solo long terminal repeat at the integration site; inverse ..... were trans- fected ... provirus between two /oxP sequences, which should leave a solo. LTR at the.
VoL 9, 381-391,
May
Cell Growth
1998
Effects of ProcoHagen C-Proteinase Growth of Cultured Rat Fibroblasts Excisable Retroviral Vector1
Michiaki Masuda,2 Hiroko and Hiroshi Yoshikura
Igarashi,
Munehide
Kano,
Department of Microbiology, Graduate Schcol of Medicine, The University of Tokyo, Tokyo 113-0033 [PA. M., H. I., M. I(j, and AIDS Research Center, National Institute of Infectious Diseases, Tokyo 1628640 [M. K., H. Y.], Japan
Abstract An excisable
retroviral
by exploiting
Cre-IoxP
integrated
TSN-lox
vector, TSN-Iox, was developed homologous recombination. An
provirus
could
be excised,
leaving
a
solo long terminal repeat at the integration site; inverse PCR, taking advantage of the solo long terminal repeat, was used to characterize cellular flanking sequences. A TSN-lox-transduced Rat2 cell clone, lox-7, was found to harbor the provirus in an intron of the procollagen C-proteinase expression
parental
enhancer protein (PCPE) gene, whose was lowered compared with that of the Rat2. When the vector provirus in Iox-7 cells
was excised, PCPE expression was elevated. The level of PCPE expression seemed to affect cell growth properties such as morphology, contact inhibition, and anchorage-independent growth. These results suggested that the excisable retroviral vector may be useful for studying the molecular basis for proviral insertion mutagenesis, and that POPE may play a significant role in controlling cell growth and differentiation. Introduction Retrovirus has been shown to affect cellular gene expression through proviral integration into host chromosomes. For example, it has been demonstrated in a variety of animal systems that proviral integration within or in the vicinity of cellular proto-oncogenes or tumor suppressor genes could cause malignant disease (reviewed in Refs. i and 2). Retroviral insertion mutagenesis has also been used for studying the function of various other cellular genes (3-8). For studying the molecular mechanism of insertion mutagenesis, it is necessary to identify the proviral integration site by deter-
Received 1/26/98; revised 4/7/98; accepted 4/7/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-
cate this fact. 1 Supported in part by research grantsfrom the Bureau of Social Security, the Japanese Ministry of Human Health and Welfare. 2 To whom requests for reprints should be addressed, at Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-i Hongo, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3812-2111, extension 3409; Fax: 81-3-5684-9374; E-mail: mmasuda0m.u-tokyo.
ac.jp.
Enhancer Revealed
Protein by an
& Differentiation
on the
mining the chromosomal sequences flanking the proviral integration site. In addition, it would be valuable if cellular gene expression inactivated by proviral insertion could be restored in a controlled manner. Therefore, a retroviral vector system that facilitates these procedures would be useful. In this study, we constructed an excisable retroviral vector designated TSN-lox by adopting the Cre-/oxP-mediated homologous recombination of bacteriophage P1 (9, 10). After transduction into the rat fibroblast cell line Rat2, the integrated provirus of the TSN-lox vector could be excised by the transfection of a Cre protein expression plasmid, leaving a solo LTR3 behind. Taking advantage of this solo LW, cellular genomic sequences flanking the upstream and the downstream of the proviral integration site were simultaneously isolated by inverse PCR. Sequence analysis of the cellular sequences flanking the proviral integration sites revealed that among TSN-Iox-transduced Rat2 cell clones, Iox-7 harbored the provirus in an intron of the PCPE gene (1 i). Compared with parental Rat2 cells, lox-7 cells expressed a lower level of PCPE mRNA and protein, due to the inactivation of one of two PCPE alleles by proviral insertion. When the TSN-lox provirus in lox-7 cells was excised by Cre-/oxP-mediated homologous recombination, leaving a solo LW, PCPE expression was up-regulated, indicating that the allele inactivated by proviral insertion had been turned on again. Interestingly, the level of PCPE expression seemed to affect cell growth properties such as morphology, susceptibility to contact inhibition, and ability to grow in soft agar in an anchorage-independent manner. Thus, active excision of the proviral sequence integrated in the PCPE intron was associated with the induction of phenotypic reversion. These results suggest that the excisable retroviral vector may be useful for studying the effects of proviral insertion mutagenesis on host cell functions, and that PCPE may play a significant role in controlling cell growth and differentiation. Results
Transduction
Vector and Its Cre-mediated Fibroblasts. The TSN-lox vector carries the HSV-i tk gene and the bacterial nec gene under the control of the 5’ LTR and the SV4O promoter, respectively Excision
of TSN-Iox
in Rat2
abbreviations used are: LTR, long terminal repeat; BMP-1, bone morphogenic protein 1; BVDU, (E)-5-(2-bromovinyl)-2’-deoxyuridlne; EST, expressed sequence tag; HAT, hypoxanthine-aminoptenn-thymidine; HSV-1, herpes simplex virus type 1; MMLV, Moloney murine leukemia virus; PCPE, procollagen C-proteinase enhancer protein; PVDF, polyvinylidene difluoride; RACE, rapid amplification of cDNA ends; RSV, Rous sarcoma virus; Uc, thymidine kinase; 0418r, G418-resistant ( defined similarly for other terms throughout article); G418, G418-sensitive (S defined similarly for other terms throughout article); GSP, gene-specific primer; DIG, digoxigenmn. 3 The
381
382
Excisable
Retroviral
Vector
and PCPE
A
Function
ture
4.5kb I
U
B
X(S)
X(S)
tk
neo
Iu
SV4O
j
LTR
orl
Io.w.P
IoxP
supernatant
infect Rat2 tion. About
3
.
‘
-ATAACTTCGTATA -TATTGAAGCATAT
UCATACAT CGTATGTA
analysis
with varying
BVDU
colonies
were
colony
Cre-1
Compatible homologous
Cre-2
and remove
Amount
Plasmid
of Transfected
0
Plasmld
1
3
(pg)
from
iB).
Southern
10
lox-5,
genes
B). To further
confirm
Fig. 1. Structure of the DNA constructs. A, MMLV-derived TSN-lox vector. The positions of the LTR, the SV4O replication origin (SV4O on) that contains the viral promoter, and the marker genes tk and nec are mdicated. Solid, open, and shaded boxes of the LTR indicate the U3, R, and U5 regions, respectively. An arrow in the U3 region indicates the direction of transcription from the viral promoter. Insertion of the loxP sequence into the Smal site within the R region of the LTR is depicted. A 4.5-kb-long interval between the Xbal sites is indicated. Additional virus-derived sequences flanking the vector and cloning plasmid pGEM-7Zf(+) are omitted. Restriction sites: B, BamHl; X, Xbal. 5, loss of the Smal site due to the insertion of the loxP sequence. The sequence of the inserted loxP oligonucleotide is also shown. Arrows, the inverted repeat at the both ends of the loxP sequence. B, the phenotype of Rat2 cells transduced with the TSN-lox vector. Cells were seeded at a density of 1 x 10’ cells/well in 24-well plates and cultured in the normal medium (-) or medium supplemented with the HAT supplement (Life Technologies, Inc.), G418 (400 tg/ml), or BVDU (100 LM). The results of Rat2, lox-7, and lox-7-derived Cre-mix, Cre-1 , and Cre-2 cells are shown. Other clones, such as lox-S and lox-9, and their derivatives showed similar results. C, generation of BVDU clones by the transfection of pcDNA-Cre. lox-7 cells seeded at a density of 3 x 10’ cells/60-mm culture dish were transfected with 0, 1 , 3, or 1 0 .tg of pcDNA-Cre or the pcDNA I/Amp vector and grown in the presence of BVDU (1 00 SM). Two weeks after transfection, cells were fixed The
transfection
results
(data
of
lox-S
and
lox-9
cells
with
pcDNA-Cre
not shown).
site
derived
PCR with
also
of the 5’ and CRE packaging
TSN-lox
DNA
and
inserted
into
3’ LTR (Fig. iA). To prepare cells (1 2) were transfected
the with
grown
G4i 8 Cre transfectants
in the were
presence pooled,
chromosomal
in these clones derivatives provirus
Chromosomal
primers
lox-7,
of
and the loss (Fig. 2, A and
was deleted
as
analysis with the
provirus between a solo LTR at the DNA
and
lox-9
of
was
the
BVDU
amplified
by
corresponding to the terminal regions (i.e. , 5’-TGAAAGACCCCACCTGTAGG-3’
and
LTR
5’-TGAAAGACCCCCGCTGACGG-3’).
of
Nucleotide
se-
quencing ture
of the 620-bp PCR product revealed that its strucis identical to an intact /oxP-containing LTR of the TSN-
lox vector
(data
was excised tion
not
the
Proviral
shown).
Therefore,
by Cre-/oxP-mediated
in a defined
tion. Both
manner,
5’ and
the
vector
homologous
Site
Flanking
Could
by Inverse
Sequences
Using
and
the
attempted
integration
to isolate
Fig. 3A; i.e. rying
the
and
cellular
site by inverse solo
enzyme,
,
genomic
digested
as Taql,
such
a circular
extracted
molecule,
primers
flanking
with
treated
from
LTR
the
a 4-base
proviral
is shown
BVDU cutter
by using
lox-9-derived
Inverse
PCR BVDU
car-
restric-
to
a pair of
(5’-GTCTCCTCTGAGTGATTGAC-3’ As
a result,
PCR products of 2.4 and i .0 kb were generated BVDU cells derived from lox-5 and lox-7, respectively 3B).
in
cells
with T4 DNA ligase
and amplified
5’-GUACTTAAGCTAGCTTGCC-3’).
and
Left
Taking advanvector excision,
PCR (i 4). The strategy
DNA
LTR was
DNA
at the Analyzed
Solo
after the Excision of the TSN-lox Provirus. tage of the solo LTR left after Cre-mediated we
recombina-
Be Isolated
PCR
provirus
recombina-
but not by illegitimate
3’ Cellular
Integration
Simultaneously
of G4i8 cul-
2E).
lox-5,
MMLV
tion
has the /oxP sequence
(Fig.
from
LTR-specitic
(Fig. 1A). The vector
g/ml).
cells do(Fig.
the presence
of the vector should leave
the
generate
(400
of the
demonstrated
region which
lox-5,
and HA
G418S
a LTR probe was used for hybridization 2, C and D). The results were compatible
integration
cloned
from
the BVDU
were
BVDU
of
Cre-/oxP-mediated the vector provirus
genes,
that the vector
deletion of the 4.5-kb two /oxP sequences,
the R region vector virus,
that excise
provirus
in their
amount
Cre-1 and Cre-2) and
analysis
and lox-9
the
of 3 x 1 0 cells about 200 BVDU
each obtained pcDNA-Cre.
and lox-9
hybridization lox-7,
of the marker
cells
similar
lox-7,
of
The efficiency
on
(designated were with
M
in Fig.
transtection
plasmid.
depended
of i 00
expected,
Vector
stained.
tk. As shown
prediction would
a single copy of TSN-lox
(Fig.
gave
presence by the
trans-
expression
in the
vector
both of the marker
rived
(i 3), were were
HSV-1
generated
formation
with the recombination
DNA of lox-5,
pcDNA-Cre
and
grown
expressing
but not the control
BVDU
BVDU clones
of the Cre protein
to cells
clones. Two BVDU clones a pooled culture (Cre-mix) lox-7, and lox-9 transfected
Iox-7
C
and
not
i B). These
amounts
pcDNA-Cre toxic
of G4i 8 but (Fig.
transtected pcDNA-Cre, and transtection with 1 0 j.tg of pcDNA-Cre generated
Cre-mix
to
presence
ic,
#{149} .
Parental
used
in the
of
Rat-2
was
for further
used
pcDNA-Cre
B
cells
to grow
BVDU
4
these
then grown under HAT selecwere generated by inoculating
Rat2 cells with 1 ml of the supernatant. Among the HAY clones, lox-5, Iox-7, and lox-9, which were able
plasmid
TATACGAAGTTAT-3’ ATATGCTTCAATA-5’
from
1 x 1 isolated
fected IoxP
harvested
cells, which were i 00 HAY colonies
failed cells
to amplify efficiently,
TaqI-digested probably
inverse
for
the (Fig.
DNA due
of
to the
Cell Growth
Iox-5 Fig. 2. Hybridization and PCR analyses of the integrated vector sequences. High molecular weight DNA (10 Lg) extracted from the cells was digested with BamHl 4-C) or EcoRl (D), fractionated by 1 .0% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with the tk gene probe (4), the nec gene probe (B), or the Xbal-Kpnl LTR fragment with loxP insertion (C and D). C: t, the signals corresponding to the DNA fragment containing the tk gene and sequence;
the
upstream
ln-5
5.0
8.0
4,0-
B 10.0-
8.07.0-
4.0-
3.0-
as-
4
5
6
7
inverse genome, downstream
primers of
the
by hybridization fragment
to
be
analysis amplified.
were
cloned
in plasmid,
determined
and
(Fig. 4). To con-
tamed
by
2
4
3
5
6
7
8
9
10
11
12
13
xkb
ykb
____,.i
a
b
C
X
11
2.5
9
9
Iox-7
10
3.9
8.2
5.5
7.6
lox-9
13
4.5
9.5
8.5
9.5
flanking
lined)
region and
primers
lox-9
PCR
products
context
Rat2
cells.
of
sequence
at the junction
was an insertion
LTR-dependent
ately
in the Rat2 cell
that this adenine
integration
upstream site
of
and lox-5
the were
Similar results of Rat2 DNA
were using
also oba pair of
of
the
upstream AA
In lox-7 an Intron
(Fig.
primers
4B, primers
represented In
are under-
are underlined).
Therefore,
the original
agreement
with
genomic
previous
studies
of 4 bases in the cellular with
flanking
the LTR (Fig. 4). In lox-7,
there
of an additional adenine residue immediof the LTR (Fig. 4B, arrowhead). It is possible residue
dinucleotide
preintegration
y
of lox-7 4C,
(Fig.
the inverse
by solo to the
(kb)
13.5
context
annealing
Bands
DeteCted
Iox-5
obtained
(data not shown). PCR amplification
*
ckb
a correct
prepared (Fig. 4A, primers are underlined) and used to amplity Rat2 genomic DNA. As expected, 300-bp products were generated
*
-
(1 5), there was a duplication
were
proviral
*
‘#{176}
Size of
PCR products
PCR
13
L#{248}-
DNA
fragment
12
r-
not shown).
PCR represented
11
wwflwTA
Instead, Tsp5091-digested DNA of lox-9-derived BVDU cells was successfully used for inverse PCR, and 1 .6-kb products
firm that the DNA
10
Cre
[7.4 kb, as determined
sequences
9
bkb
shown)])
nucleotide
8
1
not
of the
23
4.0
size
The inverse
lf.l;;t41
5.0-
large
their
D120
.
6.0-
sequence.
(data
50-
3.0-
(data
obtained
-
.
#{176} t; f.
60-
I.
i
.
-
70-
-
lox-9
lox-7
3.0-
Arrowheads in D, the specific signals in the parental lox-S, lox-7, and lox-9 cells. Asterisks in C and D, specific signals in BVDU derivatives obtained by transfection of the Cre expression plasmid. Left, the positions of size markers are indicated. Lanes 1, 5, and 9 correspond to the parental lox-5, lox-7, and lox-9 cells, respectively. Lanes 2, 6, and locorrespond to Cre-mix cells derived from lox-5, lox-7, and lox-9 cells, respectively. Similarly, Lanes 3, 7, and 1 1 correspond to Cre-i cells derived from lox-5, lox-7, and lox-9, respectively; Lanes 4, 8, and 12 correspond to Cre-2 cells derived from Iox-5, lox-7, and lox-9, respectively. DNA from the control Rat2 cells was loaded on Lane 13. E, schematic representation of the deletion of the TSN-lox vector sequences mediated by Cre-/oxP homologous recombination. An integrated TSN-lox provirus and a solo LTR left after Cre-/oxP-mediated homologous recombination are shown. Restriction sites: B, BamHl; E, EcoRl; X, Xbal. Thea, b, C, x, and y values for lox-5, lox-7, and lox-9 cells were determined from the hybridization analysis data in A-D. For each clone, x = a - 4.5, andy = b + c - 4.5.
were
(kb) 120-at. 100
4.0-
flanking
flanking
C
laO-ea 10.08.07.06.0-
383
lox-9
qoqoqI,
A
n, the signals corresponding to the DNA fragment containing the nec gene and the
downstream
lnx-7
& Differentiation
form
was derived attached
of proviral
Cells,
the Vector
of the
PCPE
locus
for
vector
integration
otide
sequences
of the
by incomplete
to
DNA
the
(16-i
Provirus
Gene.
Rat2
flanking
removal
terminus
of
the
8).
Was
Integrated
In an attempt
in the
cellular
5’
cell
to identify genome,
region
were
into the nude-
com-
384
Excisable
Retroviral
Vector
and PCPE
Function
A
B T
LTR
T
1
I
12345
I
+ +
TaqI or Tsp5091
digestion
-
Ligation
+ PCR sequences by solo LTR-dependent inverse PCR. A, strategy for analyzing cellular chromosomal sequences PCR. A solo LTR left after the deletion of the vector sequences is flanked by cellular chromosomal sequences (so!id!ines). After TaqI or TspSO9l digestion (7) and ligation, circularized cellular flanking sequences are amplified by PCR using a pair of primers annealing to the terminal regions ofthe LTR (arrows). B, DNA fragments amplified by solo LTR-dependent inverse PCR. High molecular weight DNA (1 g) extracted from lox-S-derived Cre-1 and Cre-2 cells (Lanes 1 and 2, respectively), lox-7-derived Cre-1 and Cre-2 cells (Lanes 3 and 4, respectively), and Rat2 cells (Lane 5) was digested with TaqI, treated with T4 DNA ligase, and amplified by PCR using the LTR-specific primers (5’-GTCTCCTCTGAGTGATTGAC-3’ and 5’-GTTACTTAAGCTAGCTTGCC-3’) and LA Taq polymerase. Amplified samples were subjected to 1 .0% agarose gel electrophoresis and stained with ethidium bromide. Left, the positions of size markers are indicated. An extra band of about 3.5 kb in Lane 4 was not detected reproducibly and is most likely an artifact. Fig. 3.
Amplification
flanking
the vector
of the cellular
integration
flanking
site by inverse
pared with the GenBank sequences by using the BLAST search (19). The analysis revealed that part of the flanking sequence in the upstream (- i 75 to - 1 36) of the integration site of lox-S cells has a homology to Arabidopsis thaliana genomic clone F2507 (GenBank accession number Bi 3323). Several regions of the lox-S flanking sequences were
similar
to
human
sequence-tagged
sites
(L30394,
G26698, and Z39273), a human EST (AA494428), and human and Caenorhabditis elegans high-throughput genome soquences (Z970S4 and Z92822, respectively). The lox-9 flanking sequences also contained regions homologous to human sequence-tagged sites (Gi 3523 and Gi 4854), human EST (AA236509 and AA290873), and a human high-throughput genome sequence (Z94802). However, the detected homology was too limited to unequivocally assign the integration site to a specific locus or gene within the rat genome. In contrast, a 1 09-bp-long sequence upstream of the integration site of lox-7 (Fig. 4B, - 1 69 to -61) showed striking homology
with
the
mouse
and
human
PCPE
cDNA
se-
quences (1 1 , 20) as well as a rat EST (R4707i ; Ref. 21). A cDNA clone containing this homologous region was obtained from Rat2 cell mRNA by the RACE method (Ref. 22; Fig. 5A), and its nucleotide sequence was determined (Fig. SB). The clone had an open reading frame of 1404 bases and encoded study,
a protein of 468 amino acids. During the course of this a cDNA clone of rat PCPE was isolated from a rat liver,
and its nucleotide sequence was determined (23). The nucleotide sequence of the protein-coding region of the cDNA obtained in the present study was completely identical to that of the reported sequence. There were several mis-
matches in the untranslated regions between the two clones that may be accounted for by strain-specific polymorphism (Rat2 is derived from an F344 rat, whereas the previously reported rat PCPE cDNA was isolated from a Wister rat). These results indicated that in lox-7 cells, TSN-lox vector was integrated into an intron of the PCPE gene.
PCPE Expression Was Decreased in Iox-7 cells, Assowith Altered Cell Growth Properties, and Restored by Cre-mediated Excision of the Provirus. To investigate ciated
the effects the levels and
Cre-2
of vector of PCPE cells
integration
mRNA
were
into
and protein
compared.
PCPE gene intron, in Rat2, lox-7, Cre-i,
the
Northern
hybridization
of
total RNA with a rat PCPE full-length cDNA probe revealed a major signal of 1 .5 kb corresponding to mature PCPE mRNA (Fig. 6A). A taint band of about 3.6 kb, which may represent unprocessed or alternatively spliced PCPE RNA, was also detected for these cells, including parental Rat2 (Fig.
6A).
However,
there
was
no indication
that
the
pres-
ence of the entire provirus or solo LTR led to the synthesis of unusual PCPE RNA, at least in a stable form. Photodensitometric analysis of the bands revealed that the level of PCPE mRNA in lox-7 cells was reduced by about 50% compared with that of the parental Rat2 cells (Fig. 6A, Lanes 1 and 2). In contrast, Cre-i and Cre-2 cells derived from
lox-7
showed
a high
level
(Fig. 6A, Lanes 3 and 4). Cre-2 cells seemed to be Rat2 cells. The lox-5 and cells derived from these changes
in the
amount
of PCPE
mRNA
expression
The PCPE mRNA in Cre-1 and even more abundant than that in Iox-9 cells, as well as the BVDU clones, failed to show similar of PCPE mRNA (data not shown).
Cell Growth
A
-210
A
GCATTTTGT
AGGTCCTGCT
GGG’rCCTGAG
AGCCATTCTT
CCTCAATGAC
CTTTCCTAGA
GCCTTGTCAG
GTCTCTGATA
CTGACCCTCC
CCCCAAGTGT
GCATTCCAAG
AGTc0000CT
CC&CCTGTCC
TA000CCCCA
GAACTATGGG
AGGTCTGCT
AGTCCTAGGG
ACTGTTCAGT
GGCTGGCT
GAGTAATTCT
GGGTCTTTCJIj
GSCTGGGTCC
1TGAAAGACCC
!rGccTGGTC’r
GGGTCT?GA
TCTCCCTGGA
?CCA.GCTGG
cc&GA.Acs’cc
TGTTCTTCCG
GGGTCCTGTT
CTCTGGMCO
CTGCCTAGTG
CCA.GGTCAG
. .
(LTI)
. .
61 GCCTGCCCCTAft.CCTCCCTGCTGGGGCCATTCCT1’CTAGCCTGGGTGCTGCC 121
CCGAGGCCGACCCCCMCTACACGAGACCAGTGTTCCTGTGTGGAGGGGATG1
181
GGAGTC005TTACGTGGCAAGTGAGGGTTTCCCCAA.CCTCTACCCCCCAAACAI
241
CATCTGGACAATAACGGTGCC’L’GA.GGGCCAGACTGTGTCCCTGTCCTTCCGAGI TATGGCACCCTCCTGCCGCTATGM’GCTCTGGAGGTCT!I20CTGGATC
CTCCT?GTCT
?CCCTAGGTC
CTGTTCTCT
GGGGPcCAGA
301
CTGCTCGGAA
TCOACCTCGG
CTCGGTGTTC
TGCCTAGTCT
AGG&TTCTGC
361
+210
(SA) TCTCTGCCAT
GACACCTCTT
CTGTCCTCCA
000GATGTGA
CC000GAGTC
PCPZ
:
GGGTTACGTG
llllllllllllllllllllllllllll
GCAAGTGAGG
lllllllllllllllllll
ggggacgtga
ccggggagtc
aggttacgtg
gcaagtgagg
GTTTCCCCAA
CCTCTACCCC
CCAAACMGA
AGTGCATCTG
G&CAATAMG
llW1ti! WllJ5L #{163}ULU1i!Ii (SD) TCCTCTGP
CACC&TTGCC
C5A?CAGT
CTCCCAAGCC
Us +1
AAACCTThA
fTGAAAG&.ccc
GG7ICT?1’CA
CTT&CA.GCTC
AGGGAACTG
CPCTCTTCG
GTCACAAGGG
TGGCAGGCCC
ACAGTAhCA.C
GCTCTGA&GC
ATTccAAccT
TC?CCTCTT
TGTCCACTC
CACTAC’PGGA
AA?TGAA.ccc
?GGGACTTAG
GCATATTAGG
CCAGAGCTCT
ACACTGAGGT
. .
.(LTR)
. .
GCAACTGACGAGGGCACTGGGGGACGk
481
3CTGTACAGCGGTC000CCACCTCAGGCACTGAGCACCAGTTTS’GCGGGG(
541
ACATCTCAG&
CTTT?AT?CA
TT?AGGTTTG
TTTTCGTGGT
TTGTrrGCTT
-210 GAAGCT000T
TTCCCACTGG
ACAOGTAGCC
AGGCTGGTOT
TGOCAGCAGA
GCCCGTG?CT
ccTTGTGGGA
AAGCCT?CT
TCATCTTGGA
GGAATOGA.AA
GTCTGAGTGA
CTCACTGAGT
TGGGGTcC
TGGGTAPCTG
GAcCCCCACC
GAACGCG&TG
OTOCTAGMO
TGACCTGC&G
AAACGACTQ
TTCAA?GGA -1U3 GGCCATAG.aT
. . (1Ta) . . ___________________________
U5+1 GGGTCTTTCA AGSTAATGTC
TTCT!CACTT
rGGGCCPGT
C?CT?GCATG
AkGGGGTG
?GAAGGGTTC
AGACT3CCTC
GCCTCCCTGC
AGCTCAGTGG
GCAGCcCCTC
CCTGGCTGGC
TGCTCTACAG
CTGAA.GGP
ATC?GTG
OTGTGTGCTT
GCTCTAGA&G
CCTAAACCTA
TACACPs.GTGT
GGTC.GATOG
GACACTG’20C
WCAAG&TC
Nucleotide
sequences
+210
Fig. 4.
ing the vector
of the cellular
chromosomal
721 781
CACCGAGGGAGCTCCGTCCAGTTTAATCTCAG
841
CGATGGCTTCTCAGCCTCCTACAGGACCCTGCCACGGGATGCCGTGGAAAGGJ
901 961 1021 1081 1141
Underlined
integration
on both 20-nucleotide
1261 1321 1381
AACTGGGCCCAAAGTCAM.CCACCCAGTAAGCCTAM.GTCCAGCCTGTAGAGA GGGCTCTCCTGCTACCCAGGCAACTCCAG?TGCTCCAGATGCCCCCAGCATCA ACAGTACGGTCAGCACCTrGCAGAGCAACTTTTGCTCCAGTAGCC GACAGGAACAGTGAAGGCCATGGTCCGGGGCCCAGGGGAGGGCCTCACTGTCA TCTCCT000TGTCTACAM.ACCGGAGA.CCTGGA.CCTGCC’N’CTCCAGCTAGTG4 TCTGAAGTTcTATGTGCCC?GCAAGCAGATGCCCCCCATGAAGAA&GGGCca GCTGAT0053’CAGGTGGAAGAGM.CAGAGGCCCCATCCTTCCTCCGGAGAGC’I’ GCTCTACAGGCCCAA.CCAGGA.CCAGATCCTGAGTAACCTA.GCA*GAGAAAG’11
1441
CCAGCCTAGGCCAGATGCCTGAtgtcctcgcCAgatc&gagtgtggtgCtttt
1501
tautgtttcttgactcag
Fig. 5. A, schematic representation of rat PCPE cDNA. An open reading frame flanked by the 5’ and the 3’ noncoding regions is indicated with the initiation codon (ATG), the termination codon (TGA), and the polyadenylated tail. A hatched region corresponds to the putative exon upstream of the vectorintegration site(see Fig. 4). Solldlirse.s below the cDNA structure indicate the 5’ and 3’ RACE products. A solid line corresponding to 200-bp long is shown. B, the nucleotide sequence of rat PCPE cDNA obtained in this study (GenBank accession number AF016503). Se-
quences of the protein-coding region and untranslated regions are mdicated in uppercase and lowercase letters, respectively. A canonical polyadenylation signal sequence in the 3’ untranslated region is in bold.
DNA flank-
site in lox-5 (A), lox-7 (B), and Iox-9 (C). The position of the solo LIR left after the deletion of the vector sequence is indicated as a box. Positions of nucleotides in the cellular sequence are indicated by numbering the residue in the immediate upstream and downstream of the LTR as -1 and + 1 , respectively. Duplicated tetranucleotides
ATCAGCTGTTCCTGGCACATCATTGCACCCTCAAACCAGGTGATCATGCTAAC
661
1201
C
3GAGAGGCGCAGGGA.CCCTGACCACGCCCAACTGGCCTGAGTCGGATTACC(
601
acctgtgtt
cctgtgcgga
-1 u3
421 GAcCAGTGTT
III IIIII Mouse
GTGAGAGGC
I_(A)n
B
CCGGACTGA.G
CCTGTGTGG&
TeA
-I
ctgctgctgctgctgctqttgccgctgctgctgtttCCagCaCtCCCCCtaCa
GOCCTGOTCT
-210 TCACGAGCCA
--ATO
-1U3
B
& Differentiation
sides of the integration site are shown regions correspond to the annealing
in bold. sites for
the PCR primers used to confirm that the cloned DNA represents the correct context of the Rat2 genome. B, a partial sequence of mouse PCPE cDNA (20) is shown in lowercase letters and aligned with the homologous region of the cloned DNA. Positions of the putative splice donor (SD) and acceptor sites (SA) are also indicated. Arrowhead, the insertion of an adenine residue immediately upstream of the LTR. The sequences shown in this figure are available in the GenBank database (accession numbers AF016504, AF016505, and AFO1 6506).
Immunoblot analysis of the cell lysates using an anti-PCPE antibody (24) showed that the level of the 55-kDa PCPE protein in Iox-7 cells was markedly lower than that in Rat2 cells (Fig. 6B, Lanes 1 and 2). In contrast, Gre-i and Gre-2
cells derived
from
lox-7
cells showed
a high level of PCPE
protein expression (Fig. 6B, Lanes 3 and 4). Compared with the parental Rat2 cells, Iox-7 cells showed altered
growth
properties.
Rat2 cells exhibited
flat morphol-
ogy and stopped proliferating when the culture reached confluence (Fig. 7A). On the other hand, lox-7 cells grew to a higher density, overcoming contact inhibition. The cells also exhibited spindle-shaped or round morphology. As a result, areas of clustered cells resembling transformed cell foci were generated (Fig. 7B). Cre-1 and Cre-2 cells derived from Iox-7 were highly susceptible to contact inhibition, showing flat morphology (Fig. 7, C and D). A soft agar colony formation assay revealed that Rat2 and lox-7-derived Gre-i and Cre-2 failed to grow in an anchorage-independent manner (Fig. 7, E, G, and H). In contrast, Iox-7 cells grew vigorously in an anchorage-independent manner, generating colonies in soft agar.
385
386
Excisable
Retroviral
Vector
and PCPE
Function
lox-7, rat PCPE cDNA was cloned in expression vector pSVZeo (lnvitrogen) and transfected into lox-7 cells. As shown in Fig. BA, Zeocinr cells obtained by plasmid transfection oxpressed a higher level of PCPE than lox-7 cells. These cells showed flat morphology and were subject to stringent contact inhibition (Fig. 8B). Anchorage-independent growth of these cells in soft agar was highly restricted as well (Fig. 8C). Transfection of lox-7 cells with the pSV-Zeo vector without the PCPE cDNA insert did not lead to similar changes in the
A (kb)
2
1
9.5
34
-
7.5.
4.4
-
growth
properties
(data
not
shown).
Discussion
2.4
-
PCPE
0 1 .35
Retroviruses are known to behave as insertion mutagens. It has previously been shown that the insertion of MMLV provirus into the RSV provirus that had been integrated in the rat
4
cell genome sion
-
was
associated
with
a loss of RSV RNA expres-
of the transformed
and reversion
(25). It has also been demonstrated that
malignant
with
the
disease
activation
induction
of cellular
vation
of tumor
suppressor
gration
(reviewed
in Refs.
oncogenes
genes
of the cells
animal models is associated
and/or
the
macti-
caused
by proviral
inte-
1 and 2). Insertion
mutagenesis
by
retroviral DNA and retrovirus-like transposable elements has been reported for other cellular genes as well (3-8, 26-33). For studying the effects of mutagenesis by proviral insertion,
0.24-
it would
#{149}NGAPDH
B
be
76
to develop
useful
which
reversion
easily.
In this
vector,
TSN-Iox,
bination
(kD)
of the study,
we constructed
by exploiting
-
35
-
system
also
has DNA
an additional
advantage
sequences
flanking
PCR used containing
stream
and the downstream
vector, Fig. 6. Effects of proviral integration into the PCPE gene. A, the expression of PCPE mRNA in Rat2 (Lane 1), lox-7 (Lane 2), and lox-7-derived Cre-1 and Cre-2 cells (Lanes 3 and 4, respectively). Total RNA (1 0 sg) extracted from the cells was fractionated by 1.0% agarose gel electrophoresis and hybridized with a DIG-labeled PCPE full-length cDNA probe (top panel) or control glyceraldehyde-3-phosphate dehydrogenase probe (bottom panel). Arrowhead, the position of the 1 .5-kb signal corresponding to PCPE mRNA. Left, the positions of size markers are indicated. B, the expression of PCPE protein detected by immunoblot analysis. Cell lysate (1 0 g) extracted from Rat2 (Lane 1), lox-7 (Lane 2), and lox-7derived Cre-1 and Cre-2 cells (Lanes 3 and 4, respectively) was fractionated by 1 0% SDS-PAGE, transferred to a PVDF membrane, and reacted with the anti-PCPE antibody (24). Arrowhead, the position of the signal for the SS-kDa form of the PCPE protein. Left, the positions of size markers are indicated.
the lox-7
Transfection
Resulted
Susceptibility
to Contact
age-independent of PCPE
Growth. could
in
tool
insertion manner.
and The
Flat
Morphology,
Inhibition, To affect
Cells
by cDNA
half
compared
Increased
examine
whether
the cell growth
that
genome
the inactivation
with proviral
insertion
insertion.
with
the TSN-lox
DNA
proviral
into
in the
original
Rat2
(38), the results
in an intron
of one of the two
cells.
case
the
same
Because
were compatible
PCPE
genes resulted in the synthesis of aberrant (6, 26). However, no transcripts of aberrant with PCPE exon sequences were detected of the
ai(l)
collagen
inactivated
insertion
the essential
had been disrupted of
sequences.
transduced
harbor
inverse the up-
gene
alleles
by
It has previously been shown that proviral the intron of the dilute and the Fas antigen
this is also an unlikely in lox-7. If a cis-acting
elevated
properties
with
Rat2 has a diploid
by disrupting
and Loss of Anchor-
integration
in previous studies (14, only the upstream or the
flanking to
proviral
solo LTR-dependent isolation of both
clones
shown
in characterizing
the
PCPE gene. The direction of transcription of the TSNlox provirus in lox-7 cells was the same as that of the PCPE gene, and the level of PCPE gene expression was lowered by
proviral
of PCPE in Iox-7
was
cell
recom-
as a useful
of the
the
Expression
Rat2
retroviral
homologous serve
with
be achieved
an excisable
it might
downstream flanking sequence, PCR enabled the simultaneous Among
Elevated
system
can
Cre-/oxP that
site. Although inverse 34-37) amplified DNA -
vector
mutation
to turn off cellular gene expression by proviral restore it by proviral excision in a controlled
234
45
a retroviral
specific
and demonstrated
the cellular
expression
phenotype
in various by retrovirus
position
gene,
it was
the transcriptional cis-acting
mechanism regulatory
element
also
indicated initiation
that
(39)
(40). However,
for PCPE gene inactivation element of the PCPE gene
by the TSN-lox would
RNA transcripts sizes hybridizing in lox-7 cells. In
have
provirus, abrogated
a solo LTR at PCPE
gene
Cell Growth
Rat2
lox-7
& Differentiation
387
lox-7lCre-2
lox-7/C re-i
Fig. 7. Growth properties of Rat2 (A and E), lox-7 (B and F), and lox-7-derived Cre-i (C and G) and Cre-2 (D and H). A-D, cells were seeded at a density of 3 x iO in a 60-mm plastic culture dish and grown for 2 weeks. E-H, 5 x 10” cells were seeded in 0.33% agar (Noble; Difco) with an underlay of 0.66% agar in a 60-mm culture dish and grown for 2 weeks. Photographs were taken with phase-contrast optics at x20 objective lens magnification.
expression. With regard to the inactivation RSV genome by MMLV insertion, it seemed of mRNA
mRNA
from
the RSV 5’ LTR was normal,
but src
was
severely
due to
elongation,
activation
cells.
by proviral
was restored cells.
It is also
excision
studies
were
showing
LTR,
(25).
for PCPE that
A
gene
PCPE
intran-
than that in the parental with
those
of the provirus,
reversion
in lox-7-derived
and
a
Elevated
Cre-2
of the enhancer element in studies indicated that the
presence
intron
transcription
Compared morphology
culture reached round-shaped
an
enhancer
(42,
with and
in
an
could
activate
gene
parental
confluence, morphology
Rat2 growing
lox-7 and
cells,
which
when
the
showed
flat
cells exhibited spindlereduced susceptibility
or to
cells,
lox-7
cells
grew
vigor-
anchorage-independent
such
phenotype. erties.
manner.
and
lox-9,
caused
possibility
gene
that
into
seemed
to
prop-
by the transfec-
lox-7,
and restriction
a similar
in growth
confirmed
flat morphology,
inhibition,
Rat2
did not show
the alteration
vector
expression,
transduc-
TSN-lox-transduced
was further
expression
that
by nonspe-
the levels of PCPE expression with
whereas
It is unlikely
or marker
as lox-S
to contact
which
led to
increased
sus-
of anchorage-
growth.
mechanism is unclear.
proteinase tion
inhibition,
agar. were
integration
associated
independent
The
properties
other
PCPE
ceptibility
contact
in soft
were
PCPE
elevated
to
to grow
there
Rather,
This of
sensitivity
of vector
because
identical
monolayer
Rat2 an
in growth
effects
properties
43).
stopped
citic
tion
cells
in
ability
be specifically
leaving
(4, 7, 25). Gre-i
Rat2
of the previous
the
changes
clones,
expression
and
lost
Iox-7
PCPE
Unlike agar
morphology they
tion,
cells,
soft
When the vector provirus was excised from lox-7, leaving a solo LTR, the resulting Gre-i and Cre-2 clones restored flat
these
was probably due to the effects the LTR (41), because previous of
in
sequences are unstable and In Gre-i and Cre-2 cells de-
compatible
phenotypic
of PCPE
processing
possible
that the deletion
led to
expression
from
or even higher
The results
or
probably
be responsible
containing the TSN-lox to rapid degradation.
rived
solo
termination,
could
in lox-7
affected,
inhibition.
ously
synthesis
mechanism
scripts subject
contact
production
inadequate similar
of the integrated that the initiation
by which PCPE PCPE activates
(24, 44), which to BMP-i
(45,
COOH-terminal
46).
has recently
involved
been
in developmental
PCP/BMP-i domain
affects type
is required of
type
cellular growth I procollagen Crevealed pattern
for
I procollagen
cleavage
to be forma-
of the
molecules
to
388
Excisable
Retroviral
Vector
and PCPE
A
Function
p
..
1
2
:.“
:i::
Co
:#{149} #{149}
0
:
OC Fig. 8. A, elevated expression of PCPE in representative zeer clone (lox-7/PCPE-6) of membrane, and reacted with the anti-PCPE lox-7/PCPE-6 cells on a solid surface. Cells
lox-7 cells transfected with PCPE expression plasmid. Cell lysates (1 0 g) extracted from lox-7 (Lane 1) and a lox-7 cells transfected with pSVZeo-PCPE (Lane 2) were fractionated by 10% SDS-PAGE, transferred to a PVDF antibody (24). Arrowhead, the position of the signal for the 55-kDa form of the PCPE protein. B, the growth of (3 x 1 0) were seeded in a 60-mm culture dish and grown for 2 weeks. C, the lack of anchorage-independent cells. Cells (5 x 10) were seeded in 0.33% agar (Noble; Difco) with an underlay of 0.66% agar in a 60-mm culture dish and grown were taken with phase-contrast optics at x20 objective lens magnification.
growth of lox-7/PCPE-6 for 2 weeks.
Photographs
generate mature extracellular collagen matrix. Previous studies have shown that a decrease of type I collagen synthesis is often
associated
(47-49),
and that
could
suppress
possible
malignant
the restoration
PCPE
cellular
its effects
transformation
I collagen
phenotype
affected
through
cellular
of type
the malignant
that
properties
with
synthesis
(SO). Therefore,
morphology
it is
and growth
on PCP/BMP-i
activities
and
extracellular matrix formation. Studies are in progress to examine this possibility. Due to their growth properties in vitro, Rati and its tk’ variant, Rat2, have often been used as representatives of normal rat fibroblasts. However, it was shown
that
these
cell
they
were
mice
(38). Although
been
elucidated,
transplanted
cells.
igenicity
to
that
unmask
the
was
CCI4-treated
lox-7,
induction
of liver
human
insights
Cre-/oxP
tumors and
retroviral
nature
to compare
of
the tumor-
in
PCPE
vivo.
in the fibrotic
livers
of
that it may play a role in the However,
the
level
of PCPE
viral
(Si
a Cre-/oxP-dependent
also
be useful
excision
obtained,
tered
growth
(53,
54). The the
some
properties
An analysis
of the
lead
identification
control
cell
useful
could
insertion
and
functions.
Cur-
with TSN-lox
clones
demonstrate
with parental site in these
integration
hazshowed
vector
of the
transduced
of additional
exploited reversible
study
on cellular
and
been
retroviral
of these
growth
of ma-
of potentially
effects
compared
proviral
bring
allowed
present
excisable
sequences
and
has which
52) or the removal
Rat2 clones
additional
being
systems,
for studying
of proviral
to the
,
may
progression.
recombination
sequences
that
cell lines
basis for the manifestation tumor
vector
transduction
products
PCPE expres-
of
its derivatives
and tumor
homologous
in various
rently,
nude
has not
malignant
made
(23).
on the molecular
ardous
and
to be elevated
cirrhosis
phenotypes
gene
rats
discrepancy
when
in malignant disease has not been studied oxStudies to examine PCPE expression levels in
various lignant
F344
a reduction
rats, suggesting
expression tensively.
vivo
potentially
and
shown
in
for this
are being
of Rat2,
tumorigenic
syngeneic
it seems
Attempts
expression
were
the reason
sion in vitro could Rat2
lines
cellular
differentiation.
are al-
Rat2 cells. clones may genes
whose
Materials Cell Culture.
and
Methods
packaging cell line, CRE (12), was grown in DMEM supplemented with 1 0% FCS. Rat2 is a tk mutant of rat fibroblast cell line Rati (55) and was grown in DMEM supplemented with 1 0% FCS. The transfection of plasmid DNA into cultured cells was carried out by the calcium phosphate precipitation method (56, 57) using a CelIPhect transfection kit (Pharmacia). For G418 selection, the transfected cells were grown in the presence of 400 j.tg/ml G41 8 for 2 weeks, until G41 & colonies were generated. For the soft agar colony formation assay, cells (5 x 10) were seeded in 0.33% agar (Noble; Difco) with an underlay of 0.66% agar in a 60-mm culture dish and grown for 2 weeks. Plasmids.
A MMLV-based
A DNA clone of the TSN-lox
retroviral vector (Fig. 1A) was
constructed as follows: plasmid pSV2gpt (58) was digested with PvuII; ligated with BamHl linkers; and digested with BamHI and HindIll to prepare the BamHl-Hindlll fragment containing the SV4O promoter. Plasmid pSV2neo (59) was digested with Smal, ligated with C/al linkers, and digested with HindlIl and C/al to prepare the Hindlll-Clal fragment contaming the neo gene. The BamHl-HindlIl SV4O promoter fragment and the HindIll-C/al nec gene fragment were ligated with the pGEM-7Zf(+) (Promega) that had been digested with BamHl and C/al, and a subclone plasmid containing the neo gene linked to the SV4O promoter was obtamed (plasmid 1). Plasmid pHSV-106 (Life Technologies, Inc.), which contains the HSV-1 tk gene, was digested with Bg/II, blunt-ended with Klenow enzyme, and ligated with Xhol linkers. The DNA was then digested with Xhol and AatlI, and the Xhol-AatII fragment containing the 5’ 90% of the tk gene was subcloned between the Xhol and AatIl sites of pGEM-
7Zf(+). The 3’ terminal pHSV-106
as a template
region of the tk gene was amplified and a pair of oligonucleotides,
GACGTCTrGGCCAAACGCCTC-3’
by PCR using S’-GCCTTG-
and 5’-CCATCGAlTCAGTrAGCCT-
CCCCCATC-3’ , as primers. The blunt-ended PCR product was cloned into the Smal site of pGEM-7Zf(+). The nucleotide sequence of the cloned fragment was confirmed to be identical to the reported tk gene sequence (60). The Xhol-Aatll fragment and the AatlI-C/al fragment, which contain the 5’ and the 3’ regions of the tk gene, respectively, were prepared from these subclone plasmids and ligated with pGEM-7Zf(+) digested with Xhol and C/al to construct plasmid 2. Plasmid p8.2, which contains a permutated form of MMLV proviral DNA (61), was digested with C/al and Aatll, and the LTR-containing C/aI-AatIl fragment was subcloned between the C/al and the AatIl sites of pGEM-7Zf(+). For construction of the /oxP-containing LTR, a pair of oligonucleotides, 5’-ATAACTTCGTATAATGTATGCTATACGAAG1TAT-3’ and S’-ATAACTTCGTATAGCATACAlTATACGAAGTTAT-3’ (Fig. 1A), were annealed with each other and ligated with this subclone digested with Smal to construct plasmid 3. Successful insertion of one copy of the loxP sequence into the Smal site of the LTR was confirmed by nucleotide sequencing. An infectious clone of MMLV, pArMLV48 (62), was digested with Nail, blunt-ended, and ligated with Xhol
Cell
linkers. The DNA was then digested with AatII and Xhol Aatll-Xhol fragment containing the 5’ region of the MMLV responding LTR-containing
to obtain the genome
cor-
PCPE were grown in the presence were isolated for analysis.
363-1035(63). ThleAatII-XhoI fragment and the fragment prepared from the subclone plasmid 3 were ligated with pGEM-7Zf(+) digested with C/al and Xhol to construct plasmid 4. The BarnHI-C/aI fragment containing the SV4O promoter and the nec gene, the Xhol-BamHl fragment containing the tk gene, the C/aI-AatII fragment containing the LTR, and the Nsll-XhoI fragment contaming the LTR and the 5’ region of the MMLV genome were prepared from plasmids 1 , 2, 3, and 4, respectively. These four fragments were mixed with pGEM-7Zf(+) digested with Ns,1 and AatlI, and ligation was carried out to construct the TSN-Iox vector plasmid. For construction of Cre expression plasmid pcDNA-Cre (Fig. 18), the Xhol-Sall fragment of pBS39 (American Type Cuiture Collection; Ref. 64) containing the Cre protein coding region was subcloned in the Ba/l site of
pUC19. The EcoRI-Sall fragment of the Gre-coding sequence was then excised from this subclone and cloned between the EcoRI and XhoI sites downstream of the cytomegaloviws promoter of pcDNA VAmp (Invitro-
gen). Vector Inoculation. Target cells were seeded at a density of 10 ceIls/60-mm dish on day 1 . On day 2, the medium was removed, and fresh medium, virus, and polybrene (final concentration, 5 .&g/ml) were added.
On day 3, the medium was replaced by fresh medium without polybrene. For G418 selection and HAT selection, medium containing G418 (400 .&g/m and 0.01 volume of bOx HATsupplement (LifeTechnologies, Inc.)
& Differentiation
of Zeocin (250 g/mQ,
and surviving
clones
to nucleotides CIaI-AatlI
Growth
For hybridization analysis of RNA, 5 g of total RNA by 1 .0% agarose gel electrophoresis, transferred to a
Analysis.
RNA
were fractionated
nylon membrane, and hybridized with a DIG-labeled probe. The hybridization signal was detected by anti-DIG antibody conjugated with alkaline phosphatase and CSPD using a DIG nonradloactive detection kit (Boehringer Mannheim). For the detection of PCPE mRNA, the PCPE full-length cDNA fragment was prepared from pSVZeo-PCPE and used as a hybridization probe.
Immunoblot containing
Analysis.
Cell lysates were prepared
using lysis buffer
50 mM Tris-HCI(pH
7.4), 150 mri NaCI, 1 % Triton X-iOO, 20 mri EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 g/mIaprotinin. Protein concentration in the lysates was determined by the method of Bradford (66) using a protein assay dye reagent (BiO-Rad). Cell lysates containing 10 Lg of protein were fractionated in 10% SDS-PAGE, transferred to a PVDF membrane, and reacted with a rabbit antibodyagainst mouse PCPE (24) provided by Efrat Kessler (Tel Aviv University, Tel Hashomer, Israel). The binding of anti-PCPE antibody was then detected by the enhanced chemiluminescence method using horseradish peroxidase-conjugated antirat IgG secondary antibody (Amersham).
Acknowledgments
We
thank Dr. Efrat Kessler for providing anti-PCPE antibodies. We Masako Kato, Marl Oyane, and Asako Shibamura for secretarial
greatly
was used, respectively, on day 3. DNA Analysis. For hybridization analysis of genomic DNA, 10 jg of high molecular weight DNA extracted from the cells were digested with a restriction enzyme, fractionated by 1 .0% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with a P-labeled probe. Amplification ofthe DNA sequences by PCR was carried out using
alsothank work.
a GeneAmp
strategy to identify cancer genes. Biochim. Biophys. Acts, 1287: 29-57,
Perldn-Elmer
Corp.)according to the manufacturer’s manual. Cellular genemic DNA (1 g) mixed with specific primers was amplified through 25 cycles of PCR, with each cycle consisting of danaturation at 94#{176}C for 1 mm, annealing at 50#{176}C for 1 mm, and elongation at 72#{176}C for 4 mm. For inverse PCR, genomic DNA was digested with Taql or Tsp5O9l and circularized by treatment with T4 DNA ligase. Amplification was then carried out by using LATaq polymerase (Takara Shuzo Co., Ltd.) through 30 cycles of PCR, with each cycle consisting of98#{176}Cfor 20 s and 68#{176}C for 5 mm. DNA sequencing was carried out by the dye terminator cycle sequencing method using the ABI PRISM cycle sequencing kit (Perkin-Elmer Corp.) and automated DNA sequencer 373A (Applied Biosystems). The GenBank accession numbers ofthe sequences in Fig. 6 are AF016504, AF016505, and AF016506. The GenBank accession number of the rat PCPE cDNA sequence in Fig. 7 is AF016503. Cloning and Expression of PCPE cDNA. Total RNA was extracted PCR
kit
from the cells by acid guanidinium thiocyanate-phenol chloroform extraction (65) by using an RNAzoI B kit (TEL-TEST). cDNA clones of the rat PCPE gene mRNA were Isolated from total RNA by the RACE method (22) using 3’ and 5’ RACE kits (Ufe Technologies, Inc.). For 3’ RACE, cDNA was synthesized with SuperScript II reverse transcnptase using the ohgonucleotide as a primer.
5’-GGCCACGCGTCGACTAGTACI I I I I 1 I I I I I I I I I I -3’ Thirty cycles of PCR were then carried out using a pair of ohigonucleotide primers, 5’-GGGGATGTGACCGGGGAGTC-3’ and 5’GGCCACGCGTCGACTAGTAC-3’. For 5’ RACE, cDNA was synthesized
using 5’-CACAGMGCGTCCMGTCGCTGGCC-3’
as GSP1 After RNase .
tail was added to the cDNA with terminal dedCTP. Subsequently, 30 cycles of PCR of the dCfelled cDNAwas carried out using an anchorprimer, 5’-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3’ (I = inosine), and GSP1. Then, the second round of PCR (30 cycles)was carried out using a universal primer, treatment,
oxytransferase
a poly(dC) and
5’-GGCCACGCGTCGACTAGTAC-3’, and GSP2, 5’-CCCGGATCCAGCAAAGACCTCCAGAGC-3’. Each cycle of PCR amplifications used for RACE consisted of denaturation at 94#{176}C for 30 s, annealing at 50#{176}C for 30 s, and elongation at 72#{176}C for 1 mm. Amplified DNA products were cloned into the Smal site of pUC19 for sequencing. For construction of a plasmid vector expressing rat PCPE cDNA, clones obtained by RACE were inserted in pSV-Zeo (Invitrogen)so that the full-length cDNA containing the entire protein-coding region was placed under the regulation of the 5V40 promoter. The construct was designated as pSVZeo-PCPE Cells (3 x 10) transfected with 10 g of pSVZeo-
References 1.
Jonkers,
J.,
and
Bems,
A. Retroviral
insertional
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