SPN--
The partial SPN cDNA sequence (924 bp) was used to design
SPN-specific reverse transcriptase (RT) and PCR primers for
use in a 5
-RACE system (Life Technologies Inc.) to obtain the
full-length cDNA sequence of SPN. Briefly, an antisense
SPN-specific primer (SSP-1) that was approximately 100 bp downstream
from the 5
end of the 924-bp SPN cDNA sequence was used in a
reverse transcription reaction (RT) with one µg of rat pituitary
poly(A)+ mRNA. An oligo(dC) anchor sequence was added
to the 3
end of RT reaction products with terminal
deoxynucleotidyltransferase. The oligo(dC)-tailed cDNA was
amplified by PCR using a nested antisense SPN-specific primer (SSP-2)
that was approximately 50 bp upstream of SSP-1 and the anchor primer
provided in the 5
-RACE system. PCR was performed for 50-60 cycles
with AmpliTaq DNA polymerase under standard reaction conditions (10).
Reaction products were analyzed by agarose gel electrophoresis and
selected products were excised from the gel and purified using a Qiaex
II gel extraction kit (Qiagen, Chatsworth, CA). The purified cDNA
was cloned in the pGEM-T vector (Promega, Madison, WI), and both
strands of the cDNA insert were completely sequenced. Four
iterations of this procedure were required to obtain the complete
cDNA sequence of the SPN transcript.
Preparation of Antibodies to a Peptide Deduced from the SPN cDNA-- The 120-amino acid peptide deduced from the 691-bp cDNA was analyzed for highly antigenic regions using the Hoop and Woods hydropathy index to predict antigenic regions within this peptide. One such region, a nonapeptide NH2-QRKVWKDHQ-COOH, was synthesized, purified by high pressure liquid chromatography, covalently coupled to keyhole limpet hemocyanin as a carrier immunogen (2.5 mg of peptide + 2.5 mg of keyhole limpet hemocyanin) using a standard glutaraldehyde coupling protocol (9), and then used to immunize a New Zealand White rabbit (500 µg/injection). The rabbit received booster injections at 10-day intervals. The presence of anti-peptide antibodies was assayed by enzyme-linked immunosorbent assay using the peptide covalently linked to a 96-well COBIND microtiter plate and an alkaline phosphatase-conjugated anti-rabbit IgG as the secondary Ab. Thirty days after immunization, serum was obtained from the rabbit, and the Ig fraction of the sera was purified by protein G-Sepharose affinity chromatography (9). To determine if the Abs in the postimmune Ig fraction would specifically recognize SPN, homogeneous pituitary SPN was covalently attached to COBIND microtiter wells and assayed for specific binding by the Abs in the Ig fraction using an enzyme-linked immunosorbent assay.
Expression of the SPN cDNA in Escherichia coli-- BamHI and Bpu1102I endonuclease restriction sites flanking the coding region of the SPN cDNA were constructed by PCR using the 1583-bp SPN cDNA in pGEM-T as template and Pfu polymerase. The reaction product was sequentially digested with BamHI and Bpu1102I, gel purified, and ligated in-frame in the BamHI/Bpu1102I site of expression vector pET-15b (Novagen, Madison, WI). The resulting fusion protein contained six consecutive histidine residues (His-Tag) on its amino terminus. The orientation and sequence of the SPN cDNA in the pET plasmid were confirmed by DNA sequencing. The SPN cDNA was expressed in the E. coli strain BL21 (DE53, pLysS) by growing the bacterial cells at 30 °C and following an induction protocol previously described (11); the recombinant protein was purified by metal chelation chromatography (12).
Tissue-specific Expression of SPN--
Poly(A)+
mRNA from several rat tissues were obtained commercially
(CLONTECH, Palo Alto, CA). mRNA (250 ng) from
each tissue was used as template in an oligo(dT)-primed first-strand
cDNA synthesis with Superscript RT (Life Technologies, Inc.) using the protocol provided by the manufacturer. Each reaction was treated with DNase I before the addition of RT and cDNA synthesis using a
standard protocol (9). A replicate first-strand cDNA reaction in
which RT was not added was performed for each tissue as a control for
the presence of genomic DNA. One-tenth of the first-strand cDNA
synthesis reaction was used as template with a sense SPN primer
(5-TGGAGATGTCAGAGCATCG-3
) and an antisense SPN primer (5
-TGATGGCTTCTCAGTAGAG-3
) that will amplify a 402-bp target sequence
of the SPN cDNA. The products from each set (with and without RT)
of PCR reactions were analyzed by agarose gel electrophoresis. Restriction analysis was performed on part of each reaction and yielded
the expected restriction fragments for the SPN target sequence (data
not shown).
Immunohistochemical Methods--
Rat tissues were collected and
fixed in 5% glacial acetic acid in 95% ethanol at 20 °C for
24 h before embedding in paraffin. Serial sections 4 µm thick
were cut and stained on glass slides. Staining for SPN expression was
performed using an anti-SPN monoclonal antibody (mAb) (3F10; Ref. 2).
The specificity of this anti-SPN mAb has been previously demonstrated
on intracellular SPN in rat pituitary cells (2). 3F10 binding to
intracellular SPN from rat pituitary cells is specifically blocked by
preincubation of the mAb with pure native SPN (2). Sections were
stained in PBS containing 10% goat serum/Tween 0.05% and 2 µg/ml of
anti-SPN mAb. The levels of background staining (negative control) were obtained by incubating the sections with an irrelevant isotype-matched (IgM) mAb (UAB mAb Core Facility). After incubation with the mAb (1.5 h
at room temperature), the slides were washed three times in PBS and
incubated with PBS/Tween 0.05% containing a biotinylated rabbit
anti-mouse IgM (µ-chain specific) antibody (5 µg/ml) (Pharmingen, San Diego, CA). Slides were washed three times in PBS and incubated with streptavidin coupled to fluorescein isothiocyanate at the concentration of 1 µg/ml in PBS/Tween 0.05% for 30 min at room temperature. After three washes in PBS, the slides were mounted in
ethanol and photographed using a Leitz Diaplan microscope (Leitz, Wetzlar, Germany).
Western Analysis-- GH3 cells (ATCC, Rockville, MD) were cultured in RPMI, 10% horse serum. PBMC were cultured in RPMI, 5% fetal calf serum for 24 h. Supernatants were collected and concentrated 40 times using CENTRIPREP 30 (Amicon, Beverly, MA). Cell extracts were prepared from 108 cells with standard procedures (7), and proteins were separated by electrophoresis using a 4-20% continuous gradient Tris-HCl acryl/bisacryl gel (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electroblotting under 30 mV for 16 h at 4 °C. Membranes were blocked in Tris borate saline (TBS), 3% casein, 10% goat serum, washed, and then incubated with the 3F10 mAb (2.5 µg/ml) for 1.5 h at room temperature. After three washes, membranes were incubated with a biotinylated goat antibody against mouse immunoglobulin (H + L) (0.2 µg/ml), washed again 3 times and then incubated with avidin conjugated to horse peroxidase (1 µg/ml). Membranes were washed before a 60-s incubation with the enhanced chemiluminescent substrate (Amersham Corp.) and exposed for 5 min to autoradiographic film.
Glycosylation Analysis of Native SPN-- Two hundred ng of affinity purified native rat SPN was analyzed for the presence of Asn-linked oligosaccharides by digestion with the glycosidase, PNGase F, according to the protocol provided by the manufacturer (Glyko Inc., Novato, CA). A parallel reaction in which fetuin was digested with PNGase F served as a positive control for enzyme activity. PNGase F and control reactions (without PNGase F) were performed for SPN and the control glycoprotein, fetuin, and the reaction products were analyzed under reducing conditions on 10% SDS-PAGE (13), and protein bands were stained with silver (14).
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RESULTS AND DISCUSSION |
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Isolation and Analysis of cDNA Clones-- A polyclonal anti-SPN antibody (Ab) prepared against purified bovine SPN (1) was used to screen a rat pituitary cDNA library. This Ab cross-reacts with bovine, human, mouse, and rat SPN (1, 3, 4). The initial screening (1 × 106 clones) yielded 10 independent clones after tertiary replating, and Southern analysis indicated that all clones were related. Both strands of the largest clone (clone 12) were sequenced, and it had an insert size of 691 bp. One strand of the clone 12 cDNA contained a poly(A) tail, two polyadenylation signal sequences within 50 nucleotides of the poly(A) tail, and an open reading frame (ORF) encoding a 120-amino acid peptide. The results from Northern gel analyses using rat pituitary mRNA and the clone 12 cDNA as a probe showed that this cDNA hybridized to a single mRNA species that was ~2 kilobases (Fig. 1). To identify the coding strand of clone 12, two 32P-labeled oligodeoxynucleotides complementary to each strand were synthesized and used as probes in slot analysis with rat pituitary total RNA. Only the oligodeoxynucleotide that was complementary to the sequence with the poly(A) tail and the ORF hybridized to pituitary total RNA showing that this was the coding strand of the SPN cDNA (Fig. 2). At this point we also showed that the clone 12 cDNA sequence was an authentic partial clone of the SPN cDNA. Antibodies prepared to a synthetic peptide derived from the deduced 120-amino acid ORF of clone 12 specifically bound pure native SPN in a solid phase assay (Table I).
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Nucleotide and Predicted Amino Acid Sequence of SPN--
The rat
full-length SPN cDNA isolated is 1882 nucleotides (Fig.
3), and the size of this cDNA agrees
with estimates of the size of the SPN mRNA obtained by Northern gel
analysis. The complete SPN cDNA contains an open reading frame of
1488 nucleotides that extends from the first translation consensus
sequence (15) of an initiation codon at position 56 to the first
termination codon at position 1545. The entire cDNA contains 55 and
339 nucleotides of 5- and 3
-untranslated sequences, respectively.
Translation of the cDNA results in a predicted protein of 496 amino
acids with a relative Mr of 53,113 (Fig.
4), which is less than the molecular mass
previously observed for native SPN (~63 kDa) by SDS-PAGE analysis.
Differences in the actual molecular mass of a protein and its
electrophoretic mobility in SDS-PAGE analysis could be due to either
post-translational modifications (e.g. glycosylation and/or
phosphorylation) and/or to specific regions that bind SDS anomalously
and affect electrophoretic mobility (16, 17). The results of bacterial
expression studies using the SPN coding sequence (Fig.
5) suggest that the SPN protein contains
structural regions that apparently bind SDS anomalously and affect its
electrophoretic mobility (Fig. 5). The expression of recombinant
proteins in E. coli typically leads to the production of
proteins that are not modified post-translationally. Therefore, analyses of the mass of recombinant forms by SDS-PAGE reveals proteins
of lower molecular weight compared with native proteins that are
post-translationally modified. However, SDS-PAGE analysis of SPN
produced by E. coli (Fig. 5) showed that it migrated at molecular mass higher (~63 kDa) than predicted by the deduced amino
acid sequence (~53 kDa). Consistent with the idea of structural features of SPN as a major cause of anomalous electrophoretic mobility
are the results from similar studies on the homologous protein DEAF-1.
The predicted molecular mass of DEAF-1 from the deduced amino acid
sequence is 61.5 kDa; however, SDS-PAGE analysis of both recombinant
and native DEAF-1 shows that it migrates with an apparent molecular
mass of approximately 85 kDa (7).
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Structural Features of SPN-- The first 27 amino acids of the SPN sequence is a predominantly hydrophobic region that may represent a putative secretory signal peptide sequence (18). Additionally, there is a cleavage site for a signal endopeptidase at Phe27-Ala28 which further suggests that this region may be a secretory signal sequence (18, 19). This structural feature of SPN is consistent with results of earlier metabolic labeling studies which showed that SPN is secreted by GH3 cells (somatotroph and lactotroph cell types) and also human peripheral blood mononuclear cells (1, 3, 5).
The deduced amino acid sequence of SPN contains three potential N-linked glycosylation sites (Asn-X-(Ser/Thr); Ref. 15) at Asn121, Asn239, and Asn253 (Fig. 4). To determine if SPN was glycosylated, native affinity purified pituitary SPN was incubated with PNGase F which will cleave all N-linked carbohydrate moieties. SDS-PAGE analysis of a control protein (fetuin) showed the expected reduction in molecular mass after PNGase F digestion, whereas the molecular mass of SPN did not detectably change. These results show that the native form of SPN does not undergo N-linked glycosylation (data not shown). The deduced amino acid sequence of SPN also contains a total of 23 serine and threonine residues located near basic residues which may serve as putative phosphorylation sites for serine/threonine kinases (Fig. 4). Additionally, there are tyrosine residues arrayed in the appropriate recognition sequence for tyrosine kinase. These include consensus sequences for the following kinases: 1 cAMP- or cGMP-dependent protein kinase phosphorylation site (20); 13 casein kinase II phosphorylation sites (21); 6 protein kinase C phosphorylation sites (22); 2 tyrosine kinase phosphorylation sites (23): and 1 G1 cyclin-dependent kinase (Cdk2) phosphorylation sites (24). Although we have not formally tested if native SPN is phosphorylated, the large number of possible phosphorylation sites suggests that phosphorylation of SPN is probable. Lastly, Dingwall and Laskey (25) have described a consensus bipartite nuclear localization sequence that consists of two discrete clusters of basic amino acids separated by any 10 amino acids that target proteins to the nucleus. Suppressin contains such a nuclear targeting sequence (RKKENVSCPRLVKK) at amino acids 235-248 which may facilitate its translocation to the nucleus (Fig. 4). In the results of studies presented below (see "Expression of SPN in Rat Tissues"), we show that SPN is localized to the nucleus of cells from a variety of tissues. These results are consistent with the presence of a nuclear localization sequence in the primary sequence of SPN; however, they do not indicate whether this process is dependent on the indicated nuclear localization sequence or if nuclear transport of SPN occurs by other mechanisms.Sequence Homology of SPN with Other Proteins-- The cDNA sequence of SPN is highly related to several human and mouse cDNAs of unknown function in the NCBI GenBank non-redundant expression sequence-tagged (EST) data base. Similarities are as high as 90-98% between the cDNA of rat SPN and these EST cDNAs. We assembled a contiguous sequence from the human ESTs. Sequence comparisons between this assembled human cDNA sequence and the rat SPN cDNA showed that they were approximately 80% homologous in their coding regions indicating that it represents the human SPN cDNA sequence. Additionally, we had cloned portions of the human SPN cDNA by RT-PCR using primers designed from the rat cDNA sequence, and the sequence of these human SPN cDNA fragments were identical to those contained in the EST data base. The results of the sequence comparisons between rat and human SPN confirmed in part the results of earlier immunologic and species specificity studies which indicated that human, rat, bovine, and mouse SPN were structurally conserved (1, 3, 5).
Sequence comparisons between the cDNA of SPN and the NCBI GenBankTM, EMBL, DDBJ, PDB sequence data base showed only one cDNA that was highly homologous (64%) to SPN in a 102-bp region. This cDNA belongs to a recently cloned protein in Drosophila called deformed epidermal regulatory factor-1 (DEAF-1; see Ref. 7). Further sequence comparisons between SPN and DEAF-1 at the amino acid level confirmed this relatedness and indicated that SPN represents a vertebrate cognate of DEAF-1. Specifically, SPN is highly related to DEAF-1 in two domains, one in the middle of the molecule (101 amino acids) and one in the carboxyl terminus (66 amino acids) of the molecule that are 72 and 64% homologous to DEAF-1, respectively (Figs. 4 and 6). The middle domain has been previously described in DEAF-1 as the KDWK domain, and this domain was shown to have similarity to a human brain protein (dbest R19688), a human breast protein (dbest R49909), a human nuclear phosphoprotein (26), and Caenorhabditis elegans protein CEC44f1.2 (gp Z49067). Additionally, in part of the KDWK domain (amino acids 135-205) SPN is 63% homologous to a sequence in the COOH-terminal region of a human lymphoid-specific SP100 homolog (U36500).
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Expression of SPN in Rat Tissues-- The above results show that SPN is a structurally conserved molecule across several metazoan groups. Another important question is whether within an organism SPN plays a restricted role and functions in only certain cell lineages to regulate proliferation or, alternatively, it inhibits proliferation in a variety of cells and tissues. The majority of our studies on SPN have focused on its expression and activity in pituitary cells and in lymphocytes. To determine if SPN was expressed by other cell types, we performed RT-PCR analysis on mRNA from selected tissues in the rat. The results of this analysis showed that expression of SPN is quite broad, and SPN expression was observed in every tissue examined. Specifically, SPN expression was observed in testis, spleen, skeletal muscle, liver, kidney, heart, and brain (Fig. 7).
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ACKNOWLEDGEMENTS |
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We are grateful for the technical support provided by Bernard M. Gary. We thank Dr. Denise R. Shaw for critically reading the manuscript.
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FOOTNOTES |
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* This research was supported in part by NCI Grant CA 54290 from the National Institutes of Health (to R. D. L.). Sequence analyses were performed through the computer facilities of the UAB-Center for AIDS Research supported in part by Grant P30 AI27767 from the National Institutes of Health.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) U59659.
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, BHSB 850, University of Alabama at Birmingham, Birmingham, AL 35294. Tel.: 205-934-4271; Fax: 205-934--6674.
§ Supported by a fellowship from the IPSEN Foundation of France. Present address: Unite INSERM 167, Institut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille Cedex, France.
¶ Present address: Dept. of Microbiology, University of Florida, Gainesville, FL 35294.
Present address: Dept. of Chemistry, McNeese State University,
Lake Charles, LA 70606.
1
The abbreviations used are: SPN, suppressin; RT,
reverse transcription; PCR, polymerase chain reaction; PBS,
phosphate-buffered saline; mAb, monoclonal antibody; Ab, antibody;
RACE, 5-rapid amplification of cDNA ends; bp, base pair(s); PAGE,
polyacrylamide gel electrophoresis; ORF, open reading frame; FGF-1,
fibroblast growth factor-1; EST, expression sequence-tagged; DEAF-1,
deformed epidermal autoregulatory factor-1; PBMC, peripheral blood
mononuclear cells; PNGase F,
peptide-N4-(N-acetyl-
-glucosaminyl)-asparagine
amidase.
2 R. D. LeBoeuf, M. M. Green, J. E. Blalock, and J. D. Tauber, unpublished observations.
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REFERENCES |
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