Vol. 169, No. 1, 1990 May 31, 1990
BIOCHEMICAL
DETECTION OF IAP RELATED TRANSCRIPTS
I.
RESEARCH COMMUNICATIONS Pages 222-231
AND BIOPHYSICAL
IN NORMAL AND TRANSFORMED RAT CELLS
DJAFFARl, L. DIANOUXl, S. LEIBOVICH*, L. KAPLAN3, R. EMANOIL-RAVIERI and J. PERIESI"
1 Retrovirus
et
Rgtrotransposons des Vertsb&s HBpital Saint-Louis 1 Avenue Claude Vellefaux 75010 - PARIS, FRANCE
- UPR A0043
CNRS
2 URA 1158 - I.G.RCNRS 39 Rue Camille Desmoulins 94805 - VILLEJUIF CEDEX, FRANCE 3 U.149 INSERM 123 Boulevard de Port-Royal 75014 - PARIS, FRANCE Received
April
6,
1990
Intracisternal A-particles (IAP) genes in variable copy number exist in all rodent species studied. Expression is highly repressed in Inurine normal cells except in embryonic and transformed cells. We searched for IAP related sequences expression in another rodent species cells. Using the more conserved sequence of IAP gene between mouse, Syrian hamster, and rat species (0.4 kb HindIII-PstI fragment from PMIAI4), we have been able to detect IAP related transcripts in rat cells. We found that, il IAP related transcripts since among 10 tissues tested, only the are poorly expressed in normal cells, placenta shows IAP RNA. ii) IAPs are highly expressed in all the transformed cells tested. iii) the detected transcripts appear to have similar sizes in corresponrat cells as in mouse cells (7.2 kb ; 5.4 kb). None of the probes ding to other regions of the IAP gene nor the entire sequence of PMIA14 B 1990 Academic Press, Inc. allowed us to detect such transcripts. Marine encoded
intracisternal
by members
1000 per haploid in
genome
to the
of
numerous
copies)
(IAPs) are defective
A particles family
of endogenous
proviral
retrovirions elements
(500
to
(l-5).
homologous the
particularly
rodents in
Syrian
mouse
IAPs
have
and possibly hamster
been
other (700
to
detected mammals
950)
(7)
as repetitive (6).
They
and in rat
are (500
(8) IAPs
are
system
for
gene
* Corresponding 0006-291X/90
generally
(g-11).
pathogenicity
Copyright All rights
type a large
genome)
Sequences families
of
considered The
regulation
to
IAP genes and
family
genome
author.
$1.50
0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
be unassociated
222
have
plasticity
been via
with studied
any
type
of
as a model
retrotransposition
of
Vol.
169, No. 1, 1990
proviral
BIOCHEMICAL
insertions.
retroviral
This
sequences of
IAP proviral
cations
of
IAP elements,
protein
products,
events.
Even
proliferation
cells
it
instability
during
difficult
tumor
the
cell
gene
of
population
amplifi-
clear
selection
formation
for
development
clonal
The as
retrotransposition
role
becomes
of
(12-16).
regulation,
other
a true it
transcription
particles and integrase
origin
to elucidate
in the
reverse
positive
transformation,
particularly
by
transcriptase
may be at
is
RESEARCH COMMUNICATIONS
type-A
include
and reverse
or malignant
IAP sequences
occurs
containing
insertions
which if
phenomenon
in
effects
AND BIOPHYSICAL
IAP during that
the
cell
genetic
may be influenced
phase
of
the
by
malignant
process. The as
in
IAP sequences
germ
particles
line
but
have
been
morphological In
fact,
mouse
Syrian of
fragment
of
expression
IAP
in
in different able
IAP related
to
of
detect
transcripts
tumor
has
of
IAP been
rat
used
cell
for
lines
IAP expression was done
species
homologous or tissue in
rat
by Northern
IAP types.
cells blot
not
conclusive,
to
used
related
but
than an
RNA
as probe
hamster
a
IAP.
RNA sequences the
and placenta. under
species detect
Syrian For
IAP
(17-19)
we have to
detecting
species.
in other
In order
as well
lines is
sequences
in any
cells
other cell
particles
evidenced.
sequences way
such
tumor
in
derived
related
IAP gene highly
the
in murine
expressed
rat
presence
related
here
expressed
naturally
the
hamster of murine
We report
of
not
no RNA expression
expression
we were
are
abundantly
reported
evidence
and
small
?re
stringent
first
time,
Detection
of
conditions
hybridization.
MATERIALS
AND METHODS
Cells and tissues SDVI and 111 cell lines were kindly provided by Guillouzo (Rennes, France) and were grown in Williams E Medium (Eurobio, Paris). PMC9 and ERD were kindly provided by Evrard (Paris, France) and were grown in DMEM F12 medium (Eurobio, Paris). KiNRK and XC cells have been obtained from American Type Culture Collection. Rat myogenic cells (221 were grown in DMEM H16 (Eurobio, Paris). Rat tissues (placenta, brain, liver, lung, kidney, ovary, testis, embryonic muscle) were quickly frozen in liquid nitrogen spleen, thymus, after harvesting. Electron microscopy Cells were examined with a "Philips 301" electron microscope. They were fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmic acid and embedded in epon before staining with uranyl acetate and lead citrate. At least, 250 sections were systematically scanned for each cell sample. Care was taken to avoid observed identical or proximal fields. Samples were coded prior to examination to eliminate prejudice. Morphology, size and localisation location,maturation by requirements of IAP are as follows : intracisternal diameter of 80-90 nm, two electron dense budding from the reticulum membrane, rings marged with the inner lamina of the unit membrane and no intermediate layer between the nucleoid and the external envelop. 223
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RNA isolation and blot hybridization Total cellular RNA from all the cell lines listed in table I as well as Ki-Balb cells was isolated by lysis of exponentially growing cells in Urea-LiCl according to Auffray and Rougeon (23). 50 ug of each RNA sample were denatured by glyoxal, subjected to electrophoresis in 1,l % agarose gel and transferred to nitrocellulose as previously described (24) except that baked filters were treated in boiling Tris 2OmM pH8 for 10 mn. Filters were then prehybridized and hybridized in 50 % formamide at 42°C (24). Filters were washed under stringent conditions : 2x5 mn in 2xSSC, 0.1 % SDS at 25’C and 2x20 mn in O.lxSSC, 0.1 % SDS at 50°C. Total cellular RNA from all the tissues used as well as L6 myoblasts was isolated by the guanidium thiocyanate procedure (25). The poly(A1 containing fraction of the RNA from L6 myoblasts was selected by retention on oligo(dT) cellulose under previously specified conditions (26). Gel electrophoresis, Northern blot analysis and filter washing were done as indicated above. Molecular The represents
probes PMIAl4 clone was kindly provided bv Heidman (Villejuif. France). It the entire IAP genomd(2). The 0.4 kb Hind iII/PstI as well as the 0.3 kb PvuII/EcoRI were obtained by Hind III + PstI or PvuII + EcoRI digestion of PMIA14. The PAM91 actin cDNA was cleaved with PstI to isolate a 1150 bp insert containing sequences common to all actin genes. The 1.3 kb PvuII/PvuII was obtained by PvuII digestion;1.5 kb PstI/PstI by PstI digestion and 1.4 kb Barn HI/Barn HI, by Barn HI digestion of PMIA14. The products of digestion were migrated in a 8 % acrylamide gel, and the fragments Nere eluted in 0.5M ammonium acetate, 1rMl EDTA pH8. The fragments were then labelled by random priming with a commercial kit (Boehringer, Mannheim) to achieve specific activities of about 1.109 cpm/ug.
RESULTS Heteroduplexes IAP sequences regions
of
between
rat
between
are
composed
non homology. family
formed
between
hybrid)
than
between
only
1°C and 1.5"C
were
found
sequences.
IAP
represent cells.
potential
regions
or Syrian the
genome
such
self
rat
subsets with
DNA is
hamster
the
probe
of
fact
have
that
rat
interspersed
that
the
6-C below members
hybrids)
(2).
with is
greater
Tm of the the
family
Tm of
hybrid the
self
(respectively, Similarly
and several
other
are not
known
sequences
shown
IAP sequences
of
by the
Tm of the
between
probes
divergence
indicated
low
cloned in
Tm's
rat
their
IAP
length
LTR. regions
of
or
betureen
for
detecting
probes
homology mouse
exist
IAP
and
IAP related
to
detect
between rat
the
IAP,
sequences
RNA expression
different
and, in any
of
IAP
thus, species related
cells.
1, a physical the
homology
mouse below
used
in rat figure
of
Furthermore,
isolated,
We have In
regions
some short
clones
colinear to heteroduplex
short
and rat
boundaries
sequences
analyses
of
different
Nevertheless,
rat
clone
probe
hybrids
The
and in their
and rat
memberstas
a rat
for
mouse
map of
PMIAI4
(21
35s IAP RNA and thus, represents analysis, Lueders and Kuff have
homologous
between
mouse,
Syrian 224
is
shown.
the entire shown that
hamster
and
rat
This
clone
is
IAP genome. By there were some sequences
and
Vol.
169,
No.
1, 1990
BlOCHEMlCALAND6lOPHYSlCAL
,-,
RESEARCH
COMMUNICATIONS
1Kb
.
.B-
T
C
map of mouse rMIAI4. Partial restrictlon of PMIAl4 containing gene are delimited by long terminal repeats mouse IAP probes : A: 1.3 kb PvuII/PvuII : 1.5 kb PstI/PstI F : 1.4 kb Barn HI/Barn HI D : 0.4 kb HindIII/PstI E : 0.3 kb PvuII/EcoRI Restriction sites are as follow : P, BamHI; E, EcoRI.
T
qsical
the 7.2 kb IAP. The (LTRs : 1. Fragments
Pstl;
H,
HindIII;
ends
Pv,
of used
PvuII;
the as
B,
A 1
2
3
4
5
6
7
8
91011
-
B
sctim
Figure
2 Analysis
=v
of
IAP
related
transcripts
in
normal
rat
cells
by Northern
otal cellular RNA was isolated by the guanidium thiocyanate procedure. 25 ug of each RNA sample was denatured by glyoxal, subjected to electrophoresis in l,l% agarose gel, transfered to nitrocellulose and hybridized with A : the 0.4 kb HindIII/PstI fragment from PMIA14; B : the PAM91 actin cDNA. Filters were washed under stringent conditions : 2x5 mn in 2xSSC, 0.1% SDS at 25°C and 2x20mn in O.lxSSC, O.l%SDS at 50°C. RNA sources were : lane 1 : Ki-Balb, lane 2 : Brain, lane 3 : liver, lane 4 : M.E., lane 5 : lung, lane 6 : kidney, lane 7 : ovary, lane B : testis, lane 9 : placenta, lane 10 : spleen, lane 11 : thymus. 28s and 18s ribosomal RNA markers are indicated by small dashes.
225
Vol.
169, No. 1, 1990
determined
BIOCHEMICAL
that
the
retrovirus-like
highly
elements
one
species
(8).
the
mouse
IAP full
length
104
stringency
probe
could of
other 11,
"env"
This
under
high
region
nor
the
entire
Since highly
the this
conserved
rat
stringency
IAP (0.4
IAP gene
it
(fragments
III/Pst was used
of rat
cell
the
I fragment
and
probes
rat
us to PMIAI4
following
could
could
such
experiments.
Designation
Main characteristics
Rat strain
References
SDVl Fll
Normal liver epithelial cells clones
SpragueDawley rat
Gift of Guillouzo, Rennes, France
PMC9
M.C.R.l.l Nerve cell line Immortalized with polyoma virus truncated large T sequences
Wistar
Gift of Evrard, Paris France
ERD
El.A.R.4.1. nerve cell line immortalized with adenovirus ElA sequences
KINRK
Kirsten MSV transformed rat kidney cells
De Larco, NCI, NIH, Bethesda ATCC CRL 1569
xc
RSV-induced tumor derived cell line
ATCC CCL 165
L6FBJ2
Myoblastic transfected
v-foscells
Wistar
rat
Gift of Evrard, Paris, France
Leibovitch Villejuif, France
226
to
trans-
be included
used
rat
be
in figure
1 lines
of
corresponding
detect
a
and mouse
thus
C, E indicated
allowed I from
in
the
IAP
as a probe
between
region
A, 6,
probe)
Table List
pol
the
in
non-IAP
III/Pst
conserved
the
None of
(PMIA14
Kb Hind
Kb Hind
highly
contain
homology,
conditions,
we designed
mouse
region tested
IAP mRNA only
Therefore,
not
region
sequence
background.
IAP gene 0.4
the
env
clones
Pol-containing
conditions.
mouse
overall
with
low
the
conserved
a sequence
did
stringency
of
in
in
as in all
highly
of
to
sequence
high
regions
cripts. this
the
to
low
hybridize
However,
were
as well
relatively
cross
with
RESEARCH COMMUNICATIONS
sequences
3 species, of
probe
corresponding
IAP genes. used
Because
conditions. leading
PMIAl$),
these
cross-hybridize
sequences, fragment
conserved
in
from
AND BIOPHYSICAL
in
Vol.
169, No. 1, 1990
First cells. 2).
we searched
Total The
only in
indicates
that
important rat
from
detectable (7.2
described
the
detection
RNA prepared
transcripts
is
BIOCHEMICALAND
the
mouse.
IA?
related
thymus,
note Where
similar
the
to
other
RNAs are
related
was analysed
was obtained
kbl, All
that
IAP
tissues
signal
kb and 5.4
to
of
BlOPHYSlCALRESEARCHCOMMUNlCATiONS
with the
tissues not
we were
unable
highest
level
of
in normal
on Northern
blots
placenta major
gave
expressed to
transcripts
detect
sample.
in most
(figure We found
IAP-related negative
2
transcripts results.
This
tissues.
It
any IAP RNA transcripts
in
IAP RNA is
normal
rat
detected
in
the
mouse
(27). Since wondered different
mouse if
it rat
cells
A
IAP
was true
are for
cultured
frequently rat
cells.
expressed Thus,
in
transformed
we tested
IAP
cells,
expression
we in
in vitro.
1234567 A
I
2
3
B
03 -F Total
ACTIN
04
actin
blot analysis of IAP related transcripts. cellular RNA was isolated by lysis of exponentially growing cells in LiCl-Urea. 50 ug of each RNA sample or 10 ug of Ki-Balb were electroin figure phoresed, bloted and hybridized to 82P labeled probe as described lane 2 : ERD, lane 3 : 2. Sources of RNA were as follows : lane 1 : Ki-Balb, Fll, lane 4 : PMC9, lane 5, SDVl, lane 6 : Ki NRK, lane 7 : XC. The position of migration and sizes of 285 and 18s RNA markers are indicated. A : Hybridization with the pMIA14 fragment; 8 : Hybridization with the PAM91 actin probe. orthern
Figure
4
Northern blot analysis of IAP related transcripts in L6 myoblasts, compared with v-fos-induced transformants. Total cellular RNA was isolated by the guanidium thiocyanate procedure, and selected for poly(A) containing sequences by retention on oligo(dT1 cellulose. Aliquots of 5 ug of poly(A) containing RNA were processed for gel electrophoresis and Northern blot hybridization. Sources of RNA : lane 1 : L6 LT3. Sizes of 28s and 18s RNA LA, lane 3 : clone cells, lane 2 : clone with the pMIA14 fragment; B : markers are indicated. A c Hybridization Hybridization with the PAM91 actin probe.
227
2
Vol.
169, No. 1, 1990
In
the
first
table
1. They
either
virally Total
system, from
or chemicaly
blot
detectable
In fected
ERD) (data
the
second
system,
v-fos
which
Results
presented
detected
in transformed
of
(clone
v-fos
v-fos
(clone Same
plasmid
LT3) data : the
PMIAI4
The search negative those
for
in all
that
the
control
by
rehybridization
are
gel
and subjected
to
related
RNAs were
or normal
IAP after
cells.
a longer Fll)
of
or
only
exposure immortalized
the
integrated which line,
IAP
of
an entire
the
same blots
kb HindIII/PstI
particles
(not
of
with
a
by Kuff,
NIH,
fragment
from
shown).
by electron
despite
copy copy
no IAP RNA.
provided
probe
(221. can be
a deleted
and shows of
transus
expression
has
(kindly
rat
cells
by one
line
0.4
IAP
tested,
myogenic
have cell
with
rat
studied
insert
seen with
lines
IAP related
in
and
an IAP related
which
IAP specific
those
line
a cell
obtained
cell
listed
(SW/l,
been
cells
retroviral
the
showing
rat
with
a clonal
contrary, like
signals
cornigrated
Even
in normal
4 show
obtained the
3,
cells.
previously
figure
On the
rere
containing
Bethesda)
we used
behaves
lines strains
on an agarose
figure
found
myogenic
LA).
cell
shown). had
in
in
COMMUNICATIONS
rat
several
or immortalized
transformed
not
rat
of
was loaded
were
with
6 different
tissues
As shown
no transcripts
(PMC9,
the
various
lane)
analysis.
blot,
BIOPHYSICALRESEARCH
transformed
in XC and Ki-NRK
the
cells
we used
originated
RNA (50 ug per
Northern of
BIOCHEMICALAND
extensive
microscopy
was
observation,
even
in
RNA expression. DISCUSSII)N
Numerous genome the
of
other
entire
after
copies
sequences
species
mouse
stringent
IAP-related
IAP
reported
in
implicated
the
Syrian
their
true in other
role
transcripts
could species.
in
cells
genome, divergence
and
from
transformed
and tumor benefit
from
In
study,
this another
main
(16).
(28-32)
but
Since
finding
of
we report
species,
are
Particles is
far
have from
present
rat
in has
the
genome
only
been
IAP genes
the
DNA
repetitive
might
be
understanding
expression
of
a way to detect
and estimated
were
it
(201,
of
and/or
mouse
in the
representing
particles
progression
characteristics
one another
cells
(7).
the
are
viral
found
signals
conserved
genes
of
are
probes
persistent of
IAP relate3
sequences
their
presence
species
IAP genes
labeled
These
expression hamster
of
500 IAP-related
(51.
the
Although natural
in embryogenesis
genes
to mouse with
element
confirm
sequences. the
related
by hybridization
7-kb
washings
of many species
About
of
of
IAP related IAP
related
checked it on rat species. to be present in the rat
their
heterogeneity
been clear
and their
described whether
in they
some are
rat
really
IAP. The choice relatively
low
of
the
overall
probe
was primordial
sequence
homology 228
to obtain between
this IAPs
of
signal. all
Because species,
of the
Vol.
169, No. 1, 1990
mouse
IAP full
stringency could
BIOCHEMICALAND
length
conditions.
the
to
region
(mouse,
of
to rat,
and thus
probe,
we were
such
obtained
with
normal
Thus,
highly than have
progression
of
detected
in these
normal
deleted our
genomic
case,
related
tion switch
off
nisms
proposed the
position in
and that
placenta
that
shown).
the
RT is
placenta
could
(33).
described
rat
and were in murine the
a lower
IAP
as in the transformed
cells
However
be explained
may reflect
appear during
the
from
a Wistar
rat
The
transcripts
undetectable
in
by an expression
of
myeloma
In
(20).
heterogeneity
of
IAP
genome. supports
rat
in
RNA.
ii)
in
(34).
kb)
IAP
analysis.
established
(4 kb;4.5
those
of
Thus,
Chrisotyle
transcripts
our
placenta.
The
growth
factors
of
foetus
the
genes the
(RT)
Expression
of and in
finding
that
placenta, and effecters which role
may play
retrovirus-like are these
may be an excellent
good candidates successive tissue
229
IAP-homologous an organ
way. of
to
Between
viral
embryonic differentia-
switch
on and
various
mecha-
endogenous
a crucial
role
sequences
(RTVL-HI
for
via
retroposons.
gene activations-inhibitions for
sequen-
of
of cell
involve
in a programmed
hypothetical
transcriptase (34)
line
us to
expressed
tested.
kbl
as previously
different
involved
by
poorly
expressed
transcripts 5.4
E
observations
existence
cells
kb;
C,
that
shows
blot
frequently
a cell
This
rat
to explain
events.
(7.2
smaller
development
reverse
human
likely
(not
in
many
ces,
were
are
the
transformed
probes B,
allowed
the
placenta
by Northern
induced
cells
the
course,
be more
we tested
hormones,
the
of
as in mouse
with
transcripts only
The detected
analysis
provides during
iii)
work,
in
expressed
origin,
the to
smaller
sequences
are
all seem
elements,
these
Northern-blot ces
in
tumor
cell
tested,
this
the A,
we checked
pol
same size.
agreement
IAP related
undetectable
ones.
this
mesothelial
at the
of
probe)
IAP probe,
in good
exclude,
tissues,
sizes
pleural the
: i)
transcripts
similar
are
10 tissues do not
in normal
rat
the ilsing
(fragments
(PNIA14
of
3 species
contain
None
gene
IAP gene
a cloned
species
expressed
IAP
cells
results
entire
not
cells.
IAP
probe
of PMIA14), of
conditions.
rat
mouse
IAP
sequences,
fragment did
in low
a fragment
IAP genes
stringency
and migrating
species
among
in other
mouse,
rat
mouse
these
are
in
as a probe
sequence
RNA in
the
the non-IAP
between
This
IAP
true
conditions,
HindIII/PstI
high
of
Using were
cells;
expression
detect
transcripts.
kb
conserved
under
IAP mRNAonly
Pol-containing
we designed
(8).
regions
the rat
stringency
(0.4
11, nor the
RNA transcripts Our findings
to
to
other
in figure
detect
IAP highly
be used
with
conserved
Hamster)
able to
indicated
mouse
could
corresponding
in low
Therefore,
a sequence
Syrian
region
hybridizes
highly
background.
corresponding
rat
with
high
"env"
cross
However,
cross-hybridize
leading
in
probe
BlOPHYSlCALRESEARCHCOMMUNlCATlONS
studying
them.
sequenretrotranswas It
found is
very
Vol.
169, No.1,1990
BIOCHEMICALAND
Our results with
v-fos
detected with
concerning
raise in
rat
some questions.
v-fos
genome
not
are
enhancing IAP mation
of
The
in cell The
entire
cells.
that
might
of
the
entire
expression
might
on the
elements
to
do not
in
v-fos
even
linkage if
be mediated
promoter
not
v-fos
of
exist
was
but
pattern Only
be secondary
IA?
genome
The possibility
transcription
might
transfected sequences
v-fos
integration
oncogenes
activity
features
in
strongly
reminiscent
to
transformed
cells
IAP -related
expression.
IAP genes
f we consider
expressed
help
myogenic of
that
these
COMMUNICATIONS
the
by the
activity
a cellular have
of
transfor-
any
programmed
tran sforiiidtion.
expression
expression
of
or cellular
promoter
process,
role
viral
rat
with
likely
and IAP
1 preliminary.
action
LTRs.
were
expression
sti
is
origin
cells
oncogene
studies
transfected It
the
in
Transcription
cells
genome.
is
genome-transfected between
RNA expression
myogenic
deleted
cellular
BIOPHYSICALRESEARCH
normal
of
tissues, of
a true
role
rat
IAP
often those
IAP related
define
of
related
expressed of
genes
in
for
these
mouse different
transcripts in
IAPs.
(i.e.
transformed
poorly
cells)
The discovering
of
species-specific
are
t?gUldted
cells
could
genes.
ACKNOWLEDGMENTS We thank biological work
Dr M.P.
material
and M.C. This
la Recherche
used
Poeuf
work
Leibovitch
for
sur
le
We are
here. typing
was supported Cancer)
and Dr J. the
grateful
Tobaly-Tapiero to
for
B. Boursin
providing for
some
photography
manuscript.
by a grant
from
the
"A.R.C."
(Association
pour
N" 6613.
REFERENCES 1. Lueders, 2. Lueders,
K.K. K.K.
and Kuff, and Kuff,
E.L. E.L.
(19771 (1980)
Cell I& 963-972. Proc. Natl. Acad.
Sci.
USA
77, 3571-3575. 3.Tno, M., Cole, M.D., White, A.T. and Huany, 2.C. (1980) Cell 21, 465-473. 4. Kuff, E.L., Smith, L.A. and Lueders, K.K. (1981) Mol. Cen. Biol., 1, 216-227. 5. Shen-Ong, G.L.C. and Cole, M.D. (1982) J. Virol. 42, 411-421. 6. Lueders, K.K. and Kuff, E.L. (1981) Nucleic Acids Res. 9, 5917-5930 7. Suzuki, A., Kitasato, M., Kawakami, M., and Ono, M. (T982) Nucleic Acids Res. I.& 5733-5746. 8. Lueders, K.K. and Kuff, E.L. (1983) Nucleic Acids res. 11, 4391-4408. 9. Minna, J.O., Lueders, K.K. and Kuff, E.L. (1974). J.Tatl. Cancer Inst. 52, 1211-1217. 10. Lueders, K.K.1 Fewell, J.W., Kuff. E.L. and Koch, T. (1984). Mol. Cell. Biol. Q, 2128-2135. 11. Horowitz, M., Luria, S., Rechavi, G. and Givol. D. (1984). EMBO J. 3,
2937-2941. 12.
Hawley,
(1982) Proc. 13. Rechavi, 607-611.
R.G., Natl. G.,
Schulman, M.J., Murialdo, H., Gibson, D.M. Acad. Sci. USA 2, 7425-7429. Givol, D., and Canaami, E. (1982) Nature
230
and Hozumi, (London)
N. 300,
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14. Gattoni-Celli, S., Hsiao, W.L.W., and Weinstein, 1.6. (1983) Nature (London) 306, 795-796. K.B. (1985) Mol. Cell. Biol. 5, 15. GreeGrg, R., Hawley, R., and Marcu, 3625-3628. 16. Ymer, S., Tucker, W.Q.J., Sauderson, C.J., Hapel, A.J., Campbell, H.O., and Young, I.G. (1985) Nature (London) 317, 255-258. 17. Biczysco, W., Pienkowsky, M., SoTer, D., and Koprowski, H. (1973) J. Nat1 . Cancer Inst. 51, 1041-1051. 18. Calarco, P. and%ollosi, D. (1973) Nature (London) Ned Biol. 243, 91-93. 19. Chase* D.G. and Piko, L. (1973) J. Natl. Cancer Inst. g, 1971m73. 20. Kuff, E.L. and Lueders, K.K. (1988) Advances in Cancer Research 51, 183-275. 21. Kuff, E.L. (1988) Banbury Report 30 : Eukaryotic Transposable Elements as Mutagenic Agents. Cold Spring Harbor Laboratory, 79-89. 22. Leibovitch, M.P., Leibovitch, S.A., Hillion, J., Guillier, M., Schmitz, A and Harel, J. (1987) Exp. Cell Res. 170, 80-92. 2;: Auffray, C., and Rougeon, F. (1980) Eur. J. Biochem. 107, 307-314. 24. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 52m5205. 25. Chirgwin, J.M/., Przybyla, E.A., Mac DonalcJ.R., and Rutter, W.J. (1979) Biochemistry 18, 5294. 26. Aviv, H., and Le&-, P. (1972) Proc. Natl. Acad. Sci. USA 74, 5350-5354. 27. Kuff, E.L., and Fewell, J.W. (1985) Mol. Cell. Biol. 2, 47&483. 28. Novikoff, A.B., and Biempica, L. (1966) Gann Monograph L, 65-88. 29. Weinstein, I .B., Gebert, R., Stadler, U., Orenstein, J.M., and Axel, R. (1972) Science 2, 1098-1100. 30. Orenstein, J.M., and Weinstein, I.B. (1973) Cancer Res. 33, 1998-2004. 31. Weber, H.W., Geddes, A., and Stellwagen, R.H. (1978TJ. Natl. Cancer Inst. 60, 919-923. 32. Burtonboy, G., Beckers, A., Rodhain, J ., Bazin, H., and Lamy, M.E. (1978) J. Natl. Cancer Inst. 61, 477-484. 33. Paterzon, B.M., Segal, S., Lueders, K.K., and Kuff, E.L. (1978) J. Virol.
27, 118-126. x.
Johansen,
T.,
Holm,
T.,
and Bjozklid,
231
E.
(1989) Gene 79,
259-267.
BIOCHEMICAL
DETECTION OF IAP RELATED TRANSCRIPTS
I.
RESEARCH COMMUNICATIONS Pages 222-231
AND BIOPHYSICAL
IN NORMAL AND TRANSFORMED RAT CELLS
DJAFFARl, L. DIANOUXl, S. LEIBOVICH*, L. KAPLAN3, R. EMANOIL-RAVIERI and J. PERIESI"
1 Retrovirus
et
Rgtrotransposons des Vertsb&s HBpital Saint-Louis 1 Avenue Claude Vellefaux 75010 - PARIS, FRANCE
- UPR A0043
CNRS
2 URA 1158 - I.G.RCNRS 39 Rue Camille Desmoulins 94805 - VILLEJUIF CEDEX, FRANCE 3 U.149 INSERM 123 Boulevard de Port-Royal 75014 - PARIS, FRANCE Received
April
6,
1990
Intracisternal A-particles (IAP) genes in variable copy number exist in all rodent species studied. Expression is highly repressed in Inurine normal cells except in embryonic and transformed cells. We searched for IAP related sequences expression in another rodent species cells. Using the more conserved sequence of IAP gene between mouse, Syrian hamster, and rat species (0.4 kb HindIII-PstI fragment from PMIAI4), we have been able to detect IAP related transcripts in rat cells. We found that, il IAP related transcripts since among 10 tissues tested, only the are poorly expressed in normal cells, placenta shows IAP RNA. ii) IAPs are highly expressed in all the transformed cells tested. iii) the detected transcripts appear to have similar sizes in corresponrat cells as in mouse cells (7.2 kb ; 5.4 kb). None of the probes ding to other regions of the IAP gene nor the entire sequence of PMIA14 B 1990 Academic Press, Inc. allowed us to detect such transcripts. Marine encoded
intracisternal
by members
1000 per haploid in
genome
to the
of
numerous
copies)
(IAPs) are defective
A particles family
of endogenous
proviral
retrovirions elements
(500
to
(l-5).
homologous the
particularly
rodents in
Syrian
mouse
IAPs
have
and possibly hamster
been
other (700
to
detected mammals
950)
(7)
as repetitive (6).
They
and in rat
are (500
(8) IAPs
are
system
for
gene
* Corresponding 0006-291X/90
generally
(g-11).
pathogenicity
Copyright All rights
type a large
genome)
Sequences families
of
considered The
regulation
to
IAP genes and
family
genome
author.
$1.50
0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
be unassociated
222
have
plasticity
been via
with studied
any
type
of
as a model
retrotransposition
of
Vol.
169, No. 1, 1990
proviral
BIOCHEMICAL
insertions.
retroviral
This
sequences of
IAP proviral
cations
of
IAP elements,
protein
products,
events.
Even
proliferation
cells
it
instability
during
difficult
tumor
the
cell
gene
of
population
amplifi-
clear
selection
formation
for
development
clonal
The as
retrotransposition
role
becomes
of
(12-16).
regulation,
other
a true it
transcription
particles and integrase
origin
to elucidate
in the
reverse
positive
transformation,
particularly
by
transcriptase
may be at
is
RESEARCH COMMUNICATIONS
type-A
include
and reverse
or malignant
IAP sequences
occurs
containing
insertions
which if
phenomenon
in
effects
AND BIOPHYSICAL
IAP during that
the
cell
genetic
may be influenced
phase
of
the
by
malignant
process. The as
in
IAP sequences
germ
particles
line
but
have
been
morphological In
fact,
mouse
Syrian of
fragment
of
expression
IAP
in
in different able
IAP related
to
of
detect
transcripts
tumor
has
of
IAP been
rat
used
cell
for
lines
IAP expression was done
species
homologous or tissue in
rat
by Northern
IAP types.
cells blot
not
conclusive,
to
used
related
but
than an
RNA
as probe
hamster
a
IAP.
RNA sequences the
and placenta. under
species detect
Syrian For
IAP
(17-19)
we have to
detecting
species.
in other
In order
as well
lines is
sequences
in any
cells
other cell
particles
evidenced.
sequences way
such
tumor
in
derived
related
IAP gene highly
the
in murine
expressed
rat
presence
related
here
expressed
naturally
the
hamster of murine
We report
of
not
no RNA expression
expression
we were
are
abundantly
reported
evidence
and
small
?re
stringent
first
time,
Detection
of
conditions
hybridization.
MATERIALS
AND METHODS
Cells and tissues SDVI and 111 cell lines were kindly provided by Guillouzo (Rennes, France) and were grown in Williams E Medium (Eurobio, Paris). PMC9 and ERD were kindly provided by Evrard (Paris, France) and were grown in DMEM F12 medium (Eurobio, Paris). KiNRK and XC cells have been obtained from American Type Culture Collection. Rat myogenic cells (221 were grown in DMEM H16 (Eurobio, Paris). Rat tissues (placenta, brain, liver, lung, kidney, ovary, testis, embryonic muscle) were quickly frozen in liquid nitrogen spleen, thymus, after harvesting. Electron microscopy Cells were examined with a "Philips 301" electron microscope. They were fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmic acid and embedded in epon before staining with uranyl acetate and lead citrate. At least, 250 sections were systematically scanned for each cell sample. Care was taken to avoid observed identical or proximal fields. Samples were coded prior to examination to eliminate prejudice. Morphology, size and localisation location,maturation by requirements of IAP are as follows : intracisternal diameter of 80-90 nm, two electron dense budding from the reticulum membrane, rings marged with the inner lamina of the unit membrane and no intermediate layer between the nucleoid and the external envelop. 223
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RNA isolation and blot hybridization Total cellular RNA from all the cell lines listed in table I as well as Ki-Balb cells was isolated by lysis of exponentially growing cells in Urea-LiCl according to Auffray and Rougeon (23). 50 ug of each RNA sample were denatured by glyoxal, subjected to electrophoresis in 1,l % agarose gel and transferred to nitrocellulose as previously described (24) except that baked filters were treated in boiling Tris 2OmM pH8 for 10 mn. Filters were then prehybridized and hybridized in 50 % formamide at 42°C (24). Filters were washed under stringent conditions : 2x5 mn in 2xSSC, 0.1 % SDS at 25’C and 2x20 mn in O.lxSSC, 0.1 % SDS at 50°C. Total cellular RNA from all the tissues used as well as L6 myoblasts was isolated by the guanidium thiocyanate procedure (25). The poly(A1 containing fraction of the RNA from L6 myoblasts was selected by retention on oligo(dT) cellulose under previously specified conditions (26). Gel electrophoresis, Northern blot analysis and filter washing were done as indicated above. Molecular The represents
probes PMIAl4 clone was kindly provided bv Heidman (Villejuif. France). It the entire IAP genomd(2). The 0.4 kb Hind iII/PstI as well as the 0.3 kb PvuII/EcoRI were obtained by Hind III + PstI or PvuII + EcoRI digestion of PMIA14. The PAM91 actin cDNA was cleaved with PstI to isolate a 1150 bp insert containing sequences common to all actin genes. The 1.3 kb PvuII/PvuII was obtained by PvuII digestion;1.5 kb PstI/PstI by PstI digestion and 1.4 kb Barn HI/Barn HI, by Barn HI digestion of PMIA14. The products of digestion were migrated in a 8 % acrylamide gel, and the fragments Nere eluted in 0.5M ammonium acetate, 1rMl EDTA pH8. The fragments were then labelled by random priming with a commercial kit (Boehringer, Mannheim) to achieve specific activities of about 1.109 cpm/ug.
RESULTS Heteroduplexes IAP sequences regions
of
between
rat
between
are
composed
non homology. family
formed
between
hybrid)
than
between
only
1°C and 1.5"C
were
found
sequences.
IAP
represent cells.
potential
regions
or Syrian the
genome
such
self
rat
subsets with
DNA is
hamster
the
probe
of
fact
have
that
rat
interspersed
that
the
6-C below members
hybrids)
(2).
with is
greater
Tm of the the
family
Tm of
hybrid the
self
(respectively, Similarly
and several
other
are not
known
sequences
shown
IAP sequences
of
by the
Tm of the
between
probes
divergence
indicated
low
cloned in
Tm's
rat
their
IAP
length
LTR. regions
of
or
betureen
for
detecting
probes
homology mouse
exist
IAP
and
IAP related
to
detect
between rat
the
IAP,
sequences
RNA expression
different
and, in any
of
IAP
thus, species related
cells.
1, a physical the
homology
mouse below
used
in rat figure
of
Furthermore,
isolated,
We have In
regions
some short
clones
colinear to heteroduplex
short
and rat
boundaries
sequences
analyses
of
different
Nevertheless,
rat
clone
probe
hybrids
The
and in their
and rat
memberstas
a rat
for
mouse
map of
PMIAI4
(21
35s IAP RNA and thus, represents analysis, Lueders and Kuff have
homologous
between
mouse,
Syrian 224
is
shown.
the entire shown that
hamster
and
rat
This
clone
is
IAP genome. By there were some sequences
and
Vol.
169,
No.
1, 1990
BlOCHEMlCALAND6lOPHYSlCAL
,-,
RESEARCH
COMMUNICATIONS
1Kb
.
.B-
T
C
map of mouse rMIAI4. Partial restrictlon of PMIAl4 containing gene are delimited by long terminal repeats mouse IAP probes : A: 1.3 kb PvuII/PvuII : 1.5 kb PstI/PstI F : 1.4 kb Barn HI/Barn HI D : 0.4 kb HindIII/PstI E : 0.3 kb PvuII/EcoRI Restriction sites are as follow : P, BamHI; E, EcoRI.
T
qsical
the 7.2 kb IAP. The (LTRs : 1. Fragments
Pstl;
H,
HindIII;
ends
Pv,
of used
PvuII;
the as
B,
A 1
2
3
4
5
6
7
8
91011
-
B
sctim
Figure
2 Analysis
=v
of
IAP
related
transcripts
in
normal
rat
cells
by Northern
otal cellular RNA was isolated by the guanidium thiocyanate procedure. 25 ug of each RNA sample was denatured by glyoxal, subjected to electrophoresis in l,l% agarose gel, transfered to nitrocellulose and hybridized with A : the 0.4 kb HindIII/PstI fragment from PMIA14; B : the PAM91 actin cDNA. Filters were washed under stringent conditions : 2x5 mn in 2xSSC, 0.1% SDS at 25°C and 2x20mn in O.lxSSC, O.l%SDS at 50°C. RNA sources were : lane 1 : Ki-Balb, lane 2 : Brain, lane 3 : liver, lane 4 : M.E., lane 5 : lung, lane 6 : kidney, lane 7 : ovary, lane B : testis, lane 9 : placenta, lane 10 : spleen, lane 11 : thymus. 28s and 18s ribosomal RNA markers are indicated by small dashes.
225
Vol.
169, No. 1, 1990
determined
BIOCHEMICAL
that
the
retrovirus-like
highly
elements
one
species
(8).
the
mouse
IAP full
length
104
stringency
probe
could of
other 11,
"env"
This
under
high
region
nor
the
entire
Since highly
the this
conserved
rat
stringency
IAP (0.4
IAP gene
it
(fragments
III/Pst was used
of rat
cell
the
I fragment
and
probes
rat
us to PMIAI4
following
could
could
such
experiments.
Designation
Main characteristics
Rat strain
References
SDVl Fll
Normal liver epithelial cells clones
SpragueDawley rat
Gift of Guillouzo, Rennes, France
PMC9
M.C.R.l.l Nerve cell line Immortalized with polyoma virus truncated large T sequences
Wistar
Gift of Evrard, Paris France
ERD
El.A.R.4.1. nerve cell line immortalized with adenovirus ElA sequences
KINRK
Kirsten MSV transformed rat kidney cells
De Larco, NCI, NIH, Bethesda ATCC CRL 1569
xc
RSV-induced tumor derived cell line
ATCC CCL 165
L6FBJ2
Myoblastic transfected
v-foscells
Wistar
rat
Gift of Evrard, Paris, France
Leibovitch Villejuif, France
226
to
trans-
be included
used
rat
be
in figure
1 lines
of
corresponding
detect
a
and mouse
thus
C, E indicated
allowed I from
in
the
IAP
as a probe
between
region
A, 6,
probe)
Table List
pol
the
in
non-IAP
III/Pst
conserved
the
None of
(PMIA14
Kb Hind
Kb Hind
highly
contain
homology,
conditions,
we designed
mouse
region tested
IAP mRNA only
Therefore,
not
region
sequence
background.
IAP gene 0.4
the
env
clones
Pol-containing
conditions.
mouse
overall
with
low
the
conserved
a sequence
did
stringency
of
in
in
as in all
highly
of
to
sequence
high
regions
cripts. this
the
to
low
hybridize
However,
were
as well
relatively
cross
with
RESEARCH COMMUNICATIONS
sequences
3 species, of
probe
corresponding
IAP genes. used
Because
conditions. leading
PMIAl$),
these
cross-hybridize
sequences, fragment
conserved
in
from
AND BIOPHYSICAL
in
Vol.
169, No. 1, 1990
First cells. 2).
we searched
Total The
only in
indicates
that
important rat
from
detectable (7.2
described
the
detection
RNA prepared
transcripts
is
BIOCHEMICALAND
the
mouse.
IA?
related
thymus,
note Where
similar
the
to
other
RNAs are
related
was analysed
was obtained
kbl, All
that
IAP
tissues
signal
kb and 5.4
to
of
BlOPHYSlCALRESEARCHCOMMUNlCATiONS
with the
tissues not
we were
unable
highest
level
of
in normal
on Northern
blots
placenta major
gave
expressed to
transcripts
detect
sample.
in most
(figure We found
IAP-related negative
2
transcripts results.
This
tissues.
It
any IAP RNA transcripts
in
IAP RNA is
normal
rat
detected
in
the
mouse
(27). Since wondered different
mouse if
it rat
cells
A
IAP
was true
are for
cultured
frequently rat
cells.
expressed Thus,
in
transformed
we tested
IAP
cells,
expression
we in
in vitro.
1234567 A
I
2
3
B
03 -F Total
ACTIN
04
actin
blot analysis of IAP related transcripts. cellular RNA was isolated by lysis of exponentially growing cells in LiCl-Urea. 50 ug of each RNA sample or 10 ug of Ki-Balb were electroin figure phoresed, bloted and hybridized to 82P labeled probe as described lane 2 : ERD, lane 3 : 2. Sources of RNA were as follows : lane 1 : Ki-Balb, Fll, lane 4 : PMC9, lane 5, SDVl, lane 6 : Ki NRK, lane 7 : XC. The position of migration and sizes of 285 and 18s RNA markers are indicated. A : Hybridization with the pMIA14 fragment; 8 : Hybridization with the PAM91 actin probe. orthern
Figure
4
Northern blot analysis of IAP related transcripts in L6 myoblasts, compared with v-fos-induced transformants. Total cellular RNA was isolated by the guanidium thiocyanate procedure, and selected for poly(A) containing sequences by retention on oligo(dT1 cellulose. Aliquots of 5 ug of poly(A) containing RNA were processed for gel electrophoresis and Northern blot hybridization. Sources of RNA : lane 1 : L6 LT3. Sizes of 28s and 18s RNA LA, lane 3 : clone cells, lane 2 : clone with the pMIA14 fragment; B : markers are indicated. A c Hybridization Hybridization with the PAM91 actin probe.
227
2
Vol.
169, No. 1, 1990
In
the
first
table
1. They
either
virally Total
system, from
or chemicaly
blot
detectable
In fected
ERD) (data
the
second
system,
v-fos
which
Results
presented
detected
in transformed
of
(clone
v-fos
v-fos
(clone Same
plasmid
LT3) data : the
PMIAI4
The search negative those
for
in all
that
the
control
by
rehybridization
are
gel
and subjected
to
related
RNAs were
or normal
IAP after
cells.
a longer Fll)
of
or
only
exposure immortalized
the
integrated which line,
IAP
of
an entire
the
same blots
kb HindIII/PstI
particles
(not
of
with
a
by Kuff,
NIH,
fragment
from
shown).
by electron
despite
copy copy
no IAP RNA.
provided
probe
(221. can be
a deleted
and shows of
transus
expression
has
(kindly
rat
cells
by one
line
0.4
IAP
tested,
myogenic
have cell
with
rat
studied
insert
seen with
lines
IAP related
in
and
an IAP related
which
IAP specific
those
line
a cell
obtained
cell
listed
(SW/l,
been
cells
retroviral
the
showing
rat
with
a clonal
contrary, like
signals
cornigrated
Even
in normal
4 show
obtained the
3,
cells.
previously
figure
On the
rere
containing
Bethesda)
we used
behaves
lines strains
on an agarose
figure
found
myogenic
LA).
cell
shown). had
in
in
COMMUNICATIONS
rat
several
or immortalized
transformed
not
rat
of
was loaded
were
with
6 different
tissues
As shown
no transcripts
(PMC9,
the
various
lane)
analysis.
blot,
BIOPHYSICALRESEARCH
transformed
in XC and Ki-NRK
the
cells
we used
originated
RNA (50 ug per
Northern of
BIOCHEMICALAND
extensive
microscopy
was
observation,
even
in
RNA expression. DISCUSSII)N
Numerous genome the
of
other
entire
after
copies
sequences
species
mouse
stringent
IAP-related
IAP
reported
in
implicated
the
Syrian
their
true in other
role
transcripts
could species.
in
cells
genome, divergence
and
from
transformed
and tumor benefit
from
In
study,
this another
main
(16).
(28-32)
but
Since
finding
of
we report
species,
are
Particles is
far
have from
present
rat
in has
the
genome
only
been
IAP genes
the
DNA
repetitive
might
be
understanding
expression
of
a way to detect
and estimated
were
it
(201,
of
and/or
mouse
in the
representing
particles
progression
characteristics
one another
cells
(7).
the
are
viral
found
signals
conserved
genes
of
are
probes
persistent of
IAP relate3
sequences
their
presence
species
IAP genes
labeled
These
expression hamster
of
500 IAP-related
(51.
the
Although natural
in embryogenesis
genes
to mouse with
element
confirm
sequences. the
related
by hybridization
7-kb
washings
of many species
About
of
of
IAP related IAP
related
checked it on rat species. to be present in the rat
their
heterogeneity
been clear
and their
described whether
in they
some are
rat
really
IAP. The choice relatively
low
of
the
overall
probe
was primordial
sequence
homology 228
to obtain between
this IAPs
of
signal. all
Because species,
of the
Vol.
169, No. 1, 1990
mouse
IAP full
stringency could
BIOCHEMICALAND
length
conditions.
the
to
region
(mouse,
of
to rat,
and thus
probe,
we were
such
obtained
with
normal
Thus,
highly than have
progression
of
detected
in these
normal
deleted our
genomic
case,
related
tion switch
off
nisms
proposed the
position in
and that
placenta
that
shown).
the
RT is
placenta
could
(33).
described
rat
and were in murine the
a lower
IAP
as in the transformed
cells
However
be explained
may reflect
appear during
the
from
a Wistar
rat
The
transcripts
undetectable
in
by an expression
of
myeloma
In
(20).
heterogeneity
of
IAP
genome. supports
rat
in
RNA.
ii)
in
(34).
kb)
IAP
analysis.
established
(4 kb;4.5
those
of
Thus,
Chrisotyle
transcripts
our
placenta.
The
growth
factors
of
foetus
the
genes the
(RT)
Expression
of and in
finding
that
placenta, and effecters which role
may play
retrovirus-like are these
may be an excellent
good candidates successive tissue
229
IAP-homologous an organ
way. of
to
Between
viral
embryonic differentia-
switch
on and
various
mecha-
endogenous
a crucial
role
sequences
(RTVL-HI
for
via
retroposons.
gene activations-inhibitions for
sequen-
of
of cell
involve
in a programmed
hypothetical
transcriptase (34)
line
us to
expressed
tested.
kbl
as previously
different
involved
by
poorly
expressed
transcripts 5.4
E
observations
existence
cells
kb;
C,
that
shows
blot
frequently
a cell
This
rat
to explain
events.
(7.2
smaller
development
reverse
human
likely
(not
in
many
ces,
were
are
the
transformed
probes B,
allowed
the
placenta
by Northern
induced
cells
the
course,
be more
we tested
hormones,
the
of
as in mouse
with
transcripts only
The detected
analysis
provides during
iii)
work,
in
expressed
origin,
the to
smaller
sequences
are
all seem
elements,
these
Northern-blot ces
in
tumor
cell
tested,
this
the A,
we checked
pol
same size.
agreement
IAP related
undetectable
ones.
this
mesothelial
at the
of
probe)
IAP probe,
in good
exclude,
tissues,
sizes
pleural the
: i)
transcripts
similar
are
10 tissues do not
in normal
rat
the ilsing
(fragments
(PNIA14
of
3 species
contain
None
gene
IAP gene
a cloned
species
expressed
IAP
cells
results
entire
not
cells.
IAP
probe
of PMIA14), of
conditions.
rat
mouse
IAP
sequences,
fragment did
in low
a fragment
IAP genes
stringency
and migrating
species
among
in other
mouse,
rat
mouse
these
are
in
as a probe
sequence
RNA in
the
the non-IAP
between
This
IAP
true
conditions,
HindIII/PstI
high
of
Using were
cells;
expression
detect
transcripts.
kb
conserved
under
IAP mRNAonly
Pol-containing
we designed
(8).
regions
the rat
stringency
(0.4
11, nor the
RNA transcripts Our findings
to
to
other
in figure
detect
IAP highly
be used
with
conserved
Hamster)
able to
indicated
mouse
could
corresponding
in low
Therefore,
a sequence
Syrian
region
hybridizes
highly
background.
corresponding
rat
with
high
"env"
cross
However,
cross-hybridize
leading
in
probe
BlOPHYSlCALRESEARCHCOMMUNlCATlONS
studying
them.
sequenretrotranswas It
found is
very
Vol.
169, No.1,1990
BIOCHEMICALAND
Our results with
v-fos
detected with
concerning
raise in
rat
some questions.
v-fos
genome
not
are
enhancing IAP mation
of
The
in cell The
entire
cells.
that
might
of
the
entire
expression
might
on the
elements
to
do not
in
v-fos
even
linkage if
be mediated
promoter
not
v-fos
of
exist
was
but
pattern Only
be secondary
IA?
genome
The possibility
transcription
might
transfected sequences
v-fos
integration
oncogenes
activity
features
in
strongly
reminiscent
to
transformed
cells
IAP -related
expression.
IAP genes
f we consider
expressed
help
myogenic of
that
these
COMMUNICATIONS
the
by the
activity
a cellular have
of
transfor-
any
programmed
tran sforiiidtion.
expression
expression
of
or cellular
promoter
process,
role
viral
rat
with
likely
and IAP
1 preliminary.
action
LTRs.
were
expression
sti
is
origin
cells
oncogene
studies
transfected It
the
in
Transcription
cells
genome.
is
genome-transfected between
RNA expression
myogenic
deleted
cellular
BIOPHYSICALRESEARCH
normal
of
tissues, of
a true
role
rat
IAP
often those
IAP related
define
of
related
expressed of
genes
in
for
these
mouse different
transcripts in
IAPs.
(i.e.
transformed
poorly
cells)
The discovering
of
species-specific
are
t?gUldted
cells
could
genes.
ACKNOWLEDGMENTS We thank biological work
Dr M.P.
material
and M.C. This
la Recherche
used
Poeuf
work
Leibovitch
for
sur
le
We are
here. typing
was supported Cancer)
and Dr J. the
grateful
Tobaly-Tapiero to
for
B. Boursin
providing for
some
photography
manuscript.
by a grant
from
the
"A.R.C."
(Association
pour
N" 6613.
REFERENCES 1. Lueders, 2. Lueders,
K.K. K.K.
and Kuff, and Kuff,
E.L. E.L.
(19771 (1980)
Cell I& 963-972. Proc. Natl. Acad.
Sci.
USA
77, 3571-3575. 3.Tno, M., Cole, M.D., White, A.T. and Huany, 2.C. (1980) Cell 21, 465-473. 4. Kuff, E.L., Smith, L.A. and Lueders, K.K. (1981) Mol. Cen. Biol., 1, 216-227. 5. Shen-Ong, G.L.C. and Cole, M.D. (1982) J. Virol. 42, 411-421. 6. Lueders, K.K. and Kuff, E.L. (1981) Nucleic Acids Res. 9, 5917-5930 7. Suzuki, A., Kitasato, M., Kawakami, M., and Ono, M. (T982) Nucleic Acids Res. I.& 5733-5746. 8. Lueders, K.K. and Kuff, E.L. (1983) Nucleic Acids res. 11, 4391-4408. 9. Minna, J.O., Lueders, K.K. and Kuff, E.L. (1974). J.Tatl. Cancer Inst. 52, 1211-1217. 10. Lueders, K.K.1 Fewell, J.W., Kuff. E.L. and Koch, T. (1984). Mol. Cell. Biol. Q, 2128-2135. 11. Horowitz, M., Luria, S., Rechavi, G. and Givol. D. (1984). EMBO J. 3,
2937-2941. 12.
Hawley,
(1982) Proc. 13. Rechavi, 607-611.
R.G., Natl. G.,
Schulman, M.J., Murialdo, H., Gibson, D.M. Acad. Sci. USA 2, 7425-7429. Givol, D., and Canaami, E. (1982) Nature
230
and Hozumi, (London)
N. 300,
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14. Gattoni-Celli, S., Hsiao, W.L.W., and Weinstein, 1.6. (1983) Nature (London) 306, 795-796. K.B. (1985) Mol. Cell. Biol. 5, 15. GreeGrg, R., Hawley, R., and Marcu, 3625-3628. 16. Ymer, S., Tucker, W.Q.J., Sauderson, C.J., Hapel, A.J., Campbell, H.O., and Young, I.G. (1985) Nature (London) 317, 255-258. 17. Biczysco, W., Pienkowsky, M., SoTer, D., and Koprowski, H. (1973) J. Nat1 . Cancer Inst. 51, 1041-1051. 18. Calarco, P. and%ollosi, D. (1973) Nature (London) Ned Biol. 243, 91-93. 19. Chase* D.G. and Piko, L. (1973) J. Natl. Cancer Inst. g, 1971m73. 20. Kuff, E.L. and Lueders, K.K. (1988) Advances in Cancer Research 51, 183-275. 21. Kuff, E.L. (1988) Banbury Report 30 : Eukaryotic Transposable Elements as Mutagenic Agents. Cold Spring Harbor Laboratory, 79-89. 22. Leibovitch, M.P., Leibovitch, S.A., Hillion, J., Guillier, M., Schmitz, A and Harel, J. (1987) Exp. Cell Res. 170, 80-92. 2;: Auffray, C., and Rougeon, F. (1980) Eur. J. Biochem. 107, 307-314. 24. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 52m5205. 25. Chirgwin, J.M/., Przybyla, E.A., Mac DonalcJ.R., and Rutter, W.J. (1979) Biochemistry 18, 5294. 26. Aviv, H., and Le&-, P. (1972) Proc. Natl. Acad. Sci. USA 74, 5350-5354. 27. Kuff, E.L., and Fewell, J.W. (1985) Mol. Cell. Biol. 2, 47&483. 28. Novikoff, A.B., and Biempica, L. (1966) Gann Monograph L, 65-88. 29. Weinstein, I .B., Gebert, R., Stadler, U., Orenstein, J.M., and Axel, R. (1972) Science 2, 1098-1100. 30. Orenstein, J.M., and Weinstein, I.B. (1973) Cancer Res. 33, 1998-2004. 31. Weber, H.W., Geddes, A., and Stellwagen, R.H. (1978TJ. Natl. Cancer Inst. 60, 919-923. 32. Burtonboy, G., Beckers, A., Rodhain, J ., Bazin, H., and Lamy, M.E. (1978) J. Natl. Cancer Inst. 61, 477-484. 33. Paterzon, B.M., Segal, S., Lueders, K.K., and Kuff, E.L. (1978) J. Virol.
27, 118-126. x.
Johansen,
T.,
Holm,
T.,
and Bjozklid,
231
E.
(1989) Gene 79,
259-267.
