Immunity, Vol. 7, 135±146, J uly, 1997, Copyright 1997 by Cell Press
The Role of mel-18, a Mammalian
Polycomb Group Gene, during IL-7±Dependent
Proliferation of Lymphocyte Precursors
Takeshi Akasaka,*²Koh-ichiro Tsuji,?
Hiroshi Kawahira,*²Masamoto Kanno,*²
Ken-ichi Harigaya,³Lina Hu,§Yasuhiro Ebihara,?
Tatsutoshi Nakahata,?Osamu Tetsu,*²
Masaru Taniguchi,*²and Haruhiko Koseki*²
*Core Research for Evolution Science and
J apan Science and Technology Corporation
²Division of Molecular Immunology
³Department of Pathology
§Division of Developmental Genetics
Center for Biomedical Science
School of Medicine
?Department of Clinical Oncology
The Institute for Medical Science
University of Tokyo
molecules located downstream of the IL-7 receptor ?
chain (IL-7R?) and also by the expressionand activation
of molecules required for progression through the cell
cycle (reviewed by DiSanto et al., 1995a; Leonard et al.,
1995). Disruption of the Il7, Il7Ra, Il2 common ? chain
(?c), and J ak3 gene loci, whose products are involved
in the IL-7±mediated signal-transduction pathway, re-
sults in a significant reduction in the numberof lympho-
cytes, leading to severe combined immunodeficiency
(von Freeden-J effry et al., 1995; DiSanto et al., 1995b;
Nosaka et al., 1995; Park et al., 1995; Peschon et al.,
1994; Thomis et al., 1995). However, nuclear factors
necessary for IL-7±dependent proliferation are not well
understood. Recently, it was suggested that the mam-
malian homolog of the Polycomb group (Pc-G) gene
product,Bmi-1, plays a roleinthe IL-7±dependentprolif-
eration of lymphoid precursors, although its mode of
function remains unclear (van der Lugt et al., 1994).
InDrosophilamelanogaster,the functionof Pc-G gene
products has been extensively analyzed in the context
of thetranscriptionalregulationof genes inHOM-C com-
plexes. The expression of HOM-C genes is initially in-
duced by segmentation gene products suchas Kru Èppel
and hunchback (White and Lehmann, 1986;Harding and
Levine, 1988; Irish et al., 1989; Reinitz and Levine, 1990;
Qian et al., 1991; Shimell et al., 1994). After the decay
of these proteins, the segment-specific distribution of
HOM-C genes is regulated by at least two classes of
genes, including trithorax group (trx-G) (reviewed by
Kennison, 1993) and Polycomb group (Pc-G) genes (re-
viewed by Paro, 1990; Bienz and Mu Èller, 1995). These
two groups of gene products maintain the active and
repressive states of homeotic genes in the appropriate
segments. Loss-of-function mutations of Pc-G genes
lead to the ectopic expressionof HOM-C gene products
outside their normal anterior±posterior boundaries, re-
sulting in segmental homeotic transformations (Dura
and Ingham, 1988; Glicksman and Brower, 1990; Simon
et al., 1992). Interestingly, another Pc-G gene product,
multi sex combs (msc), is required not only for the tran-
scriptional regulation of HOM-C genes but also for the
control of normal growth of hemopoietic cells (San-
tamarõ Â a and Randsholt, 1995). Thus, it is suggested that
Drosophila Pc-G gene products possess other cellular
functions, including some involved in growth control.
Recently, vertebrate homologs structurally related to
Drosophila Pc-G genes, including mel-18, bmi-1, M33,
and rae-28/Mph1, have been identified (Tagawa et al.,
1990; Haupt et al., 1991; van Lohuizen et al., 1991a;
Pearce et al., 1992; Nomura et al., 1994; Alkema et al.,
1997).The Mel-18and Bmi-1gene products shareamino
acid sequences homologous with proteins encoded by
Drosophila posterior sex combs (Psc) and its neigh-
boring gene, suppressor two of zeste (Su(z)2) (Brunk et
al, 1991; van Lohuizen et al., 1991b). M33 and rae-28/
Mph1 are thought to be murine counterparts of Poly-
comb (Pc) and polyhomeotic (ph), respectively. More-
over, not only structural butalso functionalconservation
between Drosophila Pc-G genes and their vertebrate
homologs is suggested. In mel-18-, bmi-1-, M33-, and
mel-18 is a mammalian homolog of Drosophila mela-
nogaster Polycomb group genes. Mice lacking the
mel-18 gene show a posterior transformation of the
axial skeleton, severe combined immunodeficiency,
and a food-passing disturbance in the lower intestine
due to hypertrophy of the smooth muscle layer. In this
study, the severe combined immunodeficiency ob-
served in mel-18 mutant mice is correlated with the
impaired mitotic response of lymphocyte precursors
upon interleukin-7 stimulation. Strikingly, the axial
skeleton and lymphoid phenotypes are identical in
both mel-18 and bmi-1 mutants, indicating that the
Mel-18and Bmi-1gene products might actin the same
genetic cascade. These results suggest that mamma-
lian Polycomb group gene products are involved in
cell cycle progression in the immune system.
During lymphocyte development, signals mediated
through B and T cell antigen receptor complexes play
crucial roles in the differentiation and proliferation of
lymphocyte precursors. Productive rearrangement of
the immunoglobulin heavy (IgH) chain or T cell antigen
receptor (TCR) ? chain locus and the subsequent ex-
pression of IgH or TCR? gene products allow the pro-
genitor cells to enter into the mitotically active stage
(Hardy et al., 1991; Hoffman et al., 1996). The prolifera-
tion of lymphoid precursors at this stage requires mito-
genic stimulation by interleukin-7 (IL-7). Therefore, the
acquisition of mitotic ability to respond to IL-7 is essen-
tial for the normal lymphocyte development. IL-7 re-
sponsiveness of lymphocyte precursors is thought to
be conferred by the expression of signal-transducing
rae-28/Mph1±deficient mice, posterior transformations
of the vertebral column that resemble the Pc-G mutant
phenotype in Drosophila are observed (Akasaka et al.,
1996; van der Lugt et al., 1994; Core Â et al., 1997; Y.
Takihara and K. Shimada, personal communications).
The M33 gene product is able to rescue the Pc mutant
phenotype to some extent(Mu Èlleretal., 1995).Therefore
the functions of gene products related to Drosophila
Pc-G genes may beevolutionally conserved inthe deter-
mination of segment identity along the vertebrate ante-
rior±posterioraxis. Intriguingly,Mll, a mammalianhomo-
log of Trithorax (Trx)gene products, has beenimplicated
in the normal and abnormal growth of hematopoietic
cells, suggesting the possible involvement of Pc-G and
trx-G gene products during hematopoietic cellprolifera-
tion invertebrates as wellas Drosophila (Gu et al., 1992;
Tkachuk et al., 1992; Yu et al., 1995).
The Mel-18 protein is 342 amino acids long, and the
N-terminal 102 amino acids, including the RING finger
domain, exhibit 93% identity to the Bmi-1 gene product
(Freemont et al., 1991; Ishida et al., 1993). Furthermore,
the deduced secondary structure of the Mel-18 protein
is similar not only to the Bmi-1 protein but also to the
Drosophila Psc and Su(z)2 gene products. Polymerase
chain reaction (PCR)±mediated binding site selection
reveals that the Mel-18 proteinrecognizes the 5?-GACT-
NGACT-3? motifwiththe highestaffinity, and the binding
of the Mel-18 gene product to this motif represses the
activity of juxtaposed enhancer elements (Kanno et al.,
1995). In mice lacking mel-18, posterior transformation
of the axial skeleton, defects in T and B lymphocyte
development, and hypertrophy of intestinal smooth
muscle ina restricted area leading to the intestinalatre-
sia are reproducibly observed. The axial phenotype is
correlated with alterations in Hox cluster gene expres-
sionin the developing sclerotome (Akasaka et al., 1996).
Inthis study, we investigatedthe functions of the Mel-
18 protein during lymphocyte development using mel-
18±deficient mice. The lymphoid phenotypes of mel-18
mutant mice are reminiscent of those seen in bmi-1
mutants. Defects in B and T lymphocyte development
are due to the insufficient response to IL-7 stimulation
of the lymphoid precursors. The possible involvement
of the Mel-18 protein in the cell cycle, particularly in
lymphocyte precursors, is discussed.
Figure 1. The Expression of mel-18 mRNA in Various Hematopoitic
(A) mel-18 expression in several lymphoid organs was investigated
by RNase protection assay using adult cerebellum and day 13.5 pc
totalembryonic RNA as positive controls. A 411 bp fragment(arrow),
which was protected by mel-18 mRNA, was present in all lymphoid
organs examined. ?-tubulinwith a 261 bp protected band was used
as an internal control (T).
(B) mel-18 expression in three lineages of bone marrow (BM) cells
was analyzed by RT-PCR. mel-18 expression was detectable in
CD45R?, TER119?, andMac-1?lineage cells atthe same level, while
thymocytes expressed mel-18 at a lower level. ?-actin was used as
a positive control.
(RT-PCR) using ?-actin as a control. mel-18 expression
in these lineage cells was comparable in all three lin-
eages (Figure 1B). Because the thymus is composed of
thymocytes and thymic stroma cells, mel-18 expression
in the thymocytes was investigated. mel-18 was ex-
pressed less inthe thymocytes thaninthe bone marrow
mel-18 Expression in Lymphoid Organs
The expression of murine mel-18 was investigated in
adultlymphoid organs using anRNase protectionassay.
mel-18 transcripts were present in the thymus, spleen,
bone marrow, lymph nodes, and Peyer's patches, al-
though the amounts were smaller than those seen in
adult cerebellumorin day 13.5 postcoitus (pc) embryos
(Figure 1A). Since bone marrow cells include not only
lymphoid lineage cells but also granulocyte-macro-
phage (GM) and erythroid lineage cells, the expression
of mel-18in each lineage was further investigated. Cells
from each lineage were enriched to greater than 95%
purity by a magnetic cell-sorting system and were sub-
jected to semiquantitative reverse transcriptase PCR
Selective Reduction in the Number of Lymphoid
Cells in mel-18±Deficient Mice
Mice lacking mel-18 survived until birth, but most died
between 3 and 6 weeks after birth, as described pre-
viously (Akasaka et al., 1996). The growth retardation of
the mutant pups became obvious at approximately 3
weeks of age, and the affected pups remained fragile
thereafter. The hyperplasia of the smooth muscle layer
of the lower intestine and the subsequent food-passing
problems are the likely reasons for the growth retarda-
tionobserved inmel-18±deficientmice (datanotshown).
Macroscopic abnormalities were apparent in lymphoid
organs of mel-18 mutants. The thymus and spleen were
extremely small and the lymph nodes and Peyer's
Mel-18 in Lymphocyte Development
2B). A high-magnificationviewof thewhitepulprevealed
that the accumulation of T lymphocytes inthe periarteri-
olar lymphatic sheath seen in the wild-type spleen was
impaired in mel-18±deficient mice (Figures 2C and 2D).
Inthe bonemarrow, hematopoiesis was hypoplastic and
replaced by adipocytes (Figures 2E and 2F).
To address which cell lineages are affected in the
bone marrow and spleen of mel-18 mutants, cellular
compositions were determined on cytospin prepara-
tions stained by the May-Gru Ènwald-Giemsa method
(Tables 1 and 2) (Nakahata et al., 1982). The absolute
number of lymphocytes in the bone marrow was re-
duced to approximately 10% of that in wild type, while
erythroid, granuloid, and monocyte±macrophage lin-
eage cells were reduced by half. Similar findings were
observed in the spleen. The absolute numbers of
lymphoid cells were severely reduced, whereas those of
neutrophils, eosinophils, and erythroid cells were nearly
unchanged. These results indicate that lymphocyte de-
velopment is exclusively impaired in mel-18 mutants.
Although mel-18 is ubiquitously expressed in hemato-
poietic cells, the Mel-18 gene product is functionally
required preferentially for the development of the
lymphoid cell lineage.
B cell generation in the bone marrow follows an or-
dered development and can be partitioned into several
distinct fractions (A±F) according to developmental
stage by using cell surface markers such as CD43,
CD45R (B220), heat-stable antigen, BP-1, and surface
IgM (sIgM)(Hardy et al., 1991).Inmel-18±deficient mice,
the absolute number of total CD45R?B cells was
significantly reduced, to 10%±20% compared to wild
type (Figure 3A). The number of CD45R?sIgM?mature
B cells (fractions E and F) in mel-18 mutants was about
20% of wild-type (Figure 3B). The CD45R?sIgM?CD43?
pre±B cell (fraction D) compartment in the bone marrow
showed a 20-fold reduction compared to wild type.
This fraction derives from progenitors whose pro-
liferation is dependent on IL-7 (Hardy et al., 1991).
CD45R?sIgM?CD43?pro±B cells (fractions A±C?), in-
cluding IL-7±responsive precursors (fraction C), were
also decreased about 5-fold, and large pro±B cells in
this fraction were undetectable, suggesting decreased
mitotic activity in mel-18±deficient mice (Figure 3A).
Therefore it is possible that mice that lack mel-18 have
primary defects in the expansionordifferentiationof the
pro±B cell fractions (fractions A±C?) and/or also in the
subsequent accumulation of pre±B cells (fraction D) in
the bone marrow.
The reduction of CD45R?sIgM?pre±B cells corre-
lates closely with the decreased mitotic activity in mel-
18±deficient mice (Figure 4). Sixty-eight percent of
CD45R?sIgM?pro± or pre±B cells were in the S phase
of the cell cycle in wild-type mice compared to 33% in
mel-18 mutants. CD45R?sIgM?B cells were not mitoti-
cally active in wild-type mice (3.5% in S phase), and the
numberof cells inS phase in mel-18±deficientmice was
about half of that in wild type. Therefore the impair-
ment of the mitotic activity and/or the maintenance of
CD45R?sIgM?pro± or pre±B cells is likely to be one of
the major causes of the hypoplasia of B lymphocytes.
Similarly, the absolute numbers of mature B and T
lymphocytes were also reduced to approximately 10%
Figure 2. Histological Analysis and Quantitation of Nucleated Cells
in the Thymus, Spleen, and Bone Marrow of 2-Week-Old mel-18
(A and B) Hematoxylin-eosin±stained sections of thymus (A) and
spleen (B) from mel-18 mutant (?/?) and wild-type (?/?) mice,
prepared to show their original size. The cortical region is greatly
shrunk in mel-18 mutant thymus and the white pulp is reduced in
number and size in mutant spleen.
(C and D) Higher-magnification views of primary follicles in mel-18
mutant (C) and wild-type (D) mice. The accumulation of T lympho-
cytes around the sheathic arterioles (arrows) was strongly impaired
in mel-18 mutants.
(E and F) Hematoxylin-eosin±stained sections of the bone marrow
cavity revealed reduced cellularity in mel-18 mutants (E) compared
with wildtype (F). The bonemarrow hematopoietic regionwas hypo-
plastic and replaced with adipocytes in mutants (E).
(G)The nucleated cellnumbers of thymus, spleen, and bone marrow
(BM) in mel-18 mutants were compared with wild type and found
to be about2%, 10%, and 66% of wild type, respectively. Errorbars
patches hardlyvisible in3to 4-week-oldmutants (Figure
2 and data not shown). The total numbers of nucleated
cells in the thymus, spleen, and bone marrow were re-
ducedto approximately2%, 10%, and 60% of wild type,
respectively (Figure 2G). Histological examination re-
vealed abnormal architecture due to the significant re-
ductions inlymphocytes inthe thymus,spleen, andbone
marrow. Involuted thymus due to severe hypoplasia of
the thymic cortex was seenin2-week-old mel-18mutant
mice (Figure 2A). Clusters of white pulp were signifi-
cantly reduced in numberand size in the spleen (Figure
Table 1. Cellular Composition of Nucleated Femur Bone Marrow Cells
Number Neutrophils EosinophilsMonocyte±Macrophages
Wild-type 11.8 ? 106
2.0 ? 106
1.5 ? 105
2.3 ? 105
8.8 ? 105
1.8 ? 106
5.3 ? 105
4.7 ? 105
3.5 ? 106
4.3 ? 106
2.0 ? 106
2.0 ? 106
1.8 ? 105
1.6 ? 105
1.3 ? 105
3.5 ? 105
5.9 ? 104
1.6 ? 105
2.5 ? 104
2.9 ? 104
6.4 ? 106
8.4 ? 106
2.8 ? 106
3.1 ? 106
of wild type in the spleen (Table 1, Figure 3C, and data
not shown). Interestingly, the numberof CD45R?sIgMdull
B cells was markedly increased in the spleen of mel-
18-lacking mice (Figure 3C). A similar finding was ob-
served in Il7-deficient mice (von Freeden-J effry et al.,
1995). ??T cells and NK1.1?T cells exhibited 20-fold
reduction inmel-18±deficientmice comparedto the wild
type, while natural killer (NK) cells were weakly affected
(Figure 3D). Therefore mel-18±deficientmice were more
like Il7ra-deficient than ?c- or J ak3-deficient mice, be-
cause NK cells have been shown not to develop in ?c-
or J ak3-deficient mice whereas Il7ra-deficient do have
NK cells (Cao et al., 1995, Park et al., 1995, He and
Malek, 1996). ??T cells with an intermediate level of
TCR? chain expression predominated in mel-18±
deficient mice (Figure 3D).
The thymus of3- to 4-week-old mel-18±deficient mice
was very small. In mel-18±deficient mice, exponential
outgrowth of the thymus, which usually occurs during
the neonatal stage, was totally impaired, and subse-
quently the absolute number of the thymocytes de-
creased progressively in 2- to 4-week-old mice (unpub-
lished data). The proportion of CD4?CD8?, CD4?CD8?,
and CD4?CD8?thymocytes appeared normal up to ap-
proximately 2 weeks after birth and then CD4?CD8?
thymocytes predominated (Figure 3E and data not
1995; Thomis et al., 1995; von Freeden-J effry et al.,
1995). Simultaneously, cytokine-dependent cell growth
of lymphoid, GM, and erythroid lineage cells was com-
The number of IL-7±responsive precursors and the
growth rate of each IL-7±responsive clone was exam-
ined using clonal culture in semisolid medium. Freshly
isolated nucleated cells were cultured in the presence
of IL-7, IL-7 plus stem cell factor (SCF), IL-3 plus SCF
plus erythropoietin (EPO), orIL-3 plus EPO withor with-
out fetal calf serum(FCS) (Figure 5). Lymphoid colonies
appeared in cultures containing IL-7 or IL-7 plus SCF,
and morethan90% of the cells inthese colonies derived
from wild-type and mel-18 mutant mice were positive
for CD45R expression, as reported previously (Figure
5E) (Suda et al., 1989). Therefore most cells in lymphoid
colonies were B lineage cells. In the cultures containing
30% FCS, the number of lymphoid colonies from mel-
18 mutants generated in IL-7 or IL-7 plus SCF culture
was reduced to one eighth or one quarter of that seen
in the wild-type, respectively, and the cell number in
each colony was about one tenth of wild type after 12
days of culture (Figures 5A and 5B). Therefore, the num-
ber of IL-7±responsive precursors is reduced and the
IL-7±dependent proliferation of lymphoid cells is clearly
affected in mel-18 mutants. The insufficient expansion
of pro±B cells and/or the suboptimal accumulation or
maintenance of pre±B cells in vivo is likely to be due
to the reduction in mitotic activity of IL-7±responsive
precursors in mel-18 mutants.
We also prepared serum-free cultures to exclude any
unknown mitogenic effects of FCS. In serum-free me-
dium, no IL-7±dependent colony formation or doublet
cell generation was observed in mel-18 mutants, while
IL-7±dependent precursor cells grew exponentially in
wild-type bone marrow (Figures 5C and 5D). Therefore
IL-7±responsive precursors were present in mel-18±
Altered IL-7 Responsiveness in mel-18±Deficient Mice
Since the lymphoid cell phenotypes observed in mel-
18±lacking mice partially resemble those of Il7-, Il7ra-,
?c-, orJ ak3-deficient mice, and also since the prolifera-
tion of pro±B and pro-T cells is known to depend on
IL-7, it is important to investigate the IL-7±dependent
proliferative capacity of lymphoid cells in the bone mar-
row, spleen, fetalliver, and thymus (Peschonet al., 1994;
DiSanto et al., 1995b; Nosaka et al., 1995; Park et al.,
Table 2. Cellular Composition of Nucleated Spleen Cells
Wild-type 12.6 ? 106
2.7 ? 106
3.7 ? 105
7.0 ? 105
1.3 ? 106
2.6 ? 106
5.4 ? 105
1.4 ? 106
2.6 ? 105
1.6 ? 105
2.2 ? 105
9.4 ? 105
4.2 ? 104
2.8 ? 104
4.9 ? 104
1.1 ? 104
4.2 ? 104
7.8 ? 104
2.4 ? 104
3.4 ? 104
4.2 ? 106
5.6 ? 106
1.2 ? 106
3.1 ? 106
Mel-18 in Lymphocyte Development
Figure 4. Cell Cycle Analysis of Bone Marrow Cells from mel-18
CD45R?sIgM?and CD45R?IgM?fractions were gated as indicated
by the boxes at the top. Sixty-eight percent of CD45R?sIgM?cells
were in S phase in the wild-type, while the proportion in mel-18
mutant mice was 33%.
Figure 3. Flow Cytometric Analysis of Bone Marrow Cells, Spleno-
cytes, and Thymocytes from mel-18 Mutant Mice
(A) Three-color analysis using anti-CD45R, sIgM, and CD43 was
performed using bone marrow cells from mel-18 mutant (-/-) and
wild-type(?/?) mice. (Left) Expressionof CD45R and sIgM. (Center)
CD43 expression in the sIgM negative fraction. Each fraction was
designated according to Hardy et al. (1991). (Right) Results of for-
ward scatter analysis in fractions A±C?. The number of large cells
is significantly reduced in mel-18 mutants.
(B) The frequencies of nucleated cells in each fraction were quan-
(C) The expression of CD45R and sIgM in spleen was compared
between mel-18 mutant and wild-type mice. The relative frequency
of sIgMdulllymphocytes was higher in mel-18 mutants (right).
(D) The expression of NK1.1 and TCR? chain in splenocytes from
4-week-old mel-18mutantmice. Results oftwo-colorfluorescence-
activated cell sorting analysis were shown. NK and NK T cell devel-
opment was not significantly impaired in mutant mice.
(E) Expression of CD4 and CD8 in thymus from 4-week-old mel-18
mutant (top) and litter control (bottom) mice was analyzed.
deficient mice; however, they could not proliferate upon
IL-7 stimulation without serum.
The generation of GM, megakaryocyte, erythroid
bursts (burst-forming units for erythroid colonies), and
mixed colonies in the presence of SCF plus IL-3 plus
EPO orIL-3 plus EPO was, however, not affected inmel-
18±deficient mice (Figure 5F). Therefore the develop-
ment of GM and erythroid lineage cells is intact in mel-
18±deficient mice both in vivo and in vitro. In summary,
IL-7±dependent proliferation of immature lymphocytes
is exclusively affected in the bone marrow of mel-18±
Since IL-7 promotes the accumulation of the cyto-
plasmic ? chain product in pro±B cells, we investigated
? chain expression in IL-7±responsive colonies devel-
oped in clonal culture using serum-containing medium
(Figure 5E). More than 90% of cells were negative for
sIgM expression (Figure 5E). Although the frequency of
CD45R?sIgM?cells was slightlyhigherinwild-type than
mel-18 mutants, we did not find a significant difference
in cytoplasmic ? chain expression between wild-type
and mel-18 mutants. Therefore the IL-7±dependent in-
duction of cytoplasmic ? chain±positive cells is not af-
fected in mel-18 mutants.
Day 13.5±15.5 pc fetal liver contains lymphocyte pre-
cursors, and B lymphocytes can be induced by IL-7 in
the presence of a bone marrow±derived stromal cell
line, PA6. Under these conditions, the proliferation of
CD45R?B cells was strongly impairedinmel-18mutants
compared to wild type, in which an approximately 150-
fold increase in CD45R?B cells was reproducibly seen
(Figures 5G and 5H). The subsequent accumulation of
CD45R?sIgM?CD43?pre±B cells (fraction D) was also
affected (data not shown). Therefore, it is also shown
maintenance of fetal liver lymphoid precursors requires
Mel-18 gene products.
The IL-7±dependent mitotic ability of thymocytes was
investigated in suspension culture in the presence of
phorbol-12-myristate-13-acetate(PMA) and found to be
strongly impaired in 4-week-old mel-18±deficient mice.
Proliferation of the thymocytes induced by IL-2, IL-4,
and Ca2?ionophore plus PMA were affected as well as
by IL-7 plus PMA (Figure 6A). Since IL-2 and IL-4 are
sharing the signaling pathway such as ?c/J AK3 cascade
with IL-7, it is presumed that overlapping signal-trans-
ducing pathways or downstream elements required for
the proliferation of the thymocytes might be affected in
mel-18±deficient mice. As the mixture of PMA and Ca2?
ionophore promotes cell cycle progression through the
activation of protein kinase C, the Mel-18 gene product
might function not only in the ?c/J AK3 cascade but also
in the protein kinase C pathway in thymocyte prolifera-
tion(Kim et al., 1989). Since IL-7 is knownto be required
forthe proliferationof CD4?CD8?immaturethymocytes,
Mel-18 gene products might be needed for the growth
and/or maintenance of IL-7±responsive immature lym-
phocytes (Watson et al., 1989).
The proliferative responses of mature splenic B and
T lymphocytes to lipopolysaccharide and concanavalin
A in mel-18±deficient mice were comparable to those
in wild-type mice (Figure 6B). This suggests that mature
Figure 5. Cytokine-Dependent Proliferation and Differentiation of
Bone Marrow and Fetal LiverNucleated Cells from mel-18-Deficient
(A±E) Cytokine-dependent cell growth of lymphoid, GM, and ery-
throid lineage cells of bone marrow from 4-week-old mel-18 mutant
(A) The numbers of lymphoid colonies appearing in clonal cultures
of bone marrow cells in semisolid medium containing 30% FCS and
IL-7 or IL-7 plus SCF were compared between wild-type and mel-
18 mutant mice.
(B) Cell numbers in each lymphoid colony appearing in clonal cul-
tures of bone marrow cells in semisolid medium containing 30%
FCS and IL-7 were counted.
(C) The numbers of lymphoid colonies appearing in FCS-free semi-
solid medium containing IL-7 or IL-7 plus SCF were compared.
(D) The growth responseof lymphoid precursors was investigated in
FCS-freesemisolid mediumcontaining IL-7.Sincenot evendoublets
were observed in the mutant bone marrow cell cultures, the cell
number at each time point was nominally scored as 1.
(E)Theexpressionofcytoplasmic ?chainand sIgMwas investigated
after clonal culture in the presence of 30% FCS and IL-7. Freshly
prepared thymocytes were used as negative controls for the cyto-
plasmic staining of?chain products.Freshlyprepared bonemarrow
cells were used as positive controls.
(F) The generation of GM colonies was seen in the presence of IL-7
plus SCF or IL-3 plus EPO, and burst-forming units for erythroid
(BFU-E)and mixed (MIX)colonies in the presence of IL-7 plus SCF±
and IL-3 plus EPO plus SCF±containing medium.
(G and H) IL-7 and PA6 stromal cell±dependent proliferation of fetal
liver cells from mel-18 mutant mice.
(G) Day 14.5 pc fetal liver cells from wild-type (?/?) and mel-18
mutant (?/?) fetuses were cultured on PA6 stromal cells in the
presence of IL-7 for 9 days.
(H) The proliferationof CD45R?lymphocytes inculture was severely
affected in mel-18 mutant fetal liver cells.
Mel-18 in Lymphocyte Development
Figure 6. Proliferative Response of Thymocytes and Splenocytes
from mel-18 Mutant Mice
(A)Thymocytes from 2-week-old mel-18 mutant and wild-type mice
were stimulated with IL-7, IL-2, IL-4, or Ca2?ionophore in the pres-
ence of PMA. The responsiveness to these mitogenic stimuli was
strongly affected in mel-18±deficient mice.
(B) Splenocytes from 2-week-old mel-18 mutantand wild-type mice
were stimulated with lipopolysaccharide (LPS) or concanavalin A
(ConA). No difference was observed between mutant and control
Figure 7. Expression and Functions of Molecules Involved in the
IL-7 Signaling Pathway and Proliferation of Thymocytes
(A) The expression of IL-7R? chain in CD45R?bone marrow cells
(B) The expression of Il7, ?c, and J ak3 from the bone marrow (BM)
cells and thymocytes of 4-week-old mel-18 mutantand littercontrol
mice was investigated by RT-PCR in semiquantitative fashion using
?-actin as an internal control.
(C) Activation of STAT proteins upon IL-7 stimulation was investi-
gated by electrophoretic mobility shift assay using wild-type (probe
A, lanes 1±8)and mutant(probe B, lane 9) GAS motifprobes. Whole-
cell extracts prepared from the thymocytes of 4-week-old mel-18±
deficient (M) and wild-type (W) mice were used for each binding
reaction. Normal rabbit serum was used as a control. Arrow, DNA±
STAT complexes induced upon IL-7 stimulation. F, free probe.
(D) The expressionlevelofRb gene productfrom day 16.5 pc thymo-
cytes and whole brain.
splenic lymphocytes do not require Mel-18 forprolifera-
tion, while the mitotic capacity of immature splenic lym-
phocytes to IL-7 stimulation is also impaired (data not
The IL-7 Signaling Pathway Is Not Impaired
in mel-18 Deficient Mice
To analyze the molecular basis underlying the impair-
ment of IL-7±dependent proliferation of early lympho-
cytes in 4-week-old mel-18±deficient mice, we investi-
gated the expressionand functionof molecules involved
in the IL-7 signaling pathway. The expression of Il7,
IL-7R? chain, ?c, and J ak3 proved to be unaffected in
mel-18±deficient mice in the thymus and bone marrow
(Figures 7A and 7B and data not shown). We further
examined the IL-7±induced activation of STAT proteins
in the thymocytes of mel-18±deficient mice by electro-
phoretic mobility shiftassay using wild-type and mutant
GAS motif probes because STAT proteins are known to
be activated by several cytokines including IL-2, IL-4,
IL-7,andothers (J ian-Xinetal., 1995).As showninFigure
7C, upon IL-7 stimulation, two binding complexes were
specifically induced as indicated by the arrow, and the
upperbandproved torepresentactivatedSTAT5 protein
by anti-STAT5antiserum.This resultindicatedthatfunc-
tional activation of STAT proteins including STAT5 in
thymocytes upon IL-7 stimulation was not affected in
mel-18±deficient mice. Therefore the IL-7 signal-trans-
ducing cascade functions normally in mel-18 mutant
mice although thymocytes were not capable of prolifer-
ating upon IL-7 stimulation. This result suggested that
Mel-18 might be located downstream or parallel to the
IL-7 signaling pathway.
During the mitogen-inducedproliferationof peripheral
T lymphocytes, the induction and phosphorylation of
Retinoblastoma (Rb) gene products are correlated with
the initiation of DNA synthesis (Terada et al., 1991). The
Rb gene is constitutively expressed in the thymus, pre-
sumably due to the proliferation of a certain subset of
thymocytes (Bernards et al., 1989). Thus we attempted
to investigate the expression of Rb protein in day 16.5
pc fetal thymus and brain by Western blot analysis (Fig-
ure 7D). Rb expression was strongly impaired in the
thymus of mel-18±deficient mice while the expression
in mutant brain was intact. Therefore the proliferative
defects of thymocytes in mel-18±deficient mice were
strongly correlated with a significant reduction in Rb
This observation implies that the locus-specific accu-
somes is influencedby othercis- ortrans-acting factors.
This may be important for explaining the tissue-specific
functions of Mel-18.
Functional Correlation between Mel-18 and Bmi-1
The Bmi-1 gene product is structurally similar to Mel-
18as wellas to Drosophila Psc and Su(z)2. Interestingly,
the phenotypes of mel-18± and bmi-1±deficient mice
tical. Posteriortransformations of the axial skeleton and
defects in lymphocyte development are prominent in
both mutants, while morphological and functional ab-
normalities of the cerebellum or lower intestine are ob-
served exclusively in bmi-1 or mel-18 mutants, respec-
tively (Akasaka et al., 1996; van der Lugt et al., 1996).
The ectopic expression of Hox genes in the developing
paraxialmesodermis strongly correlated with the verte-
bral phenotype in both mutants, indicating that the Mel-
18 and Bmi-1 proteins control Hox gene expression in
a similar manner. An impairment in the IL-7±dependent
proliferation of lymphocyte precursors might be the un-
derlying cause for the severe combined immunodefi-
ciency seen in mel-18 and bmi-1 mutants. Therefore, it
appears possible that the set of target genes or inter-
acting proteins for Mel-18 and Bmi-1 during paraxial
mesoderm and lymphocyte development may overlap
extensively. InDrosophila, several Pc-G gene products,
including Polycomb (Pc), Polyhomeotic (ph), and Psc
proteins are colocalized onto many but not all sites of
the polytene chromosomes. It has also been suggested
that the Pc and ph gene products are involved in a large
protein complex (Franke et al., 1992; Martin and Adler,
1993). Therefore it is likely that the Mel-18 and Bmi-1
proteins work as components of a protein complex dur-
ing axialskeletonand lymphocytedevelopmentandalso
that the loss of either Mel-18 or Bmi-1 might result in a
reduced stability of the protein complex to a similar
extent. This possibility is supported by recent observa-
tions indicating the direct interaction between Mel-18
or Bmi-1 and Rae-28/Mph1 (Alkema et al., 1997).
In mel-18±deficient mice, the development of lymphoid
lineage cells among hematopoietic cell lineages is af-
fected exclusively,althoughmel-18is equally expressed
in lymphoid, erythroid, and GM lineage cells. Significant
reductions in B and T lymphocytes in all lymphoid tis-
sues correlates strongly with defects in IL-7±dependent
cell cycle progression and/or subsequent maintenance
of immature lymphocytes.
Mel-18 Is Required for the Early
During lymphocyte development, the mitotic response
to and differentiation of lymphoid precursor cells upon
IL-7 stimulation is crucial forthe generation of anappro-
priate number of mature lymphocytes (Peschon et al.,
1994; DiSanto et al., 1995b; Nosaka et al., 1995; Park
et al., 1995; Thomis et al., 1995; von Freeden-J effry et
al., 1995). In this study, we have shown a phenotypic
resemblance of bone marrow and spleen lymphocytes
betweenmel-18mutants and micedeficientforIl7, Il7Ra,
?c, and J ak3 and also have demonstrated a significant
reduction in the mitotic activity of immature lympho-
cytes in the presence of IL-7 in mel-18 mutants. Thus, it
is strongly suggestedthat defects inthe IL-7±dependent
proliferation and/or subsequent maintenance of early
lymphocytes is the cause of the severe combined
immunodeficiency seen in mel-18 mutants. Since ?c/
J AK3/STAT5 pathway was not significantly affected,
downstream or parallel pathway required for IL-7±
dependent proliferation might be impaired in mel-18±
deficient mice. Stimulation of B cell precursors through
the IL-7R? chain is also required for the expression of
cytoplasmic ? chain via a distinct signal-transducing
pathway from the ?c/J AK3 cascade (Corcoran et al.,
1996). Since IL-7±dependent colonies derivedfrom mel-
18 mutants are positive forcytoplasmic ? chain expres-
sion, the Mel-18 gene product might not be necessary
for this process.
The cell type±specific Mel-18 function might be con-
ferred by the differential utilization of a set of down-
stream genes or interacting proteins in each lineage
or stage. In Drosophila, the binding of Psc and Su(z)2
proteins to polytene chromosomes has been shown to
be strongly influenced by another Pc-G gene product,
Enhancer of zeste [En(z)], although the accumulation of
Psc, Su(z)2, and En(z) proteins on polytene chromo-
somes does not overlap completely(Rastellietal., 1993).
Possible Roles of Pc-G Gene Products
in Cell Cycle Progression
Accumulating evidence suggests the involvement of
Mel-18 in normal and abnormal proliferation. The dimi-
nution of the Mel-18 protein in NIH3T3 fibroblasts by
expression of antisense RNA leads to the acquisition of
tumorigenic activity in nude mice, indicating a tumor
suppressor activity for Mel-18 (Kanno et al., 1995). The
overexpression of Mel-18 results in the mitotic unre-
sponsiveness ofmature resting B cells afterthe engage-
ment of B cell antigen receptor (O. T. and M. K., unpub-
lished data). Similarly, Bmi-1 is also required for the
regulated proliferation of lymphoid cells. The overex-
pression of Bmi-1 induced by retroviral insertion in E?-
myc transgenic mice or in Em-bmi-1 transgenic mice
results reproducibly in a high incidence of B lymphoma
(Haupt et al., 1991; van Lohuizen et al., 1991a; Alkema
et al., 1995). Therefore it is important to keep the
amounts of Mel-18 and Bmi-1 proteins within a certain
Mel-18 in Lymphocyte Development
wax (Sigma). Sections were cut 8 mm thick and placed onto
3-aminopropyltriethoxysilane±coated slides. The sections were de-
waxed in xylene and rehydrated first with ethanol and then with
phosphate-buffered saline. Rehydrated sections were stained with
hematoxylin-eosin according to the usual protocol.
window to maintain appropriate mitotic activity. Quanti-
tative changes in the Mel-18 or Bmi-1 proteins affect
the quantity orquality of protein complexes, resulting in
the alterationof asetof downstreamgenes orchromatin
There is now evidence that Pc-G gene products other
thanMel-18andBmi-1 are involvedinnormaland abnor-
mal cellcycle progression. InDrosophila, the Pc-G gene
product encoded by the multi sex combs (mxc) locus
functions as a tumorsuppressor gene. In mxc mutants,
uncontrolled malignant growth of blood cells as well as
homeotic transformation has previously been demon-
strated(Santamarõ Â a andRandsholt, 1995).AnotherPc-G
gene product,sexcombs onmidleg (scm), whichgeneti-
cally interacts withSu(z)2, has beenrevealed to possess
extensive similarity to the Drosophila tumor suppressor
gene l(3)mbt protein suggesting molecular interactions
between Pc-G and tumor suppressor gene products
(Bornemann et al., 1996). Recently, a direct molecular
interaction between the mammalian homolog of the
En(z) gene product, ENX-1, and the protooncogene
product Vav, which is required for antigen receptor±
mediated proliferation of B and T cells, was demon-
strated (Hobert et al., 1996). This observation suggests
that molecular complexes composed of Pc-G products
are possible links between the mitogenic signal trans-
ducing cascades and cell cycle machineries.
Phenotypic Analyses of Hematopoietic Cells
Cells from thymus, spleen, and bone marrow were prepared by
dissociation between frosted glass slides, and cell numbers were
determined microscopically. The cellularcompositions ofbone mar-
row and spleen cells were determined on cytospin preparations
according to the criteria describedpreviously (Nakahataetal.,1982).
Bone marrow cells were incubated with fluorescein-, phycoer-
ythrin-, or biotin-conjugated monoclonal antibodies and analyzed
by EPICS-XL (Coulter, Hialeah, FL) with a logarithmic amplifier. For
each sample, 1±100 ? 105cells were analyzed and viable cells
were gated out bypropium iodideexclusion. For multicoloranalysis,
cells were first incubated with anti-Fc receptor monoclonal anti-
body 2.4G2 (Unkeless, 1979) to prevent nonspecific staining. The
following antibodies were used:anti-CD45R (B220) (RA3±6B2, Phar-
mingen), anti-CD43 (S7, Pharmingen), anti-IgM (331.12, Phar-
mingen), anti-TCRab (Pharmingen), anti-NK1.1 (Pharmingen), anti-
CD4 (RM4-4, Pharmingen), and anti-CD8 (53±6.7, Pharmingen).
Cell Cycle Analysis
Bone marrow cells were first stained with fluorescence-conjugated
anti-IgM and phycoerythrin-conjugated anti-CD45R (B220), perme-
abilized with 0.004% saponine, and then stained with 1 mg/ml
7-azaactinomycin D (7AAD) for the quantitation of DNA content
(Rabinovitch et al., 1986; Schmid et al., 1991). The cell cycle in each
fraction was analyzed using the algorithm developed by Fox (1980).
Recombinant mouse IL-7 was kindly provided by Toray Industries
(Kanagawa, J apan). Recombinant mouse IL-3 and human EPO were
generously provided by Kirin Brewery (Tokyo, J apan). IL-7 ata con-
centration of 100 units/ml or a combination of 10 ng/ml IL-3 and 2
units/ml EPO was used for the lymphoid or hemopoietic progenitor
Triplicate samples of 1 ? 104bone marrow or 1 ? 105spleen
cells were incubated in with or without FCS methylcellulose culture
(Koike et al.,1988). InFCS-containing culture, 1 mlof culture mixture
containing cells, ?-medium,0.9% methylcellulose(Shin-etsuChemi-
cal, Tokyo, J apan), 30% FCS (Hyclone, Logan, UT), 1% deionized
fraction V bovine serum albumin (BSA) (Sigma, St Louis, MO), 50
?M 2-mercaptoethanol (Eastman Organic Chemicals, Rochester,
NY), and cytokines was placed in each 35 mm standard nontissue
culture dish(Nunc, Naperville, IL) and incubated at 37?C ina humidi-
fied atmosphere flushed with 5% CO2 in air. Serum-free cultures
contained components identical to those in the serum-containing
cultures except that 1% deionized crystallized globulin-free BSA,
300 ?g/ml human transferrin, 160 ?g/ml soybean lecithin (Sigma),
and 96 ?g/ml cholesterol (Nacalai Tesque, Kyoto, J apan) replaced
fraction V BSA and FCS. All cultures were scored on day 12 of
culture according to the criteria reported previously (Koike et al.,
1988). To assess the accuracy of the in situ identification of cellu-
larity in each colony, individual colonies were lifted with an Eppen-
dorf micropipette under direct microscopic visualization, spread on
glass slides using a cytocentrifuge (Cytospin II, Shandon Southern,
Sewickley, PA), and stained for morphological examination. The
following colony types were scored according to morphological
appearance: GM, megakaryocyte, granulocyte-erythrocyte-macro-
phage-megakaryocyte, blast cell, and lymphocyte colonies. Colony
size was determined onan inverted microscope when colonies con-
sisted of fewer than 500 cells. For colonies containing more than
500 cells, the counting was performed in a counting chamber.
Genotype Analyses of mel-18±Deficient Mice
Homozygous mutants were obtained bycrossing N3orN4 heterozy-
gotes backcrossed to C57BL/6. All mice were maintained under
the specific-pathogen±free condition in our animal facility at Chiba
University. Genotype analysis was performed by PCR using two
sets of primers, one for the wild-type allele and another for the
mutant. Nucleotide sequences of these primers were as follows:
MW1: 5?-ACACTTCCCAAATCTCCTCA?3? and MW2: 5?-TGCTGC
ATAGAAGTCCCGTCGCCGT?3? for wild-type allele and MM1:
5?-GAACCTGCGTGCAATCCATCTTGTTCAATG?3? and MM2: 5?-
AGCCCCCTTCTCCTGTTCCA?3? for mutant allele detection.
RNase Protection Assay and RT-PCR
Total cellular RNA was isolated from adult tissues (6 weeks after
birth) and day 13.5 pc fetuses, and RNase protection analysis was
performed as described (Akasaka et al., 1996). The ?-tubulin RNA
probe was prepared in the same way for use as an internal control
(Wang et al., 1986). Lymphoid, erythroid, and GM lineage cells in
the bone marrow were isolated with the magnetic cell-sorting sys-
tem using biotin-conjugated monoclonal antibodies against CD45R
(B220) (RA3±6B3, Pharmingen, San Diego, CA), mouse TER119/ery-
throidcells (TER119,Pharmingen),and CD11b (Mac-1)(M1/70,Phar-
mingen) as a specific surface marker, respectively. Total cellular
RNA was isolated from fractionated cells using Trizol (Life Technol-
ogy) according to the manufacturer's protocol. First-strand cDNA
was generated using an oligo dT primer and subjected to the PCR
The following primers were used to detect mel-18, ?c, J ak3, and
?-actin transcripts. For mel-18 detection, MU131: 5?-TACGCTACTT
GGAGA-3? and ML454: 5?-AAGGGGGTGAGGTGGAAGT-3?; for ?cde-
tection, IL-2RgU: 5?-ATCGAAGCTGGACGGAACTAA-3? and IL-2RgL:
5?-CATGGTGCCAACAGGGATAA-3?; forJ ak3 detection,BUS:5?-CCA
GACCAGCAGAGGGACTT-3? and BDA: 5?-CCAAGCGAACAGCAGT
AGGC-3?; and for ?-actin detection, MACT1: 5?-GAGAGGGAAA
TCGTGCGTGAC-3?and MACT2: 5?-ACATCTGCTGGAGGTGGACA-3?.
B Cell Induction Assay
Fetal livers were removed from day 14.5 pc fetuses and transferred
to dishes containing RPMI 1640 medium supplemented with 5%
FCS. Single cell suspensions were prepared by dissociation be-
tween frosted glass slides followed by passage throughnylonmesh.
Fetal thymuses were also dissected out for genotype analysis. Fetal
Thymus, spleen, and femur were dissected out and fixed overnight
in 10% formol, dehydrated with ethanol, and embedded in paraffin
liver cells were cultured in RPMI 1640 medium supplemented with
5% FCS for3 hrat37?C in5% CO2todeplete adherentcells. Floating
cells were harvested and plated onto PA6 stromal cell layers at 5 ?
104cells/ml. After 24 hr of incubation, mouse IL-7 (10 units/ml) was
added to the culture medium (Sudo et al., 1989). The cultured cells
were collected, and floating cells were isolatedfrom the PA6stromal
cells using a filter membrane. The surface expression of B lineage
markers in the floating cells was quantified as described previously
(Sudo et al., 1989).
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mocytes from wild-type and mel-18 mutant mice. Suspended thy-
mocytes were cultured at a concentration of 5 ? 104/well in 96-well
round-bottom plates and were stimulated with PMA (10 ng/ml) or
PMA plus IL-2 (20ng/ml), IL-4 (20 ng/ml), IL-7 (10 ng/ml), or A23187
(300 ng/ml) in RPMI medium supplemented with 10% FCS. Cells
were cultured for 48 hr, pulsed with 1 ?Ci of [3H]thymidine (20±40
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Electrophoretic Mobility Shift Assay
Suspended thymocytes were incubated in RPMImedium containing
10% FCS with or without 50 units/ml IL-7. After 15 minutes incuba-
tion, thymocytes were pelleted and whole-cell extracts were pre-
pared by sonification in 150 mM NaCl, 25% glycerol, 0.2 mM EDTA,
20 mM HEPES (pH 7.8), 0.5 mMDTT, 0.5mM PMSF, 10 mM NaF, 10
mM Na3VO4, and Complete (BoehringerMannheim) and subsequent
centrifugationat14,000rpmfor5min. The whole-cellextractequiva-
lent to approximately 2 ? 106cells was incubated with 15,000 cpm
32P±end-labeled oligonucleotide probes in 20 ?l of reaction mixture
containing 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1
mM DTT, 5% glycerol, and 2 ?g of poly(dI-dC). The Fc?R1 probe
lined) in lanes 1±8 (as presented in Figure 7C) and mutant Fc?R1
probe (5?-GAGCTTGTATTTCCCACAAAAGGGATC-3?, single nucle-
otide substitution in GAS motif underlined) in lane 9 were used.
GAS motif under-
Western Blot Analysis
Whole-cell extracts were prepared from the thymocyte or brain of
day 16.5 pc fetuses as described previously. The amount of protein
was assayed with the Micro bicinchoninic acid Protein Assay Kit
(Pierce). Twenty micrograms of protein was electrophoresed, and
the gel was stained with Coomassie blue to allow visual inspection
of protein amounts (data not shown). After fine adjustment of pro-
tein quantity, almostequal amounts of whole-cell extracts was sub-
jected to Western blot analysis. Western blotting was performed by
the standard method using polyvinyl difluoride membrane (Milli-
pore). The membrane was probed with polyclonal antibody against
Rb protein (14001A, Pharmingen). Detection was performed by en-
hanced chemiluminescence according to the instructions of the
manufacturer (ECL, Amersham Life Science).
Correspondence should be addressed to H. K. We are grateful to
Drs. R. Balling, A. Gossler, and A. Mu Èller for critical reading of the
manuscript; Dr. A. Nagy for providing R1 ES cells; Drs. A. Mansouri,
M. Torres, and H. Schreber for the initial instructions in ES cell
culture and aggregation techniques; Dr. H. Mossman, M. Uchida,
and S. Sugimorifor animalcare;and T. Itoh,A. Kaneko,K.Higashino,
H. Tanabe, and Y. Sawano for help in many respects. We also thank
Drs. S. Nishikawa and H. Wakao for providing the anti-IL-7R? chain
antibody and anti-STAT5 antiserum, respectively. This project was
supported by grants from the Ministry of Education, Science and
Culture (M. T.); from the Agency of Science and Technology (H. K.);
from the Ministry of Health and Welfare (M. T.), J apan; and by a
special coordination grant from Taisho Pharmaceutical Company
(M. T.). T. A. is the recipient of a J SPS Research Fellowship for
Received December 13, 1996; revised April 29, 1997.
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