CELLULAR BINDING PROTEIN HAVING AFFINITY FOR THE REGULATORY REGION OF THE HUMAN PAPILLOMA VIRUS TYPE 18

PROTEINE LIANTE D'ORIGINE CELLULAIRE PRESENTANT UNE AFFINITE POUR LA REGION REGULATRICE DU PAPILLOMAVIRUS HUMAIN DE TYPE 18

English Abstract

- 1 -
HOE 90/B 016
Abstract
Cellular binding protein having affinity for the regula-
tory region of the human papilloma virus type 18
The invention relates to a DNA sequence which codes for
the cellular DNA-binding protein RS1, which specifically
binds to the 28 bp E2 subregion of the upstream regula-
tory region (URR) of the human papilloma virus (HPV), and
to the protein RS1 and proteins which are similar to RS1.

Claims

- 15 - HOE 90/B 016
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A DNA sequence
<IMG>

- 16 -
<IMG>
which codes for the cellular DNA-binding protein RS1 or
a polypeptide which is similar to and has the biological
activity of RS1.
2. A DNA sequence which is derived from the genome of
primary human keratinocytes and which codes for the
cellular binding protein RS1 or a polypeptide which is
similar to and has the biological activity of RS1.
3. A DNA sequence which hybridizes with a DNA sequence
as claimed in claims 1 and 2, which is of natural, semi-
synthetic or synthetic origin, which is related to a DNA
sequence as claimed in claim 1 or 2 by mutation such as
nucleotide substitution, nucleotide deletion, nucleotide
insertion or inversion of nucleotide sections, and which
codes for a polypeptide which is similar to and has the
biological activity of RS1.

- 17 -
4. A recombinant DNA molecule for cloning, wherein the
DNA sequence as claimed in one of claims l to 3 is
selected.
5. A recombinant DNA molecule as claimed in claim 4,
wherein the DNA sequence is operatively connected to an
expression control sequence.
6. A recombinant DNA molecule as claimed in claim 5,
wherein the expression control sequence is selected from
an E.coli promoter system, the E.coli lac system, the
E.coli .beta.-lactamase system, the E.coli trp system, the
E.coli lipoprotein promoter, a yeast expression control
sequence or another eukaryotic expression control se-
quence.
7. A host organism which contains a DNA sequence coding
for the DNA-binding protein RS1.
8. A host organism as claimed in claim 7, which is
transformed with at least one of the recombinant DNA
molecules claimed in one of claims 4 to 6.
9. A host organism as claimed in claim 8, which is
selected from the group comprising E.coli, a different
bacterium, yeast, a different fungus, an animal or human
cell.
10. A host organism as claimed in claim 9, which is
lambda gt 11.
11. The DNA-binding protein RS1 having the following
amino acid sequences
<IMG>

- 18 -
<IMG>

- 19 -
12. A DNA-binding protein with the property of binding
specifically to the 28 bp E2 subregion of the upstream
regulatory region of the human papilloma virus HPV 18,
which subregion has the base sequence
ACCGAAAACGGTCGGGACCGAAAACGGT.
13. A DNA-binding protein having the biological activity
of RS1, which is encoded by a DNA sequence as claimed in
one of claims 1 to 3.
14. A DNA-binding protein which is prepared with the aid
of the recombinant DNA technology using a host organism
of claims 7 to 10.
15. A process for preparing RS1 or polypeptides which
are similar to and have the biological activity of RS1,
which comprises transforming a host organism with a
recombinant DNA molecule as claimed in claim 5 or 6,
cultivating the transformed organism and isolating the
RS1 or the polypeptide which is similar to RS1 after
expression.
16. A polypeptide having the biological activity of RS1,
which can be obtained by a process as claimed in claim
15.
17. A pharmaceutical composition containing the polypep-
tide RS1 or polypeptides which are similar to and have
the biological activity of RS1, as claimed in claims 11
to 14, or 16, as an agent in the treatment of cancer or
tumors.

- 20 -
18. The DNA sequence as claimed in claim 1 and
substantially as described herein.

Description

2 [);39359
BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 016 - Ma 825
ANR: 1 000 764 Dr. Lp/Wr
Description
Cellular binding protein havin~ affinity for the reoula-
tory re~ion of the human papilloma yirus tyPe 18
The invention relates to DNA sequences which code for a
DNA-binding protein (RSl), and to a novel DNA-binding
protein (RSl) which was isolated from primary human
keratinocytes with the aid of the recombinant DNA techni-
que.
The invention further relates to recombinant DNA mole-
cules for use in the preparation of RSl protein or
proteins which are similar to and have the biological
activity of RS1, and to host organisms such as bacteria,
yeasts and mammalian cells which have been transformed
with these recombinant DNA molecules.
The invention finally relates to pharmaceutical composi-
tions which contain the RSl protein or proteins which are
similar to and have the activity of RS1.
Papilloma viruses are small double-stranded DNA viruses
and are responsible for benign lesions of the skin and
the mucosal 0pithelium. However, in addition to this a
connection is also seen between the occurrence of certain
types of human papilloma viruses (HPV) and anogenital
cancer (The Papovaviridae, Vol. 2, Plenum Publishing
Corp., New York, pages 245-263). An important indication
of the connection between the development of cancer and
the presence of HPV is that in more than 90% of cervical
tumors HPV DNA sequences were found after biopsy. Most of
these tumors contain HPV type 16 or HPV type 18 DNA. HPV
18 DNA sequences have also been discovered in a number of
cells from cell lines which were obtained from cervical
tumors. Both in cervical tumors and in cell lines deriv~d

-- 2 --
X0393S~3
from such tumors the viral DNA is normally integrated in
the host genome. It is also known that the viral tran-
scription takes place in such cells. The examination of
established tumor cell lines suggested that the HPV
sequences are the sequences responsible for the develop-
ment of the cancer. There are also strong indications
that the expression of the early open reading frames
(ORF) of the E6 and E7 genes of the HPV types 1~ and 18,
which code for the transforming activity, play a decisive
role in initiating and maintaining the transformed cells
(Cancer Res. 48: 3780-3786).
Regulatory transcription elements are located in the 80-
called URR region (upstream regulatory region) of the HPV
genome which has also been termed the noncoding region or
the long-control region. This region extends over 825
base pairs from the end of the late open reading frame
(ORF Ll) to the start of the early active gene E6 in the
HPV 18 DNA. The ~PV 18 URR region contains a promoter
which is active in several tumor lines and has at least
3 different enhancer regions (J. Virol. 62:665-672,
1988).
The object of the present invention follows from the
following points:
The regulation of viral genes by cellular proteins plays
a key role in tumor development. The deregulation of the
expression of the transformed early genes E6 and E7 of
HPV 16 and 18 contributes to the process of cancer
development in the transformed cells. In this connection
the identification, the isolation and preparation of
proteins which have a binding affinity for the regulatory
region of these early genes are a decisive step in
combating the abovementioned tumors. The proteins binding
the deregulatory regions therefore possibly represent
suitable means for the preparation of an agent for
combating cancer.

-- 3 --
2039359
The present invention therefore had the ob~ect of iden-
tifying factors which can influence the regulation and
expression of viral DNA in infected human cells, and of
providing these in amounts sufficient to be able to use
them as agents against carcinoses.
This object has been achieved according to the invention
on the one hand by providing a DNA sequence, such as the
one depicted in Figure 8 and claim 1, which codes for the
cellular DNA-binding protein RSl or a polypeptide which
is similar to and has the biological activity of RSl.
On the other hand, a DNA-binding protein (RSl) having the
amino acid sequence shown in Figure 8 and claim 11, and
the property of binding specifically to the 28 bp E2
region of the HPV 18 upstream regulatory region is
provided in order to achieve the ob~ect.
The invention furthermore comprises DNA sequences which
are derived from the genome of primary human
keratinocytes and code for the cellular DNA-binding
protein RS1.
In addition, the invention comprises DNA sequences which
hybridize with one of the abovementioned DNA sequences.
These DNA sequences can be of natural, semisynthetic or
synthetic origin and are related to one of the above-
mentioned DNA sequences by mutation such as nucleotide
substitution, nucleotide deletion, nucleotide insertion
or inversion of nucleotide sections. Furthermore, these
last-mentioned DNA sequences characteristically code for
a polypeptide which is similar to and has the biological
activity of RSl.
One biological activity of RSl i8, for example, the
specific affinity (high affinity binding) of RSl for the
E2 subregion with a length of 28 bp of the upstream
regulatory region of HPV types 16 and 18. Proteins
similar to RS1 are proteins which, like the protein RSl,

2~ 3359
have an affinity for the DNA E2 subregion of the URR of
the HPV types 16-18.
The invention further comprises recombinant DNA molecules
for cloning in vectors, the DNA sequence being selected
from the group of the abovemsntioned DNA sequences which
code for the cellular DNA-binding protein RSl or proteins
which are similar to RSl.
These recombinant DNA molecules are operatively connected
to an expression control sequence.
Recombinant DNA molecules with an expression control
sequence selected from an E.coli promoter system, such as
the E.coli lac system, the E.coli ~-lactamase system, the
E.coli trp system, the E.coli lipoprotein promoter, or
with a yeast expression control sequence or another
eukaryotic expression control sequence are preferred.
The invention furthermore comprises host organisms which
are transformed with at least one of these recombinant
DNA molecules.
Preferably host organisms selected from the following
group are used: E.coli, a different bacterium, yeast, a
different fungus, an animal or human cell.
Particular preference is given to the use of a recom-
binant lambda gt 11. The DNA-binding protein RSl
according to the invention has the property of specifi-
cally binding to the 28 bp E2 region of the URR of HPVtype 18 and 16. DNA-binding proteins according to the
invention also comprise proteins having the biological
properties of RSl, ie. all proteins which are similar to
RSl and likewise have the property of binding to the E2
region of the URR of HPV type 18 and 16, or else they
have the action of RS1, namely of influencing the HPV by
specific interaction with the E2 region of the URR or
overlapping regions, such as regulating the early genes

5 - '~03935~
E6 and E7.
The proteins according to the invention are encoded by
one of the abovementioned DNA sequences according to the
invention and are produced with the aid of the recom-
binant DNA technology in a manner known per se using ahost organism according to the invention.
In these processes for preparing RSl or polypeptides
which are similar to and have the biological activity of
RSl, a host organism according to the invention is
transformed with a recombinant DNA molecule according to
the invention. The transformed organism is cultivated,
and the RS1 or the pol~peptide which is similar to RSl is
isolated from the culture medium after expression.
Proteins which can be obtained by these processes are
also proteins according to the invention.
Because of the biological activity of the proteins
according to the invention which make it possible to
influence the requlation of cells transformed with HPV
with respect to a viral expression, these proteins are
suitable for the treatment of cancer.
The present invention therefore also comprises pharmaceu-
tical compositions which contain the protein RSl or
polypeptides which are similar to RSl. These agents can
be employed in the treatment of cancer or tumors.
In the attempt to identify and isolate URR-binding
proteins, a ~trategy was used which makes it po~sible to
i~olate the factors encoded by complementary DNA (cDNAs)
on the basis of their property of sequence-specific
binding to DNA fragments. This method is based on a high
expression rate of the DNA-binding region encoding the
DNA-binding factor in bacteriophage lambda gt 11. The
interaction of this factor with double-stranded, radio-
actively labeled DNA leads to a recognition signal. The

- 6 - ~ ~39359
corresponding recombinant in the lambda gt 11 expression
library can then be isolated.
Brief descri~tion of the fi~ures:
Fig. 1:
Diagrammatic representation of the regulatory region URR
(upstream regulatory region) with human papilloma virus
HPV 18. CAAT box and TATA box are indicated by an ellipse
or by a rectangle, respectively. The E2 6ubregion which
contains the two palindromic repeats is shown underneath.
L0 The palindromic repeats are indicated by arrows. They are
the recognition region for the viral E2 proteins. The
core sequence which is common to all ~he nucleotides
(Fiq. 2) and i8 bound by the DNA-binding protein RSl has
been boxed in.
Fig. 2:
Oligonucleotides which were used in this invention:
oligonucleotides A1 to A3 contain sequences which are
derived from the E2 subregion described in Figure 1. ~hey
have a core sequence with a length of 28 base pairs
(boxed in and doubled in A3) in common.
Oligonucleotides ~ to F and G are monomers or tetramers
which are derived from other regions of the URR of the
HPV 18 or HPV 16.
Fig. 3:
Sequence of the RSl cDNA and the RSl protein. The amino
acid sequence is specified using the 3 letter code.
Isolation of the cDNA clone cRS1.
In order to isolate the cellul~r DNA-binding protein RS1
which binds to the regulatory region URR of HPV 18

- 7 - ~0393~9
(Fig. l), a lambda gt 11 expression library which con-
tains cDNA of primary epidermal keratinocytes, which are
the natural host cells of HPV, was tested. In this test,
the method originally applied by Singh et al., Cell
52:415-423, 1988 and modified by Vinson et al., (Genes
Dev. 1988 2:801-806) was used. The probe used to find
binding proteins was the oligonucleotide A3 (Fig. 2).
This oligonucleotide contains a duplication of 2 tandem
repeats which include the recognition region for the
viral E2 transactivator/ transrepressor (Nature 325:70-
73, 198~). In a first approach 500,000 pfu of the lambda
gt ll expression library were tested. Poly-dIdC, which
does not hold any biological information, was used to
prevent nonspecific binding. In a further approach, a
further 500,000 pfu were tested, denatured and sonicated
calf thymus DNA being used in this approach to prevent
nonspecific binding. Both rounds of screening led to only
one positive signal which, additionally, occurred at the
same site on the original and the filter copy. Restric-
tion analysis and partial sequencing of the cDNA frag-
ments (inserts) showed that both clones are identical and
probably are copies of the same phage. The phage which
was identified (referred to as RS1 phage) wa~ purified
in 4 round~ of plating and then analyzed in detail.
S~ecificity of binding:
The binding specificity was analyzed with the aid of
filter binding tests, 3 different oligonucleotide~ (A1,
A2 and A3) all of which contained the E2 subregion with
a length of 28 base pairs being used. As comparison
several nucleotides which are not related to the E2
subregion but are derived from the regulatory region URR
of HPV 18 and HPV 16 were used as negative controls (Fig.
2). Concentrated samples of phage (5 x 107 pfu) were
sprayed onto freshly poured E.coli Y lO90 lawn. The DNA
binding properties of RS1 and of a product of a negative
control clone which was selected from the lambda gt 11
expression library at random were analyzed with the aid

-- 8 --
2039359
of filter binding tests. In all cases, the product of the
negative control clone did not interact with any of the
tested oligonucleotides. In contrast to this, RSl bound
specifically to the oligonucleotides A1, A2 and A3. An
interaction between RSl and other control oligonucleo-
tides was not observed.
It was also found that RS1 has a high and specific
binding activity to single-stranded DNA, namely to the
noncoding (anti-sense) strand (Fig. 2) of the oligonucle-
otides Al, A2 and ~3. An affinity of RSl to the coding(sense) strand of the oligonucleotides A1, A2 and A3 or
to another single strand of the control oligonucleotides
was not observed. The negative control phage exhibited no
factor which has an activity toward single-stranded DNA.
RSl also binds to DNA at high salt concentration~ (400 mM
NaCl), which indicates a high binding specificity.
Even under high salt concentrations, exclusive binding of
RSl to oligonucleotides which contained the core sequence
with a length of 28 base pairs was observed. No
interaction with other double-stranded or single-stranded
DNA fragments was observed.
Analysis of the ~-galactosidase fusion protein encoded by
the RS1 phage:
Lysogenic RS1 phages and negative control phages were
isolated and induced to produce large amounts of their
corresponding ~-galactosidase fusion protein. Western
blot proteins of induced and noninduced lysogenic phages
were treated with anti-~-galactosidase mouse antibodies
and then coated with an anti-mouse antibody, which was
covalently bonded to alkaline phosphatase. IPTG induced
a fusion protein of approximately 150 R in the lysogenic
RSl phage.
The bindi~g properties of the ~-gal-RSl fusion protein

2C)393~;~
were tested with the aid of South-Western analysis of the
entire protein extract of induced and noninduced lysoge-
nic cultures. After the transfer, the immobilized
proteins were denatured with 7 ~ guanidine hydrochloride
for 60 min and then renatured for 24 h. Science (1988)
241:577-580. The denaturing/renaturing cycle improved the
recognition ~ignals of the proteins in the South-Western
analysis and the total protein of induced lysogenic
phages of a negative control and RSl was analyzed with
the aid of the radioactively labeled oligonucleotide A3b
probe. A single band which represents a protein with a
molecular mass of approximately 150 R was specifically
detected in the lysogenic phage RSl. The specificity of
DNA binding by this 150 K fusion protein of the South-
Western analysis additionally confirmed the binding
specificity of RSl as demonstrated in the filter binding
tests. The 150 K protein specifically binds to the
oligonucleotides Al, A2 and A3 (Fig. 2) and to their
corresponding noncoding (anti-sense) strands, and does
not bind to the control oligonucleotides.
Structure of the cDNA present in phage RSl:
The phage RSl contains a cDNA fragment with a length of
1338 nucleotides. One of the two strands starts at its
5'-end with an open reading frame tORF) of 906 nucleo-
tides followed by a 3'-nontranslated region of 432
nucleotides. The ORF with a length of 906 nucleotides,
which corresponds to 302 amino acids, is bonded in frame
to the ~-galactosidase gene at the 3'-end. The ~-galac-
tosidase-RSl fusion protein has a molecular mass of
approximately 150 K. The ~-galactosidase portion of this
fusion protein has a molecular mass of approximately 120
kD so that the cDNA-encoded portion of the fusion protein
must therefore have a molecular mass of approximately 30
kD.
These results are consistent with the molecular weight of
33.6 ~, which can be calculated for an oligopeptide with

- lo - ~0393S9
302 amino acids, which are encoded by the ORF, with a
length of 906 nucleotides, of the RSl phage cDNA. In the
ORF of 906 nucleotides a potential translation-initiation
codon i8 present at base 192. However, the surrounding
bases do not follow the Kozak consensus sequence for
eukaryotic translation/initiation sites (Cell 44: 283-
292). For this reason it may be possible that the AUG
codon in position 192 is not the original
translation/initiation codon for the RS1 gene. Two
polyadenylation consensus sequences (AATAAA) are present
in the 3~-region of the RSl cDNA and start at nucleotides
1167 and 1221 respectively. However, they are not fol-
lowed by a poly(A) tail.
Comparison of the RS1 cDNA sequence or the protein
sequence deduced therefrom with several sequence data
banks (Gene bank, Swiss Prot, Dayhoff) showed no sig-
nificant nucleotide or amino acid sequence homology of
RSl with known proteins. The amino acid sequence of RS1
shows several interesting characteristics. The protein is
predominantly rich in arginine (34 of 302 amino acid6)
and proline (35 of 302 amino acids). Proline is in part
present in the form of clusters (e.g. amino acids 278-
296). As is known, these proline clusters participate in
the activation of transcription by several transcription
factors. A region of 51 amino acids (33-84) i8 rich in
proline (20%), glutamic acid (10%), serine (10%) and
threonine (18%). Analogous regions are present in the
amino acid sequence of a group of proteins with a very
short in vivo half life ("PEST" proteins). Residues 10 to
51 have a high content of the basic amino acid arginine
(20%). RS1 also does not contain a region containing zinc
fingers, homeoboxes or leucine zippers, which repre~ent
domains, such as are present in several other DNA-binding
factors (Science 1989, 245: 371-378).

11 21~39359
Example 1
A cDNA expression library
A cDNA library of human epidermal keratinocytes, which is
cloned in the expression vector lambda gt 11, was ob-
tained from Clontech Laboratories Inc. The librarycontained approximately 1.7 x 106 independent recombinant
clones with an averaqe fragment (insert) size of 1.1 kb.
The lambda gt 11 recombinants were screened with the aid
of the host organism E.coli Y 1090. Lysogenic phages were
generated in E.coli Y 1089 or Y 1090 (in: DNA-Cloning -
A Practical Approach, Vol. 1, D.M. Glover ed. (Oxford:
IRL Press), 49-78).
Example 2
DNA probes and ~est for DNA-binding proteins
Oligonucleotides were prepared with an Applied Biosystems
380A synthesizer with the aid of the standard phosphori-
mide technique. The products were purified by means of
gel electrophoresis. Subsequently they were labeled with
(32p) dATP with the aid of polynucleotide kinase (supplied
by Boehringer Mannheim) under standard conditions.
5Onicated calf thymus DNA (supplied by Pharmacia) and
poly-dIdC (supplied by Pharmacia) were u~ed to avoid non-
specific binding.
The test for DNA-binding proteins was carried out in the
following manner:
Lambda gt 11 recombinants were distributed in a Nunc
bioassay dish in 0.7% agarose at a maximum of
1.5 x 105pfu/600 cm3. Nitrocellulose ~ilters (PA 85,
~chleicher and Schuell~ saturated with 10 ~M isopropyl-
~-D-thioglucopyranoside were, as a covering layer, placed
on top at 42C for 3 h. After incubating at 37C for a

- 12 - 2~39359
further 6 hours, the filters were removed and another
filter was placed on top at 37C for 3 h. The filters
were dried at room temperature for 10 min and then
incubated in buffer A (50 mM NaCl, 0.5 mM dithiothreitol
(DTT), 25 mM HEPES (pH 7.9) and 6 M guanidine hydro-
chloride). After slight shaking for 10 min, the solution
was replaced with the same buffer. After a further 10
min, the solution was diluted in four 5 min steps (Genes
Dev.: 1988 2:801-806) with the same volume of buffer B
(50 mM NaCl, 0.5 mM DDT and 25 mM HEPES (pH 7.9)).
Finally the filters were washed twice for 5 minutes with
buffer B and then incubated at 4C for 30 min in buffer
C (5~D Carnation nonfat dry milk, 50 mM NaCl, 0.5 mM DTT
and 25 mM HEPES (pH 7~9)). The solution was replaced by
buffer D (0.25% Carnation nonfat dry milk, 50 mM NaCl,
0.5 mM DTT and 25 mM HEP~S (ph 7.9)) and incubated for 1
min. The actual binding reaction was carried out in
buffer D which additionally contained 3 x 106 cpm/ml
(1 x 108 cpm/~g) labeled double-stranded oligonucleotide
and 10 ~g/ml nonspecific DNA (denatured sonicated calf
thymus DNA, average length: 3000 bp, or poly-dIdC,
average length: 8260 bp). After 60 min at 4C, the
filters were washed 3 times for 5 min with buffer D,
dried with the aid of 3 MN paper, and then a Rodak x-omat
AR film was exposed to the filters overnight at -70C
with the aid of an intensifying diaphragm.
Example 3:
Analysis of the ~-galactosidase fusion protein
Lysogenic phages were prepared by the method of Huynh
(DNA Cloning - A Practical Approach, Vol. 1, Oxford: IRL
Press, 49-78). The protein extracts of lysogenic phages
were prepared (Cell 1988 52:415-423) and stored at 70DC
in aliquots. A Western blot analysis was carried out with
the aid of an anti-~-galactosidase mouse monoclonal
antibody (supplied by Promega) and a phosphatase-based

- 13 - ~ ~39~5~
protoblot Western blot AP ~ystem tPromega) under the
conditions recommended by the supplier. In order to carry
out South~Western analyses, the protein extracts (50 ~g
of crude extract) were boil~d for 5 min in sample buffer
(Proc. Natl. Acad. Sci. USA 82: 6741-6744) and then
applied to a 10~ SDS PAGE gel. After the electrophoresi~,
the proteins were transferred onto nitrocellulose with
the aid of electroblotting (semi-dry system, model SDl,
CTI GmbH). The filters were then treated as described in
Science 19~8 241:577-580.
Example 4:
Sequencing:
The fragments (inserts) of the recombinant phages were
prepared with the aid of the lambda phage adsorption
system (Promega). The inserts were subcloned in M13mpl8
and then sequenced with the aid of the dideoxy method
(SEQUENASE; United States Biochemical Inc.). Both strands
were sequenced completely.
Example 5:
Northern and Southern blot hybridization:
DNA was isolated from HeLa, CG13 (tumorigenic HeLa-
fibroblast hybrids (Science 1982 215:252-259)), 444
(nontumorigenic HeLa-fibroblast hybrids), primary human
fibroblasts, SV 80 (SV 40-transformed fibroblafits), HaCat
(spontaneously immortalized keratinocytes (Journal of
Cell Biology, 1988, 106:761-771)), Caski (cervical
carcinoma), SW 480 (colon carcinoma), Wilms, Hep G2
(hepatoma) and human TR (osteosarcoma) cell lines, namely
by the method which is described in Molecular Cloning: A
Laboratory Manual: Cold Spring Harbor Laboratory. Res-
triction digests were fractionated on a 1% agarose gel
and then transferred onto GeneScreen Plus membranes (Du
Pont). The filters were prehybridized and hybridized with

- 14 - ~ ~3935
the 32P-labeled RS1 cDNA under stringent conditions.
Cytoplasmatic RNA was isolated by the method described in
Cancer Research 1988, 48: 3780-3786. Northern blot
analysis was carried out with approximately 10 ~g of
cytoplasmatic RNA which had been fractionated on a 1
agarose gel. The gel-running buffer was composed of 0.2
M morpholinopropanesulfonic acid pH 7.0; 50 mM ~odium
acetate and 1 mM EDTA pH 8Ø The fractionated cytoplas-
matic RNA was then transferred onto GeneScreen Plus
filters (Du Pont). The filters were hybridized and washed
under stringent conditions with randomly primed 32p_
labeled probes in a manner Xnown per se.
Example 6:
Filter binding test:
Concentrated (1 x 101 pfu/ml) phage solution of plaque
lysates was sprayed directly onto freshly poured E.coli
Y 1090 host cell lawn. This culture was incubated at 42C
for 2 hours and then covered with nitrocellulose filters
which were saturated with 10 ~M IPTG. The incubation was
then continued at 37C for 2 h. Subsequently, the filters
were collected and treated as described in Example 2.