Báo cáo khoa học: Interaction of an  40 kDa protein from regenerating rat liver with the )148 to )124 region of c-jun complexed with RLjunRP coincides with enhanced c-jun expression in proliferating rat liver pdf

Interaction of an  40 kDa protein from regenerating rat liver with
the )148 to )124 region of
c-jun
complexed with RLjunRP coincides
with enhanced
c-jun
expression in proliferating rat liver
Sujata Ohri*, Dipali Sharma† and Aparna Dixit
Gene Regulation Laboratory, Center for Biotechnology, Jawaharlal Nehru University, New Delhi, India
The c-jun belongs to the family of proto-oncogenes and
encodes for the protein Jun, a component of transcription
factor AP-1 involved in regulation of the expression of genes
indispensable for cell proliferation and differentiation.
While the r ole o f c-jun in the r egulation o f s uch g enes has
been well examined, the regulation of c-jun in proliferating
cells is not fully understood. We have earlier reported that
the )148 to )124 region of c-jun is involved in the positive
regulation of c-jun transcription, and interacts with a pos-
itive regulatory factor (rat liver jun regulatory protein;
RLjunRP) present in rat liver. In this investigation, we
report t hat t his region is d ifferentially recognized in prolif-
erating liver as evidenced by the formation of a complex,
different from that observed with normal liver extract. The
new c omplex appears as early as 2 h after partial hepatec-
tomy and i ts appearance coincides w ith the rise in c-jun
mRNA levels after partial hepa tectomy. In regenerating rat
liver nuclear extract, an additional protein of  40 kDa
(rRLjunRP) interacts with a pre-existing dimer o f RLjunRP
complexed with the )148 to )124 region of c-jun to f orm a
slow-migrating complex. rRLjunRP a ppears to pre-exist in
the cytosol and translocate to the nucleus as indicated by

the co ntinued p resence of t he retarded complex in nuclear
extract prepared from partially hepatectomized rats treated
with cycloheximide. UV crosslinking studies, South-West-
ern blot a nalysis, SDS/PAGE of affinity-purified factor(s),
and 2D-PAGE analys is clearly d emonstrate t hat t he addi-
tional factor in duced in response t o g rowth stimulus i s an
 40 kDa, th at binds with the dimer of RLjunRP and
enhances the c-jun transcriptio n.
Keywords: c-jun; DNA–protein interac tion; positive regula-
tory factor; r egenerating liver; t ranscriptional regulation.
Precise and coordinated control of gene expression is a
primary r equirement for normal growth, development and
function of an organism. Major control of gene expression
is exerted by regulating m RNA p roduction and involves
complex i nteractions between an array of t ranscriptionally
active proteins and s pecific regulatory DNA sequences. We
have been interested in explicating t he underlying m olecular
mechanisms of transcriptional regulation of c-jun in resting
and proliferating rat liver. The protein Jun, a major
component of the dimeric transcription factor complex
activating protein-1 (A P-1), i s e ncoded by c-jun [1–5]. Be ing
a component of AP-1, Jun is known to be involved in the
regulation of a variety of cellular processes including cellular
proliferation, differentiation, apoptosis and oncogenesis
([6–9], r eviewed i n [10]). As a n immediate early response
gene, expression of c-jun is affected by a variety of
extracellular stimuli including growth factors, cytokines,
serum phorbol esters, tumour promoters, UV radiation a nd
hormones [11–16].
Jun is known to regulate the expression of a myriad of

genes i n a variety of tissues and c ell types [17]; however,
transcriptional regulation o f c-jun itself still re mains elusive.
Among known i mportant r egulatory elements previously
identifiedinthec-jun promoter are the two AP-1 sites ()71
to )64 and )190 to )183) [18]. Pre-existing cJun homo-
dimers and cJun/ATF-2 heterodimers can bind to these two
AP-1 sites a nd activate transcription [13,18]. I nvolvement of
the )148 to )124 region of c-jun in the positive regulation o f
transcription from t he c- jun promoter through its interaction
with a pos itive r egulatory f actor (rat liver jun regulatory
protein; RLjunRP), which is present in quiescent rat liver
nuclear extract, has been reported [19]. Brach and coworkers
have earlier reported the presen ce of a factor, Nuclear
factor-jun (NF-jun), in human myeloid l eukaemia cells that
protected the )139 to )129 region of c-jun [20]. However, its
Correspondence to A. Dixit, Centre for Biotechnology, Jawaharlal
Nehru University, N ew Delhi - 110067, India.
Fax: +91 11 26198234, Tel.: +91 11 26102164 or 26704085,
E-mail: or
Abbreviations: AP-1, activating protein-1; C HX, cyclohexamide;
EMSA, electrophoretic mobility shift assay(s); IL-6 DBP, interleukin-
6 dependent DNA binding protein; NF-jun, nuclear factor-jun;
NF-jB, nuclear factor-jB; RLjunRP, rat liver jun regulatory protein;
rRLjunRP, r egenerating rat liver jun regulatory protein; RNE-d, rat
liver nuclear extract-fraction D; nRNE-d, normal rat liver nuclear
extract-fraction D; rRNE-d, r egenerating rat liver nuclear extract-
fraction D ; TFIIIA, 5S R NA gene-specific transcription factor IIIA;
TFIIIC, 5S R NA gene-specific tr anscription factor IIIC.
*Present address: Department of Pathology and Laboratory Medicine,
University of Louisville, 511 S F loyd Street, Louisville, K Y-40202,

USA.
Present address: Department o f Hematology-Oncology, W inship
Cancer Institute, E mory University Schoo l of Med icine, Clinic C,
1701 Uppergate D rive Rm # 4060, Atlanta, G A-30322, USA.
(Received 9 Septem ber 2 004, rev ised 10 O ctober 2004,
accepted 25 Octobe r 2004)
Eur. J. Biochem. 271, 4892–4902 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04458.x
activity was found to be absent from nonproliferating
diploid cells and appeared to be restricted to dividing cells
[20,21]. RLjunRP that binds to the )148 to )124 region of
c-jun, identified in our laboratory previously, differs from
NF-jun with respect to it being p resent in resting liver cells
[19]. These findings suggest that expression of c-jun is like ly
to differ from one cell type to another. Because d ifferential
interaction of factors with the cis acting elements under
different physiological conditions and in response to growth
stimuli, in part, is known to regulate differential gene
expression, it is likely that some o ther inducible factor
interacts with R LjunRP bound to the )148 to )124 region,
and further enhances c-jun transcription in proliferating
liver.
Most of the studies to gain an insight into the transcrip-
tional r egulation of c-jun in response to g rowth s timuli have
been conducted in cultured cells which do no t mimic in vivo
conditions. We have therefore chosen regenerating rat l iver
following partial h epatectomy, a s a source of proliferating
tissue to mimic in v ivo conditions. Surgical removal of two-
thirds of the liver results in regeneration of the remaining
liver lobes until t he original liv er mass is regained [22–24].
Partially hepatectomized liver has been proposed as a model

for hepatic neoplasia and is a well-suited system to study
normal regulated growth [24–26]. I t serves as a s ource of
relatively abundant quantities of homogeneous growing
tissue. P artial h epatectomy l eads to an orchestrated regen-
erative response, activating a cascade of cell signalling
events necessary for cell c ycle pro gression and proliferation
of hepatocytes. Because progression of liver proliferation
can be followed u sing this system, regenerating liver allows
us to follow c hanges in the specific factor(s) t hat may be
involved i n the initiation o f regeneration, liver growth and
development. The Jun protein has been reported to b e a
major constituent of the AP-1 com plex both i n quiescent
and e arly regenerating liver [27,28]. Activation of AP-1, in
turn, influences the expression of several genes essential for
the proliferation of hepatocytes [14,29]. It has also been
shown that the liver specific deletion of c-jun leads to
decreased hepatocyte p roliferation. Investigating regulation
of c-jun in regenerating liver is thus of significance to study
normal regulated growth in regenerating liver.
The present investigatio n was ther efore undertaken with
an attempt to e lucidate whether the )148 t o )124 r egion o f
c-jun is differentially recognized by factors present in resting
and proliferating liver, and its implication on e nhanced
c-jun expression in re generating rat liver.
Materials and methods
Animals and partial hepatectomy
Healthy female inbred rats of Wistar strain (150–170 g)
were procured from the Animal Facility, Jawaharlal Nehru
University, New Delhi, India. The rats were treated
humanly using approved procedures in accordance with

the guidelines of the Institutional Animal Ethics C ommittee
at the Jawaharlal Nehru University, New Delhi. Animals
were fed water and standard rat chow ( Hindustan Lever
Ltd, Mumbai, India) ad libitum and maintained o n a 12 h
light/dark cycle. P artial hepatectomy ( 70%) was performed
on animals as described earlier [ 30]. For the i mmediate and
early t ime points of 0 and 15 min postsurgery, the incision
was covered with sterile gauze, saturated with sterile saline,
before harvesting the liver remnant. For longer time points,
incisions were sutur ed closed until the liver was harvested.
Fractionation of nuclear extract
Experimental animals were killed at different postoperation-
al intervals. Livers were removed immediately, snap frozen in
liquid n itrogen and s tored at )80 °C until processed further
for the preparation of nuclear extract as described earlier
[31,32]. The fraction designated rat liver nuclear extract-
fraction D (RNE-d), containing maximum RNA poly-
merase II activity and essential t ranscription factors, was
used in electrophoretic mobility shift assays (EMSA).
nRNE-d refers to nuclear extract prepared from normal
rat liver and rRNE-d r efers to nuclear extract prepared from
regenerating rat liver sacrificed at 8 h after surgery (time after
partial hepatectomy at which c-jun transcription peaks in rat
liver) [ 33]. CHX-rRNE-d refers to nuclear extracts prepared
from partially hepatectomized rat livers from animals
injected with cyclohexamide (CHX in saline; 6 mg per
100 g body weight, interperitoneally) immediately after
surgery. Liver was excised at 3 h p ost surgery. Protein
estimation was carried out by the method of Bradford [34].
EMSA

EMSA using nuclear extracts prepared at different postsur-
gery intervals a nd a-
32
P-labelled oligonucleotide encompas-
sing the )148 t o )124 region of t he c-jun promoter (Jun-25)
was performed as described by Sharma et al .[19].The
binding reaction consisted of 10 lg RNE-d (preincubated
with 500 ng fragmented calf thumus DNA for 20 min), 1 n g
(0.06 p
M
) labelled J un-25 (  166.5 B q), 1 0 m
M
Tris/HCl
pH 7.5, 50 m
M
NaCl, 2.5 m
M
MgCl
2
,1m
M
dithiothreitol,
1m
M
EDTA, 0.1% (v/v) Triton X-100 and 5% (v/v)
glycerol in a final reaction volume of 40 lL unless otherwise
stated. T he complex formation was allowed to take place at
30 °C for 30 min followed by electrophoresis on a pre-
electrophoresed 6% nondenaturing polyacrylamide gel in
1· Tris/glycine buffer ( 0.0192

M
glycine, 25 m
M
Tris/HCl
pH 8.3) at 11 V Æcm
)1
for 3 h. The products were analysed
by autoradiography.
UV crosslinking of DNA–protein adducts
UV crosslinking was performed to determine t he approxi-
mate molecular mass o f DNA–protein adducts [19]. EMSA
was carried out as described above except that 100 lgof
nuclear protein and 5 ng of labelled Jun-25 were used.
Following the binding reaction, the reaction mixture on ice
was exposed to UV radiations in a UV Stratalinker
(Stratagene, La J olla, CA, USA) and auto-crosslinked
twice (2 · 60 mJ). Following UV irradiation, the mixture
was separated on a 12% SDS/PAGE as described by
Laemmli [ 35].
South-Western blot analysis
South-Western b lot analysis using nRNE-d and rRNE-d
with labelled probe (Jun-25 tetramer) was carried out as
Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4893
described earlier [ 19]. Fraction nRNE-d and rRNE-d were
separated on 12% SDS/PAGE and electrophoretically
transferred o nto a nitrocellulose membrane. The membrane
was t hen incubated i n denaturing solution (6
M
guanidine/
HCl i n 1 · binding buffer) for 10 m in. T o this, an equal

volume of 1· binding buffer was sequentially added to
dilute the guanidine/HCl in the denaturing buffer to 3
M
,
1.5
M
,0.75
M
,0.38
M
and 0.185
M
with 5 min incubation
after each addition. The m embrane w as then incubat ed in
blocking buffer [ 5% (w/v) BSA in 1 · binding buffer] for 1 h
followed by four washes (10 min each) with 1· binding
buffer. Finally, 1· binding buffer containing labelled
tetramer of Jun-25 (16650 Bq m L
)1
), fragmented calf
thymus DNA (10 lgÆmL
)1
) and 0.25% BSA was added
and allowed to incubate overnight. The strip was washed
with three changes of 1 · binding buffer over a period of
30 min and autoradiographed.
In vitro
DNase I footprinting analysis
DNase I f ootprinting analysis to identify protected region s
was p erformed as described [36]. The binding reaction was

carried out as described in EMS A with a 5¢ end-labelled
266 bp fragment of c-jun ()284 t o )18;  333 Bq) and
increasing concentration of nuclear proteins in a final
reaction volume of 50 lL containing 10 m
M
Tris/HCl,
pH 7.5, 50 m
M
NaCl, 2.5 m
M
MgCl
2
,1m
M
dithiothreitol,
1m
M
EDTA, 0.1% (v/v) Triton X-100 and 5% (v/v)
glycerol. The reaction mix w as incubated at 3 0 °Cfor
20 min. The reaction mix was then supplemented w ith
1m
M
CaCl
2
,5m
M
MgCl
2
and 50 lgoffragmentedcalf
thymus DNA followed by digestion with 0.20 UÆmL

)1
of
DNase I (Promega, Madison, WI, USA) for 90 s at 37 °C.
The reaction was terminated by addition of EDTA (30 m
M
)
and SDS (1%). The products were purified by phenol/
chloroform extraction and ethanol precipitation. The
products were dissolved i n f ormamide dye, denatur ed a t
100 °C for 2 min and separated on a pre-electrophoresed
6% urea/acrylamide sequencing gel. The gel was dried
and autoradiographed at )70 °C. A s tandard M13mp18
sequencing r eaction with a n  40mer universa l primer w as
used as a reference.
Recognition sequence DNA-affinity chromatography
Affinity purification was performed as described e arlier [19].
Radiolabelled Jun-25 concatamers were covalently bound
to CNBr-activated sepharose CL-4B. Nuclear proteins
(nRNE-d and rRNE-d) were i ncubated with the affinity
matrix (pre-equilibrated w ith 1· binding buffer excluding
Triton X-100) in the presence o f nonspecific DNA in 1 ·
binding buffer excluding Triton X-100. The proteins bound
specifically to Jun-25 were eluted with binding buffer
containing increasing concentrations of NaCl. Aliquots
from different fractions were analysed by EMSA. The
fractions showing the complex formation were analysed by
SDS/PAGE and s ilver staining [37].
Isoelectric focusing and second dimension SDS/PAGE
Affinity purified nuclear proteins were subjected to
2D-PAGE as described by Pollard [38] using a Mini-

Protean II 2-D gel apparatus (Bio-Rad Laboratories,
Hercules, CA, USA) according to the manufacturer’s
instructions. The isoelectric focussing (IEF) gel c omposition
was similar to that described by O’Farrell [39]. The
concentrations of the a mpholytes used to establish the pH
gradient were 1.6% (w/v) Bio-Lyte (pH 5–7) and 0.4%
(w/v) Bio-Lyte (pH 3–10). IEF gels were allowed to
polymerize for 1 h and prefocussed at 200 V for 10 min,
300 V and 400 V for 15 min each. Protein samples
(50–100 lg) were loaded onto the IEF gel, o verlaid w ith
20–40 lL of sample overlay buffer [9
M
urea, 1% (v/v)
ampholyte (0.8%, pH range 5–7; 0 .2%, pH range 3 –10) and
0.0025% (w/v) b romophenol blue] a nd electrofocussed a t
500 V for 1 0 min followed b y 750 V for 3.5 h . After
electrofocussing, the IEF gels were extruded from the
capillary with a gel ejector and a llowed t o e quilibrate in the
SDS e quilibration buffer [0.0625
M
Tris/HCl, p H 6 .8, 2.3%
SDS, 5% 2-mercaptoethanol (v/v), 10% glycerol ( v/v) and
0.0025% bromophenol blue] for 10 min prior to second
dimension electrophoresis. The IEF gels were then placed
on a 5% stacking gel and o verlayered with a tracking dy e.
Electrophoresis was achieved at 1 00 V through t he stacking
gel a nd at 200 V t hrough the 12% separating g el. Proteins
were visualized by silver staining [37].
Results
Establishment of differential complex formation

between factor(s) present in normal and regenerating
rat liver with the )148 to )124 region of
c-jun
In order to establish w hether the factors p resent in rRNE-d
bind to the )148 to )124 region of c-jun and form a complex
different than that observed w ith normal e xtract (nRNE-d),
EMSA was carried out using labelled Jun-25 and different
concentrations of the e xtracts prepared from normal a nd
partially hepatectomized rat livers excised at 8 and 24 h after
surgery (Fig. 1A). These t ime points were chosen based o n
our earlier studies, which showed that the c-jun mRNA level
in partially hepatectomized rat liver increased i mmediately
after p artial hepatectomy a nd attained its maximum level a t
8h.At24h,c-jun mRNA levels declined a little but still
remain significantly higher than that observed i n c ontrol
liver [33]. T he appearance of a prominent slow-migrating
complex C 2 with r RNE-d (lanes 3 and 4) can be d istinctly
seen when compared to the complex C1 formed with
nRNE-d (lanes1 and 2). A similar pattern was observed in
nuclear ext ract p repared at 24 h after surgery (lanes 5 and
6). An a lmost complete d isappearance of complex C 1 (lanes
3 and 4) suggests that an additional factor, designated as
regenerating rat liver Jun regulatory protein (rRLjunRP)
induced by partial hepatectomy may interact with RLjunRP
dimer, involved in complex C1 formation.
Specificity of the complex formation was established
(Fig. 1B,C) by incubating the rRNE-d (100 lg) with
different amounts of unlabelled Jun-25 (lanes 3–6), 100-
fold excess of n onspecific DNA, fragmented c alf thymus
DNA (lane 7) a nd pBR322 (lane 8) prior to the a ddition of

labelled oligonucleotide Jun-25. The complex formation
was found to be highly specific as established b y the
complete disappearance of the complex, when the extract
was preincubated w ith unlabelled Jun-25, w hereas, no e ffect
4894 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004
was observed when a 100- to 200-fold excess of nonspecific
DNA was used for c ompetition. Li ke the other re gulatory
proteins that bind to their specific recognition site with
higher affi nity [40], these factors c ould form a complex even
in the presence of several thousan d-fold excess o f nonspe-
cific DNA (Fig. 1C).
The optimum concentration o f m onovalent cations was
determined by carrying out EMSA in the presence of
different concentrations of NaCl. Complex formation was
observed over a range of NaCl c oncentrations, i.e.
25–100 m
M
(Fig. 1D, lanes 1–4), with maximal formation
at 5 0 m
M
NaCl(lane2).Therewasadecreaseinthe
complex formation from 100 m
M
onwards. At 250 and
500 m
M
NaCl, very little complex could b e seen (lanes 5 and
6, respectively).
The optimum concentration of MgCl
2

required for the
complex formation was titrated by c arrying out EMSA in
the presence of different concentration s of MgCl
2
(Fig. 1 E).
Complex formation could be seen in the presence o f 1 m
M
MgCl
2
(lane 1). Binding was found to be maximal in the
presence of 2.5 m
M
MgCl
2
(lane 2).
1
C2
C1




+
+
+


23 4 56
C2
C1

C2
C1
C2
C1
MgCl
2
(mM)
NaCl

(mM)
C2
C1
frag. CT DNA (µg)
frag. CT DNA (100ng)
Jun-25 (ng)
5101520
7.5% formamide
pBR 322 (200ng)
1
110
25 50 75 100 250 500
1 2.5 5 10 15 20
20 40
234
1234
56
123456
123456
78
A

B
C
D
E
Fig. 1. Spe cificity of complex formation between )148 to )124 region of
c-jun (Jun-25) and fac tors present in rRNE-d, and determination of
optimum c oncentrations of monovalent a nd divalent cations. (A) Differ-
ential complex formation of nRNE -d and rRNE-d with Jun-25. EMSA
reactions w ere c arried outin the presence of 1 ng of rad iolab elled Jun-25
and 100 lg(lanes1,3and5)and150lg (lanes 2, 4 and 6) o f nuclear
extracts. Lanes 1 and 2 r e present EM SA performed with nRNE-d, lanes
3 and 4 represen t EMSA performed with nuclear extracts prepared 8 h
after partial he patectomy an d lan es 5 and 6 represent EMSA performed
with nuclear extracts prepared at 24 h after surgery. ( B) Factors in
regenerating rat liver form sp ecific complex with the )148 to )124 re-
gion of c-jun. L ane 1 represents the i nteraction o f factor(s) present in
fraction rRNE-d with 1 n g o f rad iolabelled J un-25. E MSA r eactions
were carried out using 100 lgofrRNE-dpreincubatedwith100-fold
excess of unlabelled nonspecific DNA, namely , fragmented calf thymus
(CT) DNA (lane 7), p BR322 (lane 8 ), and i n the pr esence of various
concentrations of unlabelled Jun-25 (lanes 3–6) prior to t he addition of
labelled Jun-25. L ane 2 d e picts the b in ding reaction carried out i n the
presence of 7.5% of formamide. (C) RLjunRP can form complexes even
in the presence of a 40 000-fold excess of fragmented calf thymus DNA.
The binding reactions were carried o ut with 1 ng of labelled J un-25 and
100 lg of fractionated nuclear extracts, in the p resen ce of 1 lg(lane1),
10 lg(lane2),20lg(lane3)and40lg (lane 4) of fragmented calf
thymus DNA. (D) Titration of optimum monovalent cation concen-
trations. Binding reactions were carried out in the presence of 25, 50, 75,
100, 250 and 500 m

M
NaCl (lanes 1– 6, respectively) using 100 lgof
rRNE-d and 1 ng of labelled Jun-25. (E ) D etermination of optimum
divalent cation concentration for complex formation. EMSA were
carried ou t using 1 ng l abelled Jun-25 a nd 100 lgofrRNE-dinthe
presence of 1 m
M
(lane 1 ), 2.5 m
M
(lane 2), 5 m
M
(lane 3), 10 m
M
(lane
4), 15 m
M
(lane 5) and 20 m
M
(lane 6) MgCl
2
. C1 and C2 indicate the
two DNA–protein complexes.
Time (h)
C2
C1
C2
C1
1
0 0.25 0.5 1 2 2.5 3 4 6 83.5
23

123
4 5 6 7 8 9 10 11
Fig. 2. Appe arance of additional factor i nteracting with )148 to )124 region of c-jun after partial hepatectomy. (A)Timeofappearanceofcomplex
C2 after p artial hepatectomy. EMSA were carried out using 1 ng of labelled Jun-25 and nuclear extracts prepared from partially hepatectomized rat
livers har vested at d ifferent time intervals (ind icated on top) after surge ry. The appearance of n ew complex C2 in lanes 5–11 can b e observed. (B)
Complex f ormation in nuclear e xtract p r epared from partially hepatectomize d rat livers treated w ith CHX. EMSA was perform ed with 1 ng of
labelled J un-25 and 10 lgeachofnRNE-d,rRNE-dandCHX-rRNE-d(lanes1–3,respectively). C1 and C2 indicate the t wo complexes.
Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4895
Complex C2 appears as early as 2 h after partial
hepatectomy
It has been reported p reviously that c-jun m RNA levels in
partial hepatectomized rat liver start to r ise a pproximately
2 h after surgery, attaining its maximum level at 8 h post
partial hepatectomy [33]. Therefore, it was important to
establish whether the increase in c-jun mRNA levels could b e
correlated w ith the appearance of the factor, rRLjunRP,
involved in the f ormation of complex C2, observed with
rRNE-d, and if the time of appearance of this factor
coincides with the increase in c-jun expression induced by
partial hepatectomy i n a time dependent manner. For this
purpose, EMSA was performed with nuclear extracts
prepared from liver excised at various time intervals after
surgery. As sh own in F ig. 2 A, only c omple x C1 is seen until
1 h after partial hepatectomy (lanes 1–4). Complex C2 could
only be observed in nuclear extracts prepar ed at and after
2 h of s urgery (lanes 5–11). It h as already b een established
that RLjunRP, involved in complex C1 formation, is a
positive regulator of c-jun transcription. An increase in
complex C1 can also be observed after 2 h of surgery,
correlating with the enhanced c-jun mRNA levels reported

earlier. However, when c-jun mRNA levels peak at 8 h , t he
intensity of complex C2 remains higher than that of C1.
Factor(s) involved in the C2 complex formation pre-exist
in the cytosol
In order t o establish w hether the synthesis of the rRLjunRP
in rRNE-d, responsible for complex C2 for mation (conver-
sion of C1 observed with nRNE-d to c omplex C2 observed
with rRNE-d) is induced by partial h epatectomy or if it pre-
exists in the cytosol, EMSA was performed with CHX-
rRNE-d and labelled Jun-25 (Fig. 2B). Cycloheximide
(CHX) i s a known inhibitor of protein synthesis. Therefore,
if this factor is newly synthesized in response to a growth
stimulus, no complex at the C2 position is expected to be
present in EMSA carried out with CHX-rRNE-d. However,
no difference in the pattern of complex formation was
observed between rRNE-d (lane 2) and CHX-rRNE-d
(lane 3).
Interaction o f rRLjunRP a t the 3¢ end o f the )148 t o
)124 region of c-jun is not absolutely essential for its
interaction with R LjunRP complexed with the )148 to
)124 region of c- jun.
EMSA data (Fig. 1) u sing nuclear extracts from normal
and regenerating liver indicated that the factor rRLjunRP
induced by partial hepatectomy interacts with complex C1,
resulting in t he formation of c omplex C2. I f rRLjunRP i s
interacting with RLjunRP complexed with DNA, no
significant difference in the footprinting pattern s hould be
observed with nRNE-d a nd rRNE-d. DNase I foo tprinting
analysis using t he 5 ¢ end-labelled fragment ( )284 to )18) of
c-jun and nuclear extracts prepared from normal and

regenerating liver, w as carried out to study whether a ny
difference in the protection pattern exists betwee n t he two
extracts. Figure 3 shows that while only the central portion
of the )148 to )124 region i s protected with nRNE-d
(protected region: )140 to )131) (lanes 3–5), the protection
extends more towards the 3¢ end of this region (protected
region: )140 to )125) with rRNE-d ( lanes 6–8). This
indicates that w hile rRL junRP interacts with RLjunRP, it
must also be interacting with the 3¢ region of the )148 to
)124 region of c- jun.
C2
C1
Jun-25
12 34 5 6
Jun-25A Jun-25B
-64
-71
-87
-92
-107
-119
-124
-148
-183
-190
0
A
B
1
GAC

Protein (µg)
T
23 456 78
01020
nRNE-d rRNE-d
20 10 20 20
Fig. 3. DNase I p rotection pattern of )284 to )18 fragment of c-jun by
factors present in nRNE-d and nRNE-d, and EMSA with Jun-25 dele-
tions. (A) DNase I footprinting analysis. The 5¢ end-labelled Cfr 91-
AvaI f ragment of c-jun (encompassing )284 to )18 of c-jun)was
incubated with the indicated amounts of nRNE-d (lanes 3–5) and
rRNE-d (lanes 6– 8) foll owed by DNase I digestion. Lanes 1 and 2
represent DNase I-digested 5¢ end-labelled Cfr91-AvaI fragment of
c-jun in the absence of n uclear e xtract. GACT are the seque ncing lanes
(M13mp18 as template and  40 universal primer) electrophoresed
simultaneously as a reference. T he marked regions represent protein
binding sites identified earlier: AP-1 ()64 to )71 a nd )183 to )190),
CTF ( )87 to )92), SP-1 ()107 to )119). The )148 to )124 r eg ion of
c-jun is also shown. (B) E M SA with 5¢ an d 3¢ deletion mutants of Jun-
25. S tan dard binding reactions we re performed usin g 10 lgeachof
nRNE-d (lanes 1, 3 and 5) and rRNE-d (lanes 2, 4 and 6) and 1 ng
each of labelled Jun-25 (encompassing the )148 to )124 region of
c-jun, lanes 1 and 2), J un-25A (encompassing the )139 to )124 region
of c-jun; l an es 3 an d 4) and Jun-25B (encompassing the )148 to )134
region of c-jun). C1 and C2 indicate t he two complexes.
4896 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004
The 5¢ and 3¢ deletion mutants of Jun-25 (Jun-25A a nd
Jun-25B, respectively) were used i n EMSA to establish
whether the interaction of rRLjunRP at the 3¢ end of the
)148 to )124 region of c-jun is necessary for its interaction

with the RLjunRP dimer (Fig. 3B). It i s evident from Fig. 3
that w hen EMSA was p erformed w ith Jun-25A (encom-
passing the )139 to )124 region of c-jun), a significant
reduction in the complex formation w as observed ( lanes 3
and 4). H owever, only a slight decrease was observed o n the
complex formation in EMSA reactions carried out with
Jun-25B ( spanning the )148 to )134 reg ion of c-jun;lanes5
and 6 ), when compared to the complex formation observed
in EMSA reactions performed with control J un-25.
Complexes C1 and C2 are formed by the factor(s) binding
on the minor groove of the )148 to )124 region of
c-jun
To establish the nature of interaction b etween trans-acting
factor(s) and t he )148 to )124 r egion of c-jun,drugsthat
specifically interact with the major or minor groove of DNA
were evaluated for their ability to c ompete with trans-acting
factor(s) in EMSA. EMSA were performed with both
nRNE-d and r RNE-d i n t he presence of increasing concen-
trations of methyl green, a majo r groove b inding drug [41]
and distamycin A, a minor groove binding drug [42].
Increasing concentrations of distamycin A resulted in a
decrease in the c omplex formation both w ith nRNE-d and
rRNE-d (Fig. 4A,B, respectively) and virtually no complex
formation was seen at a c oncentration of 1 0 l
M
. Another
minor groove binding drug, actinomycin D also inhibited
complex formation both in normal and regenerating liver
extracts (Fig. 4 C,D, lanes 2 and 3 in both panels). On the
other hand, me thyl green did not affect the f ormation of the

complexes (lanes 4 and 5 ). The insensitivity of nRLjunRP
and rRLjunRP to the major groove binding drug, m ethyl
green, coupled with its sensitivity to minor groove binding
drugs, actinomycin D and distamycin A, confirms that these
are m inor gr oove binding proteins.
rRLjunRP is an  40 kDa protein that interacts with
the RLjunRP–Jun-25 adduct of  80 kDa
To assess the approximate molecular mass o f the DNA–
protein a dduct f ormed between factors present in rRNE-d
and Jun-25 and to see if there exists any difference in the
DNA–protein adduct formed between factors present in
nRNE-d and Jun-25, UV crosslinked EMSA products
were analysed on SDS/PAGE (Fig. 5A). The molecular
mass of the crosslinked complex of nRNE-d was
 80 kDa corresponding to complex C1 (lane 1). UV
crosslinking of complexes formed between rRNE-d and
Jun-25 displayed three addu cts of  40,  80, and
 120 kDa (lane 2). The  80 and  120 k Da DNA–
protein adducts correspond to the complexes C1 and C2,
respectively. A diffused c omplex at  40 kDa (lane 2)
seems to be formed between rRLjunRP and Jun-25. This
is in line with our footprinting data where an a dditional
protection at the 3¢ end of the )148 to )124 region of
c-jun was observed (Fig. 3, lanes 6–8). These data suggest
that rRLjunRP is also able to interact with Jun-25
independently. However, this interaction seems to be very
weak as a smaller c omplex was not observed in E MSA.
Thus, partial hepatectomy results in the a ppearance of an
additional factor of  40 kDa that complexes with
RLjunRP dimer bound to Jun-25.

This was further confirmed by South-Western blot
analysis of nRNE-d and rRNE-d using radiolabelled
Jun-25 (Fig. 5 B). A single hybridized band of  40 kDa
was observed w ith both nRNE-d (lane 1) and rRNE-d (lane
2). We h ave reported p reviously that the trans-acting f actor
RLjunRP, present in nRNE-d, is a p rotein of  40 kDa [19]
that binds to its recognition sequence as a dimer. UV
crosslinking and South-Western blot analysis using rRNE-d
and Jun-25 collectively suggest that an additional factor of
 40 kDa is present only in rRNE-d, and binds to the
 80 kDa DNA–protein adduct corresponding to complex
C1 to give rise to the DNA–protein adduct of  120 kDa
corresponding to the slow-migrating complex C2.
Affinity purification of
trans
-acting factor(s) from rRNE-d
The trans-acting factors present in r RNE-d interacting
with Jun-25 were purified by recognition s equence affinity
chromatography for further characterization. Major p e ak
fractions eluted between 0.1
M
and 0.5
M
NaCl (Fig. 6A)
did not show any complex formation with Jun-25 in
EMSA. T he factors interacting with the )148 to )124
region of c-jun eluted in 1
M
NaCl as evidenced from the
Dist. A (mM)

- 0.1 1.0 2.5
-


-
0.5 1.5
1.50.5
123 45 1 2 345
-


-
0.5 1.5
1.50.5
- 0.1
12341234567
1.0 2.510 25 50
Act. D (mM)
Methyl Green (mM)
A
C
B
D
Fig. 4. Determination o f the bind ing site of of
factors int eracting with the )148 to )124 region
of c-jun. (A,B) Effect of distamycin A (Dist. A)
on complex f ormation. Standard EMSA
reactions were carried o ut us ing 1 ng of
labelled Jun-25 and 10 lg of nRNE-d (A) and
rRNE-d ( B) in the presence of varying con-

centrations of dist amyc in A (indicated on
top). (C,D) E ffect of actinomycin D (Act. D)
and methyl g reen on c omplex formation.
EMSA we re p erformed using 1 ng of labelled
Jun-25 an d of 10 lgrRNE-d(C)andrRNE-d
(D) in t he pr esence of 0.5 and 1.5 m
M
of
actinomycin D (lanes 2 and 3) and methyl
green (lanes 4 and 5).
Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4897
formation of complexes with Jun-25 (Fig. 6B). Residual
complex formation could also be s een in t he fraction
eluted with 2
M
NaCl. A nalysis o f f ractions eluting in 1
M
NaCl (Fraction s 42 and 45, lanes 1 and 2, r espectively) on
SDS/PAGE revealed a prominent band of  40 kDa
(Fig. 6C). These data further c onfirm that the additional
factor rRLjunRP, p resent in rRNE-d, is o f  40 kDa. The
interaction between rRLjunRP and RLjunRP appears to
be very strong as both C1 and C2 complexes are observed
in the s ame fraction. If rRLjunRP was weakly bound to
RLjunRP, it would have dislodged at lower concentra-
tions and only complex C1 would be observed in these
fractions.
When RLjunRP was purified from nRNE-d, it eluted at
2
M

NaCl [19] whereas the factors from rRNE-d e luted a t
slightly lower concentration of NaCl, i.e. 1
M
(Fig.6A).This
indicates that RLjunRP alone has higher affinity to the
recognition c omplex than when it is complexed with t he
additional factor in duced by partial hepatectomy. This is
also su pported b y t he effect of NaCl on complex formation
with nRNE-d and rRNE-d. Although a decrease in the
complex formation with increasing salt concentration was
seen with both t he extracts, nRNE-d retained the complex
formationevenat250m
M
NaCl [19] whereas very little
complex c ould be s een between factors present in rRNE-d
and Jun-25 at this concentration ( Fig. 1D) .
kDa
97
66
55
42
40
31
14
21
31
40
42
55
66

97
kDa
12
12
F
A
B
Fig. 5. UV c rosslinking and S outh-Western blot analysis. (A) Deter-
mination of th e mole cular mass of the complex between rRLjunRP–
RLjunRP and the )148 to )124 region of c-jun by UV crosslinking.
Complex between RLjunRP (lane 1) wit h it s c ognate sequence was
formed un der standard cond itio ns using 100 lgofrRNE-dand5ng
of labelled Jun-25 f ollowed by U V irradiation (2 · 60 mJ) in a UV
crosslinker. D NA–protein complex was separated from free DNA by
electrophoresis o n S DS /PAGE. Autoradiography revealed the p res-
ence of complex (shown by arrows). Numbers represent protein
molecular mass markers. F indicates free labelled Jun-25. (B) South-
Western b lot analysis o f fraction r RNE-d with J un- 25. Fifty mic ro-
grams of n RNE-d and rRNE-d were fractionated on SDS/PAGE
(lanes 1 and 2), transferred onto a nitrocellulose sheet and probed with
radiolabelled tetramer of Jun-25 oligonucleotide. The m olecular ma ss
(kDa)ofthemarkersisshownontheleftside.
FT
2
5
5
0.05
0.05
0.1
0.2

0.25
0.3
0.5
1.0
2.0
8
18
10 15
20
20
25
25
Fraction
30
30
38
42
52
35
40 45 50
1.5
0.5
1
1
C2
C1
kDa
210
134
82

40
32
ML12
L
Absorbance 280 nm
0
P
1
P
2
(0.5M)
(1.0M)
A
B
C
Fig. 6. Affin ity purification of factors interacting with the )148 to )124
region of c-jun from regenerating ra t liver. (A) Spectrophotometric
elution p rofile: rR NE-d was subjected to sequence-specific affi nity
column chroma tography and a ll the f ractions obtained were analysed
spectrophotometrically. Absorbance a t 280 nm was measured a nd
plotted. (B) Assessment of c omplex formation ability of eluted f rac-
tions from DNA affinity column. P resence o f factors in different
fractions ob tained b y a ffin ity ch romatography w as checked using
EMSA with labelled )148 to )124 oligonucleotide fragment of c-jun.L
represents EM SA re action with the lo aded f raction and the num bers
on top represent the fractio n numbers. The nu mbers at the bottom
represent the salt co ncentration in t he re spective fraction. ( C) S DS/
PAGE analysis of fractions positive for the complex formation with
Jun-25. The fractionated nuclear extract, rRNE-d fraction (L) and the
peak fractions number 42 (showing D NA binding ability in EMSA)

and 45 eluting in 1
M
NaCl were subjected to S DS/PAGE a nd silver
stained (la nes 1 and 2, respectively ). M represents the m id-ran ge
molecular m ass m arkers.
4898 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004
2D gel electrophoresis confirms the presence of rRLjunRP
in addition to RLjunRP in regenerating rat liver
EMSA and U V crosslinking data have indicated the
presence of an additional factor-rRLjunRP in rRNE-d
in addition to RLjunRP. Because only a single band of
 40 kDa could be seen in South-Western b lot analysis,
affinity purified factors f rom nRNE-d a nd rRNE-d were
analyzed by 2D gel electrophoresis to verify the presence of
rRLjunRP. As evident from Fig. 7A, a single spot at
 40 kDa positio n was observed w ith RLjunRP s uggesting
that it binds to Jun-25 as a homodimer. In affinity purified
rRLjunRP, two s pots t ailing e ach o ther were visualized at
the affinity purified RLjunRP position (indicated by an
arrowhead). In addition, two s pots close to each other a t a
slightly lower position than t hat of R LjunRP and to wards
acidic pI (indicated by an arrow, Fig. 7B) were also
observed. Thus, both RLjunRP a nd rRLjunRP in regen-
erating liver appear to have two isoforms. These isoforms
could a rise due to differential phosphorylation or any other
post-translational modification of t he factors. Thus, the 2D-
PAGE data clearly d emonstrate t hat RLjunRP b inds to
Jun-25 as a homodimer (complex C 1) and an additional
protein having app roximately t he same molecular mass but
different pI value complexes with C 1 to give a heavier

complex ( C2) i n the case of affi nity pur ified factor(s) from
regenerating rat liver.
Discussion
In eukaryotes, expression of g enes is differentially regulated
in response to a complex set of environmental and
developmental cues. The stable association of multiple
transcription factors with eukaryotic genes has been
described in vitro and in vivo. The significance of such
stable interactions is that, i n many cells, a stable pattern of
gene activity is maintained for long periods of time and, in
the case of t ermin ally differentiated cells, until cell death.
However, for genes like c-jun, that require their transcrip-
tional activity t o b e m odulated, the transient association
and dissociation of transcription factor s is a dvantageous.
c-jun belongs to a class of cellular g enes, t ermed e arly
response or immediate early response genes, which are
characterized by a rapid and transient activation of
transcription in response to growth stimulus. Expression
of c-jun is positively autoregulated by AP-1 [3,43]. However,
sites further upstream o f the AP-1 site may play an
important role in transcriptional regulation of c-jun
[15,44]. A positive r egulatory trans-acting factor, RLjunRP,
in rat liver has b een identified that interacts with the )148 to
)124 region of c-jun [19]. The present investigation led to the
identification of yet another factor, rRLjunRP in rat liver
induced in response to p artial hepatectomy t hat interacts
with RLjunRP complexed with the above region, indicating
the key role this element m ay play in differential regulation
of c-jun tran scription at different stages of h epatic regener-
ation. Further, it is noted that although c-jun expression is

maximum at 8 h after surgery, the new factor rRLjunRP
appeared as early a s 2 h post surgery and i ts appearance
coincided w ith the reported increase in c-jun mRNA levels
in rat liver [33]. Thus, the interaction of this f actor involved
in C2 formation may, i n p art, be attributed to the increased
c-jun mRNA levels after p artial hepatectomy. The p resence
of a diffused  40 kDa protein–DNA adduct i n UV
crosslinking studies and e xtended protection i n DNase I
footprinting analysis using regenerating liver nuclear extract
indicate that rRLjunRP can weakly interact with the 3¢ of
the )148 to )124 region of c-jun. However, this interaction
was not found to be absolutely essential f or its interaction
with RLjunRP c omplexed with Jun-25. The interaction of
rRLjunRP with t he 3 ¢ end of Jun-25 might be stabilizing i ts
interaction with RLjunRP bound to Jun-25. Extended
protection could also be due to the larger protein complex in
regenerating liver bo und to the t arget site.
An increase in the C 1 complex formation can also be seen
2 h after partial hepatectomy, indicating in creased
RLjunRP concentrations. RLjunRP that binds to the
)148 and )124 region even in resting liver appears to b e
involved in controlling both b asal and inducible transcrip-
tion of c-jun. rRLjunRP that appears in response to partial
hepatectomy is likely to play the key regulatory role in vivo
in modulating c-jun expression in response to partial
kDa
IEF IEF
SDS
SDS
kDa

134
82
40
32
18
7
210
134
82
40
32
18
7
BA
210
Fig. 7. 2D electrophoresis of affinity purified factors from nRNE-d and rRNE-d. Affinity purified proteins interacting with the )148 to )124 region of
c-jun from nRNE-d (A) a nd rRNE-d (B) were s eparated in the first dimension by IEF using ampholytes pH 3–10 (from le ft to right). The second
dimension was SD S(12%)/ PAGE followed by detection by silve r staining. Arrowhead points to the single spot of RLjunRP in (A). A rrowhead and
arrow in (B) indicate the two spots c orresponding to R LjunRp a n d r RLjunRP, respectiv ely. The insets show 1.5· magnification of the spots.
Ó FEBS 2004 Expression of c-jun in regenerating rat liver (Eur. J. Biochem. 271) 4899
hepatectomy. RLjunRP concentrations appear to be
important for d ifferential c-jun expression. The activation
of c-jun expression by binding of rRLjunRP to RLjunRP
complexed to the target site is transient. Availability of
abundant RLjunRP, involved in the formation of complex
C1, a llows r RLjunRP to b ring about maximal activation of
c-jun transcription. A similar relationship in t he concentra-
tions of factors 5S RNA gene-specific transcription factor
IIIA (TFIIIA) and the 5S RNA gene-specific transcription
factor II IC (TFIIIC), involved in the regulation of 5S RNA

in developing Xenopus oocytes has been reported ( [45],
reviewed in [46]). The binding of TFIIIC and activation of
5S RNA is facilitated by an increase in TFIIIA concentra-
tion. Without TFIIIA being bound, TFIIIC cannot recog-
nize a 5S RNA gene specifically [47]. Like TFIIIC,
rRLjunRP cannot b ind t o t he cis-acting e lement present
within the )148 to )124 region a nd only e levates transcrip-
tion from t he c-jun promoter by its interaction with
RLjunRP occupying the element present within the )148
to )124 region of c-jun. T he fact that the rRLjunRP–
RLjunRP–DNA complex has lower affinity than
RLjunRP–DNA complex, suggests the transient role that
rRLjunRP must play in the activation of c-jun transcription.
Thus, t hese factors i nteracting with the )148 to )124 region
of c-jun are involved i n differential expression o f c-jun in
liver cells that are induced to proliferate.
Persistence of complex C2 even with the extracts
prepared from animals t reated with CHX s uggests that
the factor rRLjunRp pre-exists in the cytosol. Upon
growth stimulus by partial hepatectomy, it possibly
undergoes s ome modification(s) resulting in its transloca-
tion from the cytosol to the nucleus. However, the
appearance of complex C2 only at 2 h post surgery
indeed indicates that a cascade of s ignalling events m ight
be occurring, preceding to the translocation o f rRLjunRP
from cytosol t o nucleus, a nd converting its i nactive form
to an active one. The activation of rRLjunRP is
independent of protein synthesis, which suggests that
modification of a pre-existing molecule is sufficient for its
activation as has been reported for nuclear factor-jB

(NF-jB), interleukin-6 dependent DNA b inding protein
(IL-6 D BP), AP-1 and NF-jun [ 20,48,49]. Large scale
purification of rRLjunRP for further characterization
and cDNA cloning of the gene encoding the same are
under p rogress t o help in understanding its structural
and functional aspects.
Presence of a nuclear factor NF-jun, recognizing the )139
to )129 region o f c-jun only in rapidly pro liferating cells is
reported by B rach et al. [20]. Its p resence was not detectable
in nonproliferating diploid lung fibroblasts, b lood mono-
cytes, granulocytes or resting T-cells. Our studies with rat
liver indicate that although, like NF-jun, pre-existing
rRLjunRP is t ranslocated i n response t o s ignals transduced
after p artial hepatectomy, it binds t o RLjunRP, a factor
also present in normal liver, precomplexed with this
element, and facilitates c-jun transcription. rRLjunRP is
different from N F-jun; this is evident from t he fact that it
is an  40 k Da protein that binds to the RLjunRP
homodimer of  80 kDa giving r ise to a n  120 kDa
DNA–protein adduct, whereas NF-jun is reported to f orm
DNA–protein adducts of 55 and 125 kDa.
This study thus provides an insight into one of the many
molecular m echanisms that c ould b e involved i n differential
gene regulation of c-jun expression in quiescent and
proliferating r at liver. The role of the )148 t o )124 r egion
of c-jun in transcriptional regulation of c-jun in rat liver is
established and two f actors, RLjunRP and rRLjunRP
present in normal [19] and proliferating liver, which
recognize this element have been identified. The f actors
binding to this region are in addition to the already known

regulatory factors that mediate induction response to
growth stimu lus. B ased on these results, a hypothetical
model for regulation of c-jun expression mediated by the
)148 to )124 region in normal and regenerating liver by
these factors is proposed (Fig. 8). According to this,
RLjunRP is involved in controlling both basal and inducible
transcription of c-jun. I nduction of rRLjunRP upon partial
hepatectomy apparently medi ates the interaction between
RLjunRP and the factors of the i nitiation m achinery to
form more actively transcribing initiation complexes. Signal
transduction leading to differential phosphorylation o f
factors a fter partial hepatectomy could i n p art m odulate
the activity o f these factors. Differential recognition o f this
region by these factors indicates the role this element may
play in regulating c-jun expression. Unlike other response
elements, namely those t hat m ediate induction by gluco-
corticoid hormone [50,51], the cis-acting element present
within the )14 8 to )124 region of c-jun has c onsiderable
basal activity, by virtue of being bound by the positive
regulator RLjunRP in resting liver prior t o s timulation by
partial hepatectomy. Recognition of the same region by
NF-jun, which is present only in p roliferating c ells, does
indeed indicate that the transcriptional r egulation of c-jun is
very complex, differing from one cell type to another and
involves different cell and tissue specific factor(s) that b ind
to their cognate recognition sequences bringing about
modulated c-jun expression.
-124
-148
-148

-124
2xnRLjunRP
rRLjunRP
P?
+ 1
+ 1
Initiation Complex
A
B
Fig. 8. Sch ematic model for the ac tivation of transcription o f c-jun in
normal and regenerating rat liver b y factors interacting with the )148 to
)124regionofc-ju n. (A) RLjunRP dimer is prebound to the )148 to
)12 4 region of c-jun in normal liver. ( B) Partial hepatecomy r esults in
the t ranslocation of rRLjunRP to the nucleus which then facilitates the
interaction of transactivating domains with the facto rs of the initiation
complex that re sults in more prod uctive initiation complexes. Differ-
ential phosphorylation ( P?) may p lay a key ro le in m odulating th e
activities of these factors. The transcription start site is denoted by +1.
4900 S. Ohri et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Acknowledgements
Council of Scientific and Industrial Research (CSIR), India is duly
acknowledged for t he Senior Research Fellowships to S .O. and D.S.
The animal work included in this paper had the approval of
Institutional Animal Ethic s Committee, J. N. U. (IAEC- JNU Proje ct
Code no. 27/1999).
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