Isolation and Characterization of a Pituitary Tumor-Transforming Gene (PTTG)
Lin Pei and
Shlomo Melmed1
Division of Endocrinology and Metabolism Cedars-Sinai Research
Institute-UCLA School of Medicine Los Angeles, California 90048
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ABSTRACT
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Pathogenesis of tumor formation in the anterior
pituitary has been intensively studied, but the common mechanism
involved in pituitary cell transformation and tumorigenesis remains
elusive. In this study, we used mRNA differential display PCR to
identify mRNAs that are differentially expressed in rat pituitary tumor
cells compared with normal pituitary tissue. An mRNA exclusively
expressed in pituitary tumor but not in normal pituitary was
characterized. Using this pituitary tumor-specific PCR product as a
probe to screen a cDNA library constructed from rat pituitary tumor
GH4 cells, a cDNA of 974 bp was isolated. This
cDNA encodes a novel protein of 199 amino acids, which contains no well
characterized functional motifs. The mRNA of this cDNA is detected in
normal adult testis and in embryonic liver, where the transcript is
about 300 bp shorter and expressed at a much lower level than that
detected from pituitary tumor cells. Overexpression of this protein in
mouse 3T3 fibroblasts shows that it inhibits cell proliferation and
induces cell transformation in vitro. Injection of
transfected 3T3 cells into athymic nude mice resulted in tumor
formation within 3 weeks in all animals. These results indicate that
pituitary tumor cells express a unique and potent transforming gene
(PTTG), which may play a role in pituitary tumorigenesis.
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INTRODUCTION
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Anterior pituitary tumors are mostly benign hormone-secreting or
nonfunctioning adenomas arising from a monoclonal expansion of a
genetically mutated cell (1, 2, 3). The most well characterized oncogene
in pituitary tumors is gsp, a constitutively active Gsa resulting from
activating point mutations in this gene (4, 5). Gsa mutations occur in
about 40% of GH-secreting tumors, and constitutively activated cAMP
response element binding protein is also found in a subset of these
tumors (6). Although point mutations of Ras oncogene (7, 8), loss of
heterozygosity near the Rb locus on chromosome 13 (9, 10, 11, 12, 13, 14), and loss of
heterozygosity on chromosome 11 (15, 16, 17) have been implicated in some
pituitary tumors, the mechanism that causes pituitary cell
transformation remains largely unknown.
To clarify the molecular mechanisms involved in pituitary
tumorigenesis, we used differential display PCR (18, 19) to identify
mRNAs differentially expressed in pituitary tumor cells. This technique
has been successfully used to identify mdm2 oncogene amplification in
murine uterine adenocarcinomas (20) and melanin-concentrating hormone
in the hypothalamus of ob/ob mice (21). We show here isolation and
characterization of a pituitary tumor-derived gene
(PTTG)2 that induces cell transformation
in vitro and tumor formation in nude mice.
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RESULTS
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Molecular Cloning of Pituitary Tumor-Transforming Gene
(PTTG) from Rat Pituitary Tumor Cells
Using 120 pair primers in the PCR, 11 DNA bands
that appeared to be differentially expressed in pituitary tumor cells
were identified. These bands were evaluated further by Northern blot
analysis, using the PCR products as probes. Pituitary tumor-specific
signals were detected for two bands. DNA sequencing analysis revealed
that one sequence was homologous with insulin-induced growth response
protein, while the other showed no homology to known sequences in the
GenBank at the time of isolation. This 396-bp fragment (Fig. 1
, a and b) detected a highly expressed mRNA of about 1.3
kb in pituitary tumor cells, but not in normal pituitary or in
osteogenic sarcoma cells (Fig. 1c
).

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Figure 1. Identification of PTTG by RNA Differential Display
PCR
A, PCR of RNA from pituitary tumor cells and normal pituitary using 5'
AAGCTTTTTTTTTTTG 3' as the anchored primer and 5' AAGCTTGCTGCTC 3' as
an arbitrary primer. The differentially displayed band is indicated by
an arrow. B, PCR reamplification of the differentially
displayed band in panel A. C, Northern blot analysis using reamplified
PCR product in panel B as the probe. GC and GH4, GH- and
PRL-secreting pituitary tumor cells, respectively; Pit, normal
pituitary; UM108, ostegenic sarcoma cells. Equal loading of RNA is
indicated by 28S and 18S ribosomal bands.
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To characterize this pituitary tumor-specific mRNA further, a cDNA
library was constructed using mRNA isolated from rat pituitary tumor
cells. Using the 396-bp PCR fragment as a probe, a cDNA clone of 974 bp
was isolated and characterized. This cDNA was designated as PTTG. The
sequence of PTTG contains an open reading frame for 199 amino acids
(Fig. 2
). The presence of an in-frame stop codon
upstream of the predicted initiation codon indicates that PTTG contains
the complete open reading frame. This was verified by demonstrating
both in vitro transcription and in vitro
translation of the gene product. As shown in Fig. 3
, translation of in vitro transcribed PTTG sense mRNA results
in a protein of approximately 25 kDa on SDS-PAGE, whereas no protein
was generated in either the reaction without added mRNA or when PTTG
antisense mRNA was used.

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Figure 2. DNA and Amino Acid Sequence of PTTG
The translation initiation codon is underlined and
italicized, and the stop codons are
underlined.
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Figure 3. In vitro Transcription and
Translation of the PTTG
In vitro transcribed and translated PTTG product was
analyzed by SDS-PAGE. The PTTG protein is indicated by an
arrow; mol wt marker is indicated.
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A subsequent data base search using BLAST EST program showed some
homology between the coding sequence of PTTG and several partial
transcripts of unknown function expressed during mouse embryonic
development and in ovarian cancer. The highest homology was to a mouse
embryonic cDNA sequence (mi66d08. rl). Protein profile analysis (BLAST
Program search of databases of the National Center for Biotechnology
Information), however, indicated that PTTG shares no homology with
known protein sequences, and it is highly hydrophilic and contains no
well recognized functional motifs.
Tissue Distribution of PTTG
The tissue expression pattern of PTTG mRNA was studied by Northern
blot analysis. Figure 4A
shows that among adult tissues
examined, testis is the only tissue that expresses PTTG mRNA, and the
testis expression level is much lower (2 mg polyA+ mRNA,
24 h exposure) than in pituitary tumor cells (20 mg total RNA,
6 h exposure, Fig. 2
). PTTG is also expressed at low levels in
embryonic liver (Fig. 4B
). Hybridization to ß-actin control probe
revealed appropriate transcripts for all RNA samples. Interestingly,
the transcript in both testis and fetal liver (
1 kb) is shorter
than the transcript in pituitary tumors (1.3 kb), suggesting that the
mRNA may be either differentially spliced or uses alternate promoters
or polyadenylation sites in these tissues, and that the 1.3-kb
transcript is specific for pituitary tumor cells.

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Figure 4. PTTG mRNA Expression in Normal Tissues
Panel A, PolyA+ RNA from eight different rat tissues
(indicated at the top of the figure) were used for
Northern blot analysis. Top panel shows hybridization to
PTTG probe (indicated by an arrow).
Bottom panel shows hybridization to the control
ß-actin probe. Molecular weight marker is shown on the
side. Exposure time was 24 h for the PTTG probe and
2 h for the actin probe. Panel B, PolyA+ RNA from four
different human fetal tissues (indicated at the top of
the figure) were used for Northern blot analysis. Hybridization to PTTG
and ß-actin probes are indicated by arrows.
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Overexpression of PTTG in NIH 3T3 Cells Inhibits Cell
Proliferation
It is difficult to predict the function of PTTG because the
predicted protein sequence of PTTG does not contain recognizable
motifs. Because PTTG mRNA is overexpressed in pituitary tumor cells, we
initially sought to determine whether this protein exerts an effect on
cell proliferation and transformation. Thus, an eukaryotic expression
vector containing the entire coding region of PTTG was stably
transfected into NIH 3T3 fibroblasts. Expression levels of the PTTG
were monitored by immunoblot analysis using a specific polyclonal
antibody directed against the first 17 amino acids of the protein.
Expression levels of individual clones varied (Fig. 5
),
and clones that expressed higher protein levels were used for further
analysis (Fig. 5
, lanes 3, 4, 8, 9, and 10). NIH 3T3 cells seem to
express low level endogenous PTSG protein, as a faint band is
detectable by anti-PTTG antibody (Fig. 5
, lane C).

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Figure 5. Overexpression of PTTG in NIH 3T3 Fibroblast Cells:
Western Blot Analysis
The specific band detected by an anti-PTTG polyclonal antibody is
indicated by an arrow. C, 3T3 cells transfected with
pCMV vector without the insert; lanes 310 indicate individual clones
transfected with PTTG expression vector. Molecular weight markers are
indicated.
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A nonradioactive cell proliferation assay (22, 23) was used to
determine the effect of PTTG protein overexpression on cell
proliferation. Figure 6
shows that growth rate of 3T3
cells expressing PTTG protein (assayed by cellular conversion of
tetrazolium into formazan) was suppressed 25 to 50% as compared with
3T3 cells expressing the pCMV vector alone, indicating that PTTG
protein inhibits cell proliferation. The suppression of cell
proliferation by PTTG protein is not surprising in view of the fact
that pituitary adenomas are invariably slow-growing both in rat (24)
and in humans (3).

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Figure 6. Effect of PTTG Expression on Cell Proliferation
Cell growth rate is expressed as absorbance at 595 nm. Error
bars represent SEM (n = 6). Three independent
experiments were performed.
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PTTG Induces NIH 3T3 Cell Transformation and Generates Tumors in
Nude Mice
The transforming property of PTTG protein was demonstrated by its
ability to form foci in monolayer cultures and show
anchorage-independent growth in soft agar (Fig. 7
and
Table 1
). As primary pituitary cells are an admixture of
multiple cell types and they do not replicate in vitro, NIH
3T3 cells were employed. As shown in Fig. 7
, NIH 3T3 parental cells and
3T3 cells transfected with pCMV vector do not form colonies on soft
agar (panels A and B), whereas 3T3 cells transfected with PTTG form
large colonies (panel C). In addition, focal transformation is observed
in cells overexpressing PTTG protein (panel E), but cells expressing
pCMV vector without the PTTG insert showed similar morphology to the
parental 3T3 cells (panel D). PTTG significantly induced the efficiency
of colony formation up to 1.32% as compared with 0.013% for
vector-only transfectants (Table 1
).

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Figure 7. PTTG Induces Morphological Transformation and the
Soft-Agar Growth of NIH 3T3 Cells
Cells (104 per dish) were plated as described in
Materials and Methods. a, b, and c, Colony growth of the
parental, pCMV-transfected, and pCMV-PTTG-transfected NIH 3T3 cells,
respectively. d and e, Morphology of the pCMV vector alone and
pCMV-PTTG-transfected NIH 3T3 cells, respectively.
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To determine whether PTTG is tumorigenic in vivo,
PTTG-transfected 3T3 cells were injected subcutaneously into athymic
nude mice. All injected animals developed large tumors (13 g) within
3 weeks (Fig. 8
and Table 2
). No mouse
injected with vector-only transfected cells developed tumors. Thus,
PTTG is a potent transforming gene in vivo.

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Figure 8. PTTG Induces Tumor Formation in Nude Mice
Cells (3 x 105) were injected subcutaneously into
athymic nude mice. Photograph was taken 3 weeks after injection. PTTG,
Mouse injected with 3T3 cells transfected with PTTG expression vector;
C, mouse injected with 3T3 cells transfected with pCMV vector alone.
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DISCUSSION
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Mechanisms for pituitary tumorigenesis involve a multistep cuscade
of recently characterized molecular events (25). Although the
importance of GSa mutant proteins in the development of GH-secreting
pituitary tumors is well established (4, 5), only about one third of
these tumors contains these mutations, indicating the presence of
additional transforming events in pituitary tumorigenesis. Despite
efforts at screening for mutations of a variety of oncogenes and tumor
suppressor genes in pituitary tumors, no other transforming gene has
heretofore been identified.
In this study, we have taken a different approach to identify genes
specifically expressed in pituitary tumor cells utilizing a recently
developed RNA differential display assay (18, 19). We chose established
GH- and PRL-secreting rat pituitary tumor cell lines to eliminate the
admixture of normal tissues present in surgically excised human
pituitary tumors or solid experimental rat tumors. Upon screening about
30% of expressed mRNA, a pituitary tumor-derived gene (PTTG) was
identified and characterized. PTTG encodes for a protein of 199 amino
acids that contains no characterized functional motif, suggesting that
PTTG is a novel protein.
The pituitary tumor-specific expression of PTTG was shown by Northern
blot analysis. Testis and fetal liver are the only normal tissues other
than pituitary tumor cells that show PTTG expression. Interestingly,
PTTG mRNA in testis and fetal liver is about 250 bp shorter than that
of pituitary tumor, suggesting that it may represent a PTTG-splicing
variant or alternative usage of promoters or polyadenylation sites. In
view of the partial homology of PTTG to several mouse embryonic and
ovarian cancer cDNAs, these observations suggest that PTTG may paly a
role in fetal development and tumor formation in the ovary.
The importance of PTTG in tumorigenesis was illustrated by its ability
to transform 3T3 fibroblasts when overexpressed in these cells, as
shown by morphological change and anchorage-independent growth of PTTG
transfectants in soft agar. Furthermore, nude mice injected with
PTTG-expressing 3T3 cells developed large tumors within 3 weeks at all
injection sites. These data show that PTTG alone is capable of cellular
transformation, without the requirement of a complimentary oncogene,
and that it is potently tumorigenic in vivo. Generally,
full-cell transformation requires two complementary oncogenes (26, 27, 28).
However, overexpression of a single oncogene may be sufficient to
induce cellular transformation as shown in Rat-1 cell transformation by
overexpression of Ras alone (29). Interestingly, PTTG does not
stimulate but rather inhibits cell proliferation (within 72 h of
assaying time) in cultured cells. This antiproliferative effect is
similar to that seen with transforming growth factor-ß, which exerts
potent inhibition of cell growth (30). It is possible, however, that
once cells are transformed, cell proliferation is accelerated, which
results in rapid growth of tumors in nude mice. It is unlikely that
PTTG represents an activated oncogene due to point mutations in its
coding region, as the sequence of the normal testicular transcript is
identical to the transcript of pituitary tumor cells in the coding
region (L. Pei and S. Melmed, unpublished data). It is, however,
possible that enhanced tissue expression of PTTG by an as yet
unidentified mechanism, may contribute to its oncogenic function.
In summary, we have isolated a novel cDNA (PTTG) that is overexpressed
in rat pituitary tumor cells, induces cellular transformation when
overexpressed in NIH 3T3 fibroblasts, and is tumorigenic in nude mice,
suggesting that PTTG may play a role in pituitary cell transformation
and tumor formation. PTTG represents the first isolated transforming
gene highly expressed in pituitary tumor cells. Its unique sequence and
the presence of a shorter transcript in testis and fetal liver suggest
that it may belong to a new family of transforming genes. Further
characterization of PTTG and its related genes will provide more
insights into molecular mechanisms for tumorigenesis in the anterior
pituitary.
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MATERIALS AND METHODS
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RNA Isolation, PCR Differential Display, and Northern Blot
Analysis
GC and GH4 pituitary tumor cell lines and an
osteogenic sarcoma cell line UM108 were grown in DMEM supplemented with
10% FBS. Normal Sprague-Dawley rat pituitaries were freshly excised.
Total RNA was extracted from cultured cells and pituitary tissue using
RNeasy kit (QIAGEN, Chatsworth, CA) according to manufacturers
instructions. Trace DNA contamination in RNA preparations was removed
by DNaseI (GenHunter Corp., Boston, MA) digestion. Complementary DNA
was synthesized from 200 ng total RNA using MMLV reverse transcriptase
(GenHunter) and one of three anchored primers (GenHunter). The cDNA
generated was used in the PCR display. Three downstream anchored
primers AAGCT11X (where X may be A, G, or C), were used in
conjunction with 40 upstream arbitrary primers for PCR display. We used
120 primer pairs to screen mRNA expression in pituitary tumors
vs. normal pituitary. One tenth of the cDNA generated from
the RT reaction was amplified using AmpliTaq DNA polymerase (Perkin
Elmer, Norwalk, CT) in a total volume of 20 µl containing 10
mM Tris, pH 8.4, 50 mM KCl, 1.5 mM
MgCl2, 0.001% gelatin, 2 µM deoxynucleoside
triphosphates, 0.2 µM each primer and 1 µl
[35S]dATP. PCR cycles consisted of 30 sec at 94 C, 2 min
at 40 C, and 30 sec at 72 C for 40 cycles. The products were separated
on 6% sequencing gels, and dried gels were exposed to Kodak film for
2448 h. After development, The DNA fragments from pituitary tumor and
normal pituitary were compared. Bands unique to pituitary tumor were
excised from the gel, and DNA was extracted by boiling in 100 ml water
and precipitated with ethanol in the presence of glycogen (GenHunter).
DNA was reamplified using the original set of primers and the same
thermal cycling conditions except that the deoxynucleoside triphosphate
concentration was increased to 20 mM. Reaction products
were run on 1% agarose gel and stained with ethidium bromide. Bands
were excised from the gel, eluted (QIAGEN), cloned in to TA vectors
(Invitrogen, San Diego, CA) and sequenced using sequenase (USB,
Cleveland, OH). For Northern blot analysis, 20 µg total RNA were
fractionated on 1% agarose gel, blotted on to nylon membrane, and
hybridized with random primed probe using Quickhyb solutions
(Stratagene, La Jolla, CA). After washing, membranes were exposed to
Kodak films for 672 h. A rat multiple tissue and a human fetal tissue
Northern blot were purchased from Clontech. Approximately 2 µg poly
A+ RNA per lane from eight different rat tissues were run
on a denaturing formaldehyde 1.2% agarose gel, transferred to nylon
membrane, and UV-cross linked. The membrane was first hybridized to the
full-length PTTG cDNA probe and was stripped and rehybridized to a
human ß-actin cDNA control probe. Hybridization was performed at 60 C
for 1 h in ExpressHyb hybridization solution (Clontech). Washing
was twice for 15 min at room temperature in 2x NaCl-sodium citrate,
0.05% SDS, and twice for 15 min at 50 C in 0.1% NaCl-sodium citrate,
0.1% SDS.
cDNA Library Construction, Screening, and DNA Sequencing
Poly A+ RNA was isolated from pituitary tumor GH4
cells using mRNA isolation kit (Stratagene, La Jolla, CA) according to
manufacturers instructions, and was used to construct a cDNA library
in ZAP Express vectors (Stratagene). The cDNA library was constructed
using ZAP Express cDNA synthesis and Gigapack III gold cloning kit
(Stratagene) after manufacturers instructions. The library was
screened using the 396-bp differentially displayed PCR product (cloned
into TA vector) as the probe. After tertiary screening, positive clones
were excised by in vivo excision using helper phage. The
resulting pBK-CMV phagemid containing the insert was identified by
Southern blotting analysis. Unidirectional nested deletions were made
into the DNA insert using EXOIII/Mung bean nuclease deletion kit
(Stratagene) after manufacturers instructions. Both strands of the
insert DNA were sequenced using Sequenase (USB).
In Vitro Transcription and Translation
The sense and antisense PTTG mRNAs were in
vitro transcribed using T3 and T7 RNA polymerase (Stratagene),
respectively. The excess template was removed by DNase I digestion. The
in vitro transcribed mRNA was translated in rabbit
reticulocyte lysate (Stratagene). Reactions were carried out at 30 C
for 60 min, in a total volume of 25 µl containing 3 µl in
vitro transcribed RNA, 2 µl [35S]methionine
(Dupont, Wilmington, DE) and 20 µl lysate. Translation products were
analyzed by SDS-PAGE (15% resolving gel and 5% stacking gel), and
exposed to Kodak film for 16 h.
Overexpression of PTTG in NIH 3T3 Cells and Western Blot
Analysis
The entire coding region of the PTTG was cloned in frame into
pBK-CMV eukaryotic expression vector (Stratagene) and transfected into
NIH 3T3 cells by calcium precipitation. Forty eight hours after
transfection, cells were diluted 1:10 and grown in selection medium
containing 1 mg/ml G418 for 2 weeks when individual colonies were
isolated. Cell extracts were prepared from each colony, separated on
15% SDS-polyacrylamide gels, and blotted onto nylon membrane. A
polyclonal antibody was generated using the first 17 amino acids of
PTTG as epitope (Research Genetics, Huntsville, AL). The antibody was
diluted 1:5000 and incubated with the above membrane at room
temperature for 1 h. After washing, the membrane was incubated
with horseradish peroxidase-labeled secondary antibody for 1 h at
room temperature. The hybridization signal was detected by enhanced
chemiluminescence (ECL detection system, Amersham, Arlington, IL).
Cell Proliferation Assay
Cell proliferation was assayed using CellTiter 96 nonradioactive
cell proliferation assay kit (Promega, Madison, WI) according to the
manufacturers instructions. Five thousand cells were seeded in
96-well plates (six wells for each clone in each assay), and incubated
at 37 C for 2472 h. At each time point, 15 µl of the dye solution
were added to each well and incubated at 37 C for 4 h. One hundred
microliters of the solubilization/stop solution were then added to each
well. After 1 h of incubation, the contents of the wells were
mixed, and absorbance at 595 nm was recorded using an ELISA reader.
Absorbance at 595 nm correlates directly with the number of cells in
each well.
PTTG Transformation in Vitro and in
Vivo
For soft agar assay (31), 60-mm tissue culture plates were
coated with 5 ml soft agar (20% 2x DMEM, 50% DMEM, 10% FBS, 20%
2.5% agar, melted and combined at 45 C). Two milliters of cells
suspended in medium were then combined with 4 ml agar mixture, and 1.5
ml of this mixture were added to each plate. Cells were plated at a
density of 104 cells per dish and incubated for 14 days
before counting the number of colonies and photography. Cells (3
x 105) of either PTTG or pCMV vector alone transfected
cells were resuspended in PBS and injected subcutaneously into nude
mice (five animals for each group). Tumors were excised from animals at
the end of the third week and weighed.
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FOOTNOTES
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1 Address requests for reprints to: Shlomo Melmend, Division of
Endocrinology and Metabolism, Cedars-Sinai Research Institute-UCLA
School of Medicine, 8700 Beverly Boulevard, B131 Los Angeles,
California 90048. 
Supported by NIH Grants DK-42742 (S.M.) and DK-02346 (L.P.) and the
Doris Factor Molecular Endocrinology Laboratory.
2 The GenBank accession number for PTTG is:
U73030. 
Received for publication October 28, 1996.
Revision received January 17, 1997.
Accepted for publication January 21, 1997.
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