NOT, A Human Immediate-Early
Response Gene Closely Related to
the Steroid/Thyroid
Hormone
Receptor NAKI /TR3
Hans W. Magest,
Richard
Olaf Rilke*,
Rodrigo
Bravo,
Gabriele
Senger,
and
A. Kroczekt
Max-Planck-Society
Research Unit for Immunology/Rheumatology
(H.W.M., OR., R.A.K.)
91054 Erlangen, Germany
Bristol-Meyers Squibb Pharmaceutical Research Institute (R.B.)
Princeton,-New Jersey
Imperial Cancer Research Fund (G.S.)
London, United Kingdom
the induction of specific target genes, as is the case
with classical steroid receptors. (Molecular
Endocrinology 6: 1563-1591,1994)
By analyzing the early genetic response of human T
cells following mitogenic
activation we have identified NOT, a member of the steroid/thyroid
hormone
family of receptors.
NOT has all structural features
of steroid/thyroid
hormone
receptors
(C2C2 zincfinger domain, ligand binding domain), but is rapidly
and only very transiently
expressed
after cell activation, which is clearly at variance with classical
steroid receptors such as glucocorticoid
or estrogen
receptors. NOT gene induction is independent
of de
nova protein synthesis, defining NOT as an immediate-early
response
gene. Short-lived
NOT mRNA
(4.2 kilobases) expression
could be observed in vitro
in a greater number of tissue types following
activation by a variety of distinct stimuli. In viva, NOT
mRNA expression
was detected
exclusively
in the
brain, where a very strong signal was observed. By
immunoblot
analysis of human T cell lysates with
NOT specific antisera two activation-dependent
protein bands (66 and 59 kilodaltons)
could be detected.
NOT gene was localized
to human chromosome
2q22-q23. Sequence
comparison
revealed that NOT
is the human homolog of the murine NURRl and rat
RNR-1. Moreover
NOT is closely related to NAKl/
TR3, a previously
identified
human orphan steroid
receptor. Several lines of evidence indicate that NOT
and NAKl/TR3
form a distinct
and exclusive
subgroup
of orphan
steroid
receptors,
whose
expression characteristics
in vitro and in vivo resemble the expression
of nonsteroid
immediate-early
transcription
factors such as jun and fos. NOT and
NAKl/TR3
thus may function as general coactivators of gene transcription
rather than participate
in
INTRODUCTION
In resting cells such as peripheral blood (PB) T cells or
primary fibroblasts, Go to G, transition of the cell cycle
can be initiated in vivo and in vitro by a variety of
growth-promoting
agents including polypeptide growth
factors, antigens (in the case of T cells), and diverse
mitogens. Interaction of these reagents with their specific receptors induces a cascade of intracellular biochemical events resulting in a direct transcriptional
activation of a large number of new genes. Genes
transcribed within the first few hours of cell activation
not requiring de novo protein synthesis represent the
immediate-early
genes. These genes are coding for
secretory proteins and growth factor receptors and also
for protooncogenes
and transcription
factors involved
in the activation of secondary genes. Typically, immediate-early genes are up-regulated quickly, have a short
half-life, and are primarily transcribed in the first phase
of cell activation. The immediate-early
genes thus initiate a complex network of biochemical events leading
to cell cycle progression and cell growth (l-5).
When analyzing a collection of immediate-early genes
transcriptionally
up-regulated
after mitogenic T cell activation, we identified a zinc-finger protein, designated
NOT (nuclear receptor of T cells), with significant similarity to steroid/thyroid
hormone receptors. This family
of nuclear receptors can be divided into two major
groups. The classical steroid receptors (e.g. glucocorticoid or estrogen receptor) are ligand-activated
tran-
0888-8810/94$03.00/0
Molecular
Copyright
Endocdnorogy
0 1994 by The EndoW-@
society
1583
MOL ENDO. 1994
Vol8No.
11
scription factors with characteristic
structural features
(C2C2 zinc-finger domain, ligand binding domain). Classical steroid receptors are involved in the development,
differentiation,
and cell homeostasis
by positively or
negatively regulating the expression of specific target
genes. The second group, the orphan receptors, have
all structural features of steroid receptors. However, by
definition, both the corresponding
ligands and the function of orphan receptors are not known (6-9). In contrast to immediate-early
genes, steroid receptor and
orphan receptor genes are often transcribed in resting
cells, their mRNA has a longer half-life, and changes in
the level of transcription are less pronounced and often
only gradual (H. W. Mages, 0. Rilke, and R. A. Kroczek,
unpublished).
In the present report we describe the
identification and characterization
of the mitogen-inducible immediate-early
receptor NOT.
RESULTS
Cloning and NOT cDNA Sequence
NOT was first identified as a gene being rapidly but
transiently induced after mitogenic stimulation in T cells.
For sequence determination
several NOT cDNA clones
were isolated from a XgtlO cDNA library constructed
from activated PB T cells. The nucleotide sequence of
the largest cDNA clone (Fig. 1 A) comprises 3427 nucleotides (nt) and contains a major open reading frame of
1794 nt starting with an initiation methionine at nt 318,
whose flanking sequence is in accordance
with the
Kozak consensus
sequence for translation
initiation
(Fig. 1B and Ref. 11). The open reading frame is followed by an 3’untranslated
region of 1316 nt containing several ATTTA elements known to confer mRNA
instability, a property of many immediate-early
mRNAs
(12). A typical polyadenylation
signal is present 17 nt
upstream of the poly(A) tail. In the Y-untranslated
region of the cDNA clone (317 nt in length) several in
frame stop codons can be found indicating that the
whole coding region has been cloned (Fig. 1 B).
Structural Features of the NOT Protein: Homology
to Steroid/Thyroid Hormone Receptors
NOT cDNA encodes a protein with a calculated M, of
66.6 kDa. Comparison
of the NOT amino acid (aa)
Fig. 1. NOT cDNA and Derived Protein 8equence
A, Schematic representation of a full length NOT cDNA. B,
Nuclectide sequence of NOT and deduced aa sequence. Numbers on the /eft and right indicate nt and aa residues, respectively. The zinc-finger region is shaded, the cysteines involved
in the zinc-finger formation are boxed. The putative leucine
zipper domain is framed (the leucines of the zipper are encircled). In the 3’-untranslated region the four AllTA signals are
underlined.
Consensus polyadenylation signals are boxed. In
the 5’-untranslated region the in-frame stop ccdons are in
bold italics.
ImmediateEarly Receptor NOT
1585
sequence with known protein sequences revealed that
NOT has significant similarity in sequence and structure
to steroid/thyroid
hormone receptors and is the human
homolog of the mouse NURRl and rat RNR-1 gene
(13,14). The human and the mouse gene products both
consist of 598 aa with nearly identical aa composition
(99.5% identity) differing in only three positions (aa 131
T/S, 134 G/S, 354 E/D). The RNR-1 protein consists
of 597 aa and is 97.6% identical to the NOT protein.
Like all members of the steroid/thyroid
hormone receptor family, NOT contains two zinc-fingers of the C2C2type at aa position 263-318.
This region is highly
homologous
to the zinc-finger region of the human
NAKl/TR3
orphan receptor (90% at the aa level, Fig.
2; Refs. 15 and 16). NAKl/TR3
is a previously described member of the steroid/thyroid
hormone receptor family also known as nur77/NlO in the mouse (17,
18) and NGFI-B in rat (19). The zinc-finger region of
NOT is also related to the zinc-fingers of several members of the steroid receptor family (Fig. 2). The carboxyterminal part of the NOT protein (aa 409-598)
which
corresponds
to the ligand binding domain of several
steroid/thyroid
receptors (6, 20, 21) is again highly
homologous
to NAKl/TR3
(71% at the aa level) and
related to RARa (30% at the aa level; Fig. 3). This
region of the protein (aa 430-451)
also contains a
leucine-zipper,
a motif known to mediate protein-protein
interactions (Figs. 1B and 3; Refs. 22 and 23). This
structural feature, which is unusual for steroid receptors, is also present in the NAKl /TR3 receptor (15, 16).
P.bax
“leucine-zipper”
Fig. 3. Comparison of the Ligand Binding Domains of NOT,
NAKl /TR3, Retinoic Acid Receptor-a, Chicken Ovalbumin Up
stream Promotor Transcription Factor, Estrogen Receptor,
and Retinoid X Receptor-a
The putative leucinezipper in NOT and NAKl/TR3 is indicated.
The amino-terminal
part of the NOT protein (aa l-262)
is highly acidic (charge = -7) and contains glutaminerich (15%, aa 80-l 38) and prolinerich (16,8%, aa 127233) domains; another proline-rich area (22%, aa 346391) is located in the carboxy part of the protein. The
NOT protein thus has structural features that have been
associated with the capability of DNA binding proteins
to activate gene transcription (24). In addition, the NOT
protein sequence contains several potential protein kinase C and casein kinase II phosphorylation
sites (25)
as well as an N-glycosylation site (aa position 66) which
might be important for the regulation of the biological
activity of the NOT receptor.
Induction of NOT Gene Expression
in Human T
Cells and Primary Human Fibroblasts
Fig. 2. Comparison of the Zinc-Finger Region of NOT with the
Zinc-Finger Regions of Representative Members of the Steroid/Thyroid Hormone Receptor Family
The cysteines involved in zinc-finger formation are printed
in bold. Amino acids conserved in all proteins are indicated by
an asterisk. The P and D boxes are indicated. References:
NAKl/TR3, (15, 18); RXR& human retinoic X receptor ,3 (46);
RXRa, human retinoic X receptor-a (47); ERR2 and ERRl,
estrogen receptor-related proteins 1 and 2 (48); TR2 (49);
COUP-TF/ear3, chicken ovalbumin upstream promotor transcription factor (50, 51); ARP-1, human apoAl regulatory protein-l (52); RARa, human retinoic acid receptor-cu (53, 54);
ER, human estrogen receptor (55. 56).
The induction of NOT gene expression during the GoG, transition of the cell cycle was investigated by Northern blot analysis using two different cell systems. In
quiescent PB T cells NOT mRNA is only barely detectable. Stimulation of these cells by the plant lectin phytohemagglutinin
(PHA-P), which mainly acts via the T
cell receptor (26) resulted in a drastic increase in NOT
mRNA (4.2 kilobases) levels within 2 h. Thereafter the
mRNA decreased and reached resting levels 8 h after
stimulation (Fig. 4). In contrast, mRNA for the glucocorticoid receptor was already detectable in quiescent
cells. The glucocorticoid
receptor signal was not influenced by PHA-P during the first 4 h of stimulation and
was down-regulated
thereafter (Fig. 4). The NOT gene
could also be induced by the Ca’+-ionophore
A23187
or the tumor promoter phorbol 12-myristate 13-acetate
Vo18No.11
MOL ENDO. 1994
1588
NOT
4.2 kb
200 116 97 66-
-
GR
Fig. 4. Expression Kinetics of NOT in PB T Cells
PB T cells were either left unstimulated or were stimulated
for 2 h, 4 h, 8 h, and 12 h with PHA-P. NOT gene induction
was followed by Northern blot analysis. For comparison, glucocorticoid receptor gene expression is shown.
&a FCS FCS + CHX
Fig. 5. Time Course and Superinduction of NOT Gene Expression in Primary Human Fibroblasts
Growth-arrested fibroblasts were stimulated with 20% FCS
in the absence or presence of CHX for the time periods
indicated. Messenger RNA levels were determined by Northern
blot analysis.
(PMA), which activates protein kinase C (27) demonstrating that NOT can be activated via different signal
transduction pathways (data not shown). In growtharrestedfibroblastsNOT mRNA was already detectable
30 min after serum stimulationreaching maximumlevels at 3 h. mRNA could no longer be detected after 24
h. Superinduction of NOT mRNA in the presence of
cycloheximide(CHX) indicated that no de novo protein
synthesis is required for NOT gene expression, thus
defining NOT as an immediate-early response gene
(Fig. 5).
Expression of the NOT Protein in Human PB T Cells
A NOT region-specificantiserum generated against a
NOT fusion protein was used to characterize the NOT
protein expressionin vivo. PB T cells were left unstimulated or were stimulated with Ca*+-ionophore and
PMA, and cell lysateswere analyzed by immunoblotting
as describedin Materials and Methods. The antiserum
reacted predominantly with two activation-dependent
protein bands (Fig. 6). The 66 K band corresponds in
size to the predicted NOT protein. The protein band
Fig. 0. Time Course of NOT Protein Expression in Primary
Human T Cells Analyzed by lmmunoblotting
Lysates of PB T cells were prepared from unstimulated cells
or from cells stimulated for 4 h with Ca*+-ionophore and PMA.
Equal amounts of protein were analyzed by 12% SDS-polyacrylamide electrophoresis and immunoblotted with NOT-specific antiserum as described in Materials and Methods.
The
position of molecular size markers, in kilodaltons, are indicated
on the left.
with an apparent size of approximately 59 K could ba
an alternative translationproduct usingone of the possibledownstreamtranslation initiation sitesof the NOT
mRNA. The diffuse nature of the two NOT protein
bands may result from posttranslationalmodifications
as suggested by the presence of multiple putative
phosphorylation and glycosylation sites in the NOT
protein sequence.Posttranslationalmodificationswere
identified as the cause for a similar heterogeneity in
protein size in the related NGFI-B protein, the rat homolog of NAKl /TR3 (28).
Expression of NOT in Various Cell Types
Cell type specificity of NOT gene expression was analyzed by Northern blotting. In unstimulatedhumancell
lines a very weak basal NOT mRNA signal was observed (Fig. 7A). After stimulationwith Ca*+-ionophore
+ PMA, strong NOT gene expression could be observed in a numberof ceil types (PEER, B958, Hs913T,
MRC5, HeLa, Fig. 7B). In unstimulatedprimary murine
tissues a strong signal (comparableto activated HeLa
cells) was observed in the brain. The other tissues
tested (thyroid gland, thymus, kidney, spleen,and lung)
were negative. NOT mRNA was also detected in primary calvaria osteoblastsusing high stringency conditions (Fig. 7C).
Chromosomal Localization of the Human NOT
Receptor Gene
The chromosomal localization of the NOT gene was
determined by fluorescence in situ hybridization on
replication R-banded human metaphase spreads.
1587
ImmediateEarly Receptor NOT
B
A
unstimulated
PMA + A33187
+ CHX
Fig. 7. Northern Analysis of NOT Gene induction in Various
Human Cell Lines and Different Murine Tissues
A, Unstimulated cell lines. B, Cell lines stimulated for 3 h by
Ca*+-ionophore A231 87 and PMA in the presence of CHX. C,
RNA from mouse thyroid gland, thymus, brain, kidney, spleen,
lung (5 pg total RNA), and primary calvaria osteoblasts (2 pg
polyA+ RNA) was hybridized to a human NOT-specific probe
under high stringency conditions.
D
chr. 3 hamster chr. 2
Twenty metaphaseswere examined, and two hybridization sites were observed. Fluorescent signalswere
found on chromosome2 in the region 2q22-q23and on
chromosome3 band 3~21.3 in 37 (93%) and 29 (73%)
of the 40 possiblehybridization sites, respectively (Fig.
8, A, B, and C).
To discriminateon which of the two chromosomes
the NOT gene is localized Southern blots were performed with genomic DNA from human/Chinesehamster somatic cell hybrids containing human chromosome3 (GM/NA10253) or humanchromosome2 (GM/
NA10826B)(Fig. 8D). In the cell hybrid containingchromosome2 the restriction pattern contained the bands
found in humangenomicDNA. In contrast, the analysis
of the cell hybrid containinghumanchromosome3 gave
only a restriction pattern correspondingto the hamster
background pattern. Thus, both in situ hybridization
and somatic cell hybrid mappingdemonstratesa localization of the NOT gene on chromosome2, region q22q23.
In several casesof non-Hodgkin lymphoma,deletion
of this locushasbeen reported (29). In addition, deletion
of 2q23-33 as well as a translocation t(2;14)
(q23;q32.3)has beendescribedas the sole abnormality
in patients with acute nonlymphocytic leukemia (30,
31). It will be interesting to determine whether the
expression or structure of the NOT gene is altered in
these neoplasms.
In the present report we have described the structure
andexpressioncharacteristicsof NOT, a steroid orphan
1 2 3
4 5 6
7 8 9
human
101112
-
HEB
HEB
HEB
23130
-
9416
-
65.57
-
4361
-
2322
2027
HEB
Fig. 8. Chromosomal Localization of the NOT Gene by Fluorescence in Situ Hybridization and Somatic Cell Hybrid Mapping
A, Replication R-banded metaphase with hybridization signals on chromosome band 2q22q23 (arrowhead) and on band
3~21.3 (arrow). B, The same metaphase stained with 4,6diamidino-2-phenylindole-dihydrochloride.
C, Ideograms of
chromosome 2 and 3 with oars showing the hybridization
sites. D, Southern blot analysis showing restriction patterns
of genomic DNA digested with the restriction enzymes HindIll
(H), EcoRl (E), and BarnHI (B). Lane 1-3, human/Chinese
hamster somatic cell hybrid GM/NA10253 (containing human
chromosome 3); lane 4-6, Chinese hamster control; lane 7-9,
human/Chinese hamster somatic call hybrid GM/NA10826B
(containing human chromosome 2); lane 10-12, human control.
receptor. Sequence comparison with known proteins
shows that NOT is the human homologof a receptor
recently describedboth in the mouse(NURRl , Ref. 13)
and in the rat (RNR-1, Ref. 14). In fact, NOT differs
from the NURRl protein only by 3 aa (99.5% identity)
and shows 97.6% identity to RNR-1. This very high
MOL
1588
ENDO.
1994
evolutionary conservation
of the sequence suggests
that all domains of the molecule have a critical functional
role. NOT contains all structural elements of steroid
receptors: an amino terminus fulfilling the criteria of a
transactivation
domain, a C2C2-zincfinger domain
including a P box and a D box, which have been shown
to be critical for DNA binding properties
of steroid
receptors, and a ligand binding domain (6-9). In addition, a leucine-zipper
motif can be identified in the ligand
binding domain, a feature usually not observed with
steroid receptors. This motif has been shown previously
to participate in protein-protein
interactions (22, 23).
Interestingly, the zinc-finger and the ligand-binding
domains of NOT are highly homologous
to another
identified orphan receptor termed NAKl/lR3
in the
human system (15, 16) nur77/NlO
in the mouse (17,
18) and NGFI-B in the rat (19); even the leucine-zipper
motif is conserved. In addition, a motif located carboxyterminal of the zinc-finger domain identified to be essential for DNA recognition (A-box, Ref. 32) is identical
in NOT and NAK/TRS. When the sequence and structure of NOT and NAKl/TR3
are compared with the
remaining steroid receptors it is apparent that NOT and
NAKl/TR3
form a closely related subgroup within the
orphan receptors, whereas a greater number of receptors are more distantly related.
The functional role of NOT and NAKl/TR3
in vivo is
as yet unknown; only the expression characteristics
of
both genes provide some clues at present. We have
observed that NOT is expressed in a number of cell
lines of T cell, B cell, and fibroblast origin; however, in
vitro significant expression is seen only following cell
activation. Primary T cells expressed NOT after stimulation with a Ca*‘-ionophore
or a phorbol ester, agents
that utilize distinct activation pathways, and primary
fibroblasts responded to serum stimulation in the state
of quiescence. Thus it is apparent that various types of
stimuli have similar effects on NOT gene expression. In
regard to the function, a conspicuous
finding was the
very strong mRNA signal obtained in brain tissue, when
we analyzed NOT expression in the mouse. Substantial
amounts of NOT mRNA could also be detected in
mouse primary osteoblasts;
all other tissues tested
were negative. It is apparent when all these observations are taken together that NOT has a rather broad
tissue distribution and is expressed during the transition
from the Go to the G, phase of the cell cycle. However,
the detection of NOT mRNA in the brain indicates that
NOT gene expression
is not mandatorily associated
with cell cycle progression, since cells do not proliferate
at this anatomical site.
Interestingly, the expression pattern of NAKl /TR3/
nur77/NlO/NGFI-B
(15-19) seems to be rather similar
to our observations
with NOT. Altogether,
the described expression characteristics
of NOT and NAKl/
TR3 are reminiscent of several well characterized
immediate-early genes, such as members of the fos and
jun families of transcription
factors. These immediate-
V0l8N0.11
early genes are involved in the transition from quiescence to proliferation
in very many cell types in response to a broad spectrum of stimuli and are also
expressed at high levels in the brain following sensory
stimulation (33, 34). It will be of great interest to determine whether expression of NOT and NAKl in the brain
follows similar patterns and thus resembles the function
of jun and fos transcription factors in vivo.
Regarding the expression kinetics it is apparent that
NOT is a typical immediate-early
gene. We could demonstrate that the NOT gene is activated within 1 h of
stimulation independent
of de novo protein synthesis.
The data obtained at mRNA level, the substantial increase of mRNA signal in the presence of CHX, and
the short-lived expression of NOT mRNA all indicate a
short half-life of the molecule in vivo. However, unlike
jun and fos, which are down-regulated
within 1 h, NOT
expression kinetics both at mRNA and protein levels
are more protracted.
Classical steroid receptors have a clearly different
expression pattern. Although also broadly expressed,
they are often transcribed at substantial levels in resting
cells, and the changes in gene expression follow a more
gradual course (H. W. Mages, 0. Rilke, and R. A.
Kroczek, unpublished).
In T cells we have, in fact,
observed a fairly strong glucocorticoid
receptor mRNA
signal in the resting state that declined following cell
activation.
Taken together, all observations
suggest that NOT
and NAKl form a separate subgroup within the family
of orphan steroid receptors.
Although
structurally
closely related to classical steroid receptors their tissue
expression pattern and kinetics resemble the expression characteristics of typical immediate-early
transcription factors. The concept of a distinct functional role of
NOT and NAKl/TR3
within the orphan receptor family
is strongly supported by the observation that NOT and
NAKl/TR3
were the only steroid receptor-like
molecules found in a collection of 100 distinct immediateearly T cell activation genes that we have established
and analyzed. The challenge of future work will be to
determine whether NOT and NAKl/TR3
are general
coactivators of gene transcription,
e.g. jun or fos, or
whether they participate in the activation of a specific
set of genes, as is the case with classical steroid
receptors. In this context it is of interest that nur77/
NAKlpR3
plays a preeminent
role in activation-induced apoptosis
of T cell hybridomas.
These very
recent findings demonstrate that functional inactivation
of nur77/NAKl
/TR3 totally abrogates apoptosis in this
model (35, 36).
MATERIALS
AND METHODS
Cells
Peripheral
blood mononuclear
cells were isolated
coats obtained from blood donors by Ficoll-Hypaque
from buffy
gradient
Immediate-Early
Receptor
1589
NOT
centrifugation.
PB T cells were isolated by passage of peripheral blood mononuclear
cells over a nylon-wool
column,
resulting in a 94-98%
pure T cell population
as judged
by
immunofluorescence
flow cytometry
using an anti-CD3
antibody. The following cell lines were obtained from the American
Type Culture Collection
(Rockville,
MD): Jurkat (human T cell
lymphoma),
MOLT-4
(human acute lymphoblastic
T cell lymphoma),
895-8
(EBV-transformed
marmoset
leukocytes),
U937 (human histiocytic
lymphoma),
Hs913T (human fibrosarcoma),
MRC-5
(human
lung fibroblasts),
IMR-32
(human
neuroblastoma),
HepG2
(human
hepatocellular
carcinoma),
and HeLa (human epitheloid
carcinoma).
The immature
human
T cell lymphoma
PEER (37) was kindly provided
by C. M.
Niemeyer
(Freiburg,
Germany).
Cell Culture
and Cell Treatment
PB T cells and the various
cell lines were cultured
in RPM1
1640 medium (GIBCO
BRL, Gaithersburg,
MD) supplemented
with 10% heat-inactivated
FCS, 25 mM HEPES buffer, 2 mM
L-glutamine,
100 U/ml penicillin, and 100 U/ml streptomycin,
50 PM 2-mercaptoethanol
at 37 C in a humidified
5% CO*
atmosphere.
PB T cells (2.5 x 106/ml) and cell lines were treated
for
indicated time periods with PHA-P (1:400, Difco, Detroit,
Ml)
or with Ca*+-ionophore
A231 87 (125 rig/ml) and PMA (20 ng/
ml) both from Sigma (St. Louis, MO) in the absence or presence
of CHX (10 pg/ml, Serva, Heidelberg,
Germany).
Primary
human foreskin fibroblasts
were cultured
in Dulbecco’s
modified
Eagle’s medium (including
10% FCS) to confluence.
The cells
were made quiescent
by incubation
in 1% FCS overnight
and
subsequently
stimulated
with 20% FCS in the absence
or
presence of CHX.
RNA Isolation
and Northern
Analysis
Total cellular RNA was isolated according
to Chirgwin
(38) and
Glisin (39). Northern
analysis was performed
with 5 pg total
RNA as described
previously
(40). Equal loading and transfer
of the RNA to the membrane
support
were controlled
by UVfluorescence
(41). Utilized probes were nick-translated
to a
specific activity of 2 x 10’ cpm/pg DNA. Blots were pretreated
with 0.1 x sodium citrate (SSC), 0.5% SDS for 10 min at 65
C. Hybridization
was in 50% formamide,
10% SDS, 250 mM
NaCI, and 120 mM Na2HP04 (pH 7.0) at 47 C for 24 h. Blots
were washed including a final stringency
step with 0.1% SSC,
0.1% SDS at 50 C for 30 min.
NOT cDNA
Cloning
NOT was isolated from a collection
of T cell activation
genes
established
as follows. A XgtlO cDNA library was constructed
using poly(A+) RNA from PB T cells activated
for 2 h by Ca2+ionophore
A231 87 (125 rig/ml) and PMA (20 rig/ml) in the
presence
of CHX (10 pg/ml).
cDNA clones (2 x 1 OS) were
transferred
in duplicate
to nitrocellulose
filters and hybridized
with 32P-labeled
cDNA obtained
from poly(A+)
RNA isolated
from quiescent
or activated
cells. By this differential
screening
procedure
clones preferentially
hybridizing
with cDNA of activated cells were isolated.
Redundant
clones were eliminated
by cross-hybridization
resulting
in a collection
of 100 independent
activation
genes. NOT full length clones were obtained by rescreening
the original Xgtl 0 cDNA library.
DNA Sequencing
and Sequence
Analysis
NOT cDNA clones were subcloned
in both orientations
into
pBluescript
II SK(+) (Stratagene,
La Jolla, CA). Serial nested
deletions
were created
with the Exo/Sl
system
from Pharmacia (Piscataway,
NJ). Single strand DNA was prepared
and
the nt sequence
was determined
using the Sequenase
Kit
from United States Biochemicals
(Cleveland,
OH).
Nucleotide
and aa sequence
analysis were carried out using
the UWGCG
Sequence
Analysis Software
Package.
Generation
and lmmunoaffinity
Specfic
Antisera
Purification
of NOT
A NOT specific region (aa position
159-235)
was subcloned
into pSEM3
(42) and expressed
as a @galactosidase
fusion
protein in Escherichia
co/i BMH 7118 cells (43) by induction
with 1 mM isopropyl-/3-o-thiogalacto-pyranoside.
Purification
of
inclusion bodies was according
to Marston
(44). The inclusion
bodies were dissolved
in 8 M urea and then desalted on a PD10 column (Pharmacia).
Approximately
500 pg of the fusion
protein
were mixed at a 1:l (vol/vol)
ratio with complete
Freund’s
adjuvant and injected into rabbits SC at multiple sites.
After several
boosts with incomplete
Freund’s
adjuvant
the
rabbits were bled and NOT specific antisera obtained.
For affinity purification
of the antiserum,
20 mg purified NOT
fusion protein were covalently
linked to 1 ml Affi-Gel
10 according to the instructions
of the manufacturer
(BieRad
Laboratories,
Richmond,
CA), except that linkage was performed
in 6 M urea. The Affi-Gel column was loaded with antiserum
and incubated
for 2 h at room temperature.
After washing, the
antibodies
were directly
eluted with elution
buffer (0.05 M
glycine buffer,
0.15 M NaCl [pH 2.31) into 0.5 M sodium
phosphate
(pH 7.7).
lmmunoblotting
PB T cells were either left unstimulated
or were stimulated
for
4 h with Cap+-ionophore
(125 rig/ml) and PMA (20 rig/ml). Cells
were pelleted, lysed in SDS-sample
buffer (62.5 mM Tris/HCI
[pH 6.81, 20% glycerol, 2% SDS, 0.1 M dithiotreitol,
0.0025%
bromphenol
blue [wt/vol])
and heated at 95 C for 4 min. The
samples were sonicated
for 30 set (Labsonic
2000, B. Braun
Biotech, Allentown,
PA), and equal amounts
(10 pg) of protein
were separated
on a 12% SDS-polyacrylamide
gel and transferred to a polyvinylidenfluorid
membrane
(Millipore,
Bedford,
MA). The membrane
was treated with blocking buffer [0.2% IBlock reagent (Tropix,
Bedford,
MA), 0.1% Tween 20 detergent in PBS] and incubated
with affinity-purified
rabbit antiNOT antiserum
diluted 1:500 in blocking buffer for 1 h followed
by incubation
with goat anti-rabbit
immunoglobulin
G alkaline
phosphatase
(Tropix) diluted 1 :l 0000 in blocking buffer for 15
min. After several washing
steps the membrane
was treated
with Nitro-Block
reaaent
(Trooix.
1:20 dilution) for 5 min and
incubated
in substraFe sol;tion
[0.05 mM CSPO (Tropix) in 0.1
M diethanolamine,
1 mM MgC12 (pH lO.O)] for 5 min. After
treatment
the membrane
was exposed
to x-ray film (XAR,
Kodak).
Chromosomal
Localization
of the NOT Receptor
For chromosomal
localization
a genomic NOT clone was isolated from a human placenta
genomic library (Lambda
Fix II,
Stratagene)
using a NOT cDNA probe. The identity of the 11
kilobase insert of the genomic clone was verified by restriction
analysis and Southern
blotting. Fluoresence
in situ hybridization was performed
as described
previously
(45). Briefly, metaphase spreads
were obtained
from normal PB lymphocytes
after incorporation
of bromodeoxyuridine
during the first half
of the S phase. The qenomic
NOT probe was labeled with
biotin-14-dATP
by nick-translation
using the Bionick kit (GIBCO
BRL). Probe DNA (200 na) orecioitated
with 5 ua C,t 1 DNA
(GIBCO
BRL) was disso&d
in ‘20 ~1 hybridi;a%onsolution
(50% formamide,
2 x SSC, 10% dextran
sulfate,
pH 7.0),
denatured
for 3 min at 75 C and preannealed
for 30 min at 37
C to suppress
repetitive
sequences.
The probe mixture
was
MOL
1590
ENDO.
1994
then applied to the previously
denatured
chromosome
preparation and hybridized
for 72 h at 37 C. After posthybridization
washes at 42 C in 50% formamidex SSC the hybridized
probe was detected
with Texas Red-conjugated
avidin with
one round of signal amplification
using biotinylated
antiavidin
and a second layer of avidin Texas Red. Reverse banding was
obtained
by incubation
with a fluorescein-coupled
anti-bromodeoxyuridine
monoclonal
antibody
(mAb). The slide was
mounted
in antifading
buffer (Citifluor)
containing
0.2 pg/ml
4,8diamidino-2-phenylindoledihydrochloride
as counterstain.
Digitized images were obtained
with a Zeiss Axioscop
microscope (Cart Zeiss, New York, NY) equipped
with a cooled
CCD camera (Photometrics,
Tucson, AZ). Separate
gray scale
images of probe signals,
banding
pattern,
and counterstain
were captured,
merged, and further processed
using software
developed
by T. Rand and D. C. Ward (Yale University,
New
Haven, CT). Computer
images were printed with a Sony UP
D7000E digital color printer.
Southern
blots of genomic
DNA from human PB T cells,
Chinese hamster
cells, and genomic
DNA of human/Chinese
hamster
somatic
cell hybrids
GM/NAl0826B
and GM/
NA10253
(NIGMS
Human
Genetic
Mutant
Cell Repository,
Coriell Institute
for Medical
Research,
Camden,
NJ) were
performed
with NOT cDNA probe using standard
procedures.
Pretreatment,
hybridization,
and washing
of Southern
blots
was as described
for Northern
analysis.
Acknowledgments
We thank Dr. Ian Williams for a blot with murine osteoblast
mRNA. Dr. Andrew Cato for the glucocorticoid
receptor
probe,
and Dr. Miguel Beato for critical reading the manuscript.
Received
March
28, 1994. Re-revision
received
July 7,
1994. Accepted
July 19, 1994.
Address requests
for reprints to: Richard A. Kroczek,
Molecular Immunology,
Robert
Koch Institute,
Nordufer
20, D13353 Berlin, Germany.
This work was supported
by a grant from the Sander
Foundation
(to R.A.K.).
‘The NOT cDNA sequence
was deposited
in the EMBL
databank
(accession
number:
X7591 8).
tPresent
address:
Robert Koch-Institute,
Molecular
Immunology, Nordufer
20, 13353 Berlin, Germany
fPresent
address:
Universitatsklinikum
der Technischen
UnNersitat,
lnstitut fur Biologie,
Fetscherstrasse
74, 01307
Dresden,
Germany.
V0l8N0.11
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