Note: Descriptions are shown in the official language in which they were submitted.
~ 7=
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BRE~ST Sr~l~C G~S AJ~D PRO~l~S
This invention relates to newly identified
polynucleotides, polypeptides encoded by such
polynucleotides, and the use of such polynucleotides and
polypeptides for detecting disorders of the breast,
particularly the presence of breast cancer and breast cancer
metastases. The present invention further relates to
;nh;b;ting the production and function of the polypeptides o~
the present invention. The twenty breast specific genes of
the present invention are sometimes hereina~ter referred to
as "BSG1~, "BSG2" etc.
The m~mm~ry gland is subject to a variety of disorders
that should be readily detectable. Detection may be
accomplished by inspection which usually consists o~
palpation. Un~ortunately, so few periodic self ~ m; n~tions
are made that many breast masses are discovered only by
acci~ent~l palpation. Aspiration o~ suspected cysts with a
fine-gauge needle is another fairly rQmmon diagnostic
practice. Md,.-.oy.d~hy or xeroradiography (so$t-tissue x-ray)
of the breast of yet another. A biopsy of a lesion or
suspected area is an extreme method of diagnostic test.
There are many types of tumors and cysts which affect
the m~mm~ry gland. Fibro~nom~s is the most commo~ benign
breast tumor. As a pathological entity, it ranks third
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h~h; n~ cystic disease and carcinoma, respectively. These
tumors are seen most fre~uently in young people and are
usually readily recognized because they feel encapsulated.
Fibrocystic disease, a benign condition, is the most ro~on
disease of the ~emale breast, occurring in about 20% of pre-
menopausal women. Lipomas of the breast are also co~ ~n and
they are benign in nature. Carr; n~- of the breast is the
most c~ malignant condition among women and carries with
it the highest fatality rate o~ all cancers affecting this
sex. At some during her li~e, one of every 15 women in the
USA will develop cancer of the breast. Its reported Ann~
incidence is 70 per 100,000 females in the population in
1947, rising to 72.5 in 1969 for whites, and rising ~rom 47.8
to 60.1 for blacks. The ~nnll~l mortality rate from 1930 to
the present has l ; n~ fairly constant, at d~lo~imately 23
per 100,000 female population. Breast cancer is rare in men,
but when it does occur, it usually not recognized until late,
and thus the results of treatment are poor. In women,
carcinoma of the breast is rarely seen before age 30 and the
incidence rises rapidly after menopause. For this reason,
post-menopausal breast masses should be considered cancer
until proved otherwise.
In accordance with an aspect of the present invention,
there are provided nucleic acid probes comprising nucleic
acid molecules of sufficient l-ength to specifically hybridize
to the RNA transcribed from the human breast specific genes
of the present invention or to DNA corresponding to such RNA.
In accordance with another aspect of the present
invention there is provided a method of and products for
diagnosing breast cancer formation and breast cancer
metastases by detecting the presence of RNA transcribed from
the human breast specific genes of the present invention or
DNA corresponding to such RNA in a sample d~rived ~rom a
host.
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In accordance with yet another aspect of the present
invention, there is provided a method of and products for
diagnosing breast cancer formation and breast cancer
metastases by detecting an altered level of a polypeptide
correspo~; ng to the breast specific genes of the present
invention in a sample derived from a host, whereby an
elevated level of the polypeptide indicates a breast cancer
diagnosis.
In accordance with another aspect of the present
invention, there are provided isolated polynucleotides
encoding hllm~n breast specific polypeptides, including mRNAs,
DNAs, cDNAs, genomic DNAs, as well as antisense analogs and
biologically active and diagnostically or therapeutically
useful fragments thereof.
In accordance with still another aspect of the present
invention there are provided human breast specific genes
which include polynucleotides as set forth in the sequence
listing.
In accordance with a further aspect of the present
invention, there are provided novel polypeptides encoded by
the polynucleotides, as well as biologically active and
diagnostically or therapeutically useful fragments, analogs
and derivatives thereof.
In accordance with yet a further aspect of the present
invention, there is provided- a process for producing such
polypeptides by recomhin~nt techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells,
containing a polynucleotide of the present invention, under
conditions promoting expression of said proteins and
subsequent recovery of said proteins.
In accordance with yet a further aspect of the present
invention, there are provided antibodies specific to such
polypeptides, which may be employed to detect breast cancer
cells or breast cancer metastasis.
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In accordance with another aspect o~ the present
invention, there are provided processes ~or using one or more
o~ the polypeptides o~ the present invention to treat breast
cancer and ~or using the polypeptides to screen ~or compounds
which interact with the polypeptides, ~or example, compounds
which ; nh; h; t or activate the polypeptides o~ the present
invention.
In accordance with yet another aspect o~ the present
invention, there is provided a ~creen for detecting compounds
which ~ nh; h; t activation o~ one or more o~ the
polynucleotides and/or polypeptides of the present invention
which may be used to therapeutically, for example, in the
treatment of breast cancer.
In accordance with yet a ~urther aspect o~ the present
invention, there are provided processes ~or utilizing such
polypeptides, or polynucleotides encoding such polypeptides,
~or in vitro purposes related to scienti~ic research,
synthesis o~ DNA and manu~acture o~ DNA vectors.
These and other aspects o~ the present invention should
be apparent to those skilled in the art ~rom the teAch;ngs
herein.
The ~ollowing drawings are illustrative o~ embo~;m~nts
o~ the invention and are not meant to limit the scope o~ the
invention as encompassed by the cl ~;mc,
Figure 1 is a ~ull length cDNA sequence o~ breast
speci~ic gene 1 o~ the present invention.
Figure 2 is a partial CDNA sequence and the
corresponding deduced amino acid sequence o~ breast speci~ic
gene 2 of the present invention.
Figure 3 is a partial cDNA sequence and deduced amino
acid sequence o~ breast speci$ic gene 3 of the invention.
Figure 4 is a partial CDNA seguence and the
corresponding deduced amino acid sequence o~ breast speci~ic
gene 4 o~ the present invention.
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Figure 5 is a partial cDNA sequence of breast specif ic
gene 5 of the present invention.
Figure 6 is a partial cDNA and deduced amino acid
sequence of breast specif ic gene 6 of the present invention .
Figure 7 is a partial cDNA sequence of breast specif ic
gene 7 of the present invention.
Figure 8 is a partial CDNA sequence of breast specif ic
gene 8 of the present invention.
Figure 9 is a partial CDNA sequence of breast specif ic
gene 9 of the present invention.
Figure 10 is a partial CDNA sequence of breast specific
gene 10 of the present invention.
Figure 11 is a partial CDNA se~uence of breast specific
gene 11 of the present invention.
Figure 12 is a partial cDNA sequence of breast specif ic
gene 12 of the present invention.
Figure 13 is a partial cDNA sequence of breast specif ic
gene 13 of the present invention.
Figure 14 is a partial CDNA sequence of breast specif ic
gene 14 of the present invention.
Figure 15 is a partial CDNA sequence of breast specif ic
gene 15 of the present invention.
Figure 16 is a partial CDNA sequence of breast specif ic
gene 16 of the present invention.
Figure 17 is a partial CE)NA sequence of breast specific
gene 17 of the present invention.
Figure 18 is a partial CDNA seguence of breast specif ic
gene 18 of the present invention.
Figure 19 is a partial CDNA sequence of breast specific
gene 19 of the present invention.
Figure 20 is a partial cDNA sequence of breast specific
gene 2 0 of the present invention .
The term ~breast specif ic gene " means that such gene is
primarily expressed in tissues derived from the breast, and
such genes may be expressed in cells derived f rom tissues
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other than ~rom the breast. However, the expression o~ such
genes is signi~icantly higher in tissues derived ~rom the
breast than ~rom non-breast tissues.
In accordance with one aspect o~ the present invention
there is provided a polynucleotide which ~nco~s the mature
polypeptides having the deduced amino acid sequence o~ Figure
1 (SEQ ID NO:1) and ~ragments, analogues and derivatives
thereof.
In accordance with a further aspect o~ the present
invention there is provided a polynucleotide which encodes
the same mature polypeptide as a human gene having a coding
portion which ~ont~; n~ a polynucleotide which is at least 90
identical (pre~erably at least 95% identical and most
pre~erably at least 97~ or 100% identical) to one of the
polynucleotides o~ Figures 2-20 (SEQ ID NO:2-20) , as well as
~ragments thereo~.
In accordance with still another aspect o~ the present
invention there is provided a polynucleotide which encodes
~or the same mature polypeptide as a human gene whose coding
portion includes a polynucleotide wnicn is at ieas~ 9û~
identical to (pre~erably at least 95~ identical to and most
preferably at least 97~ or 100% identical) to one o~ the
polynucleotides included in ATCC Deposit No. 97175 deposited
June 2, 1995.
In accordance with yet -another aspect o~ the present
invention, there is provided a polynucleotide probe which
hybridizes to mRNA (or the corresponding cDNA) which is
transcribed ~rom the coding portion o~ a human gene which
coding portion includes a DNA sequence which is at least 90~
identical to (pre~erably at least 95~ identical to) and most
preferably at least 97~ or 100~ identical) to one o~ the
polynucleotide sequences o~ Figures 1-20 (SEQ ID NO:1-20) .
The present invention ~urther relates to a mature
polypeptide encoded by a coding portion o~ a human gene which
coding portion includes a DNA sequence which is at lest 9û~
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identical to (preferably at least 95% identical to and more
preferably 97~ or 100~ identical to) one of the
polynucleotides of Figures 2-20 (SEQ ID N0:2-20), as well as
analogues, derivatives and fragments of such polypeptides.
The present invention also relates to one of the mature
polypeptides of Figure 1 (SEQ ID NO:1) and fragments,
analogues and derivatives of such polypeptides.
The present invention further relates to the same mature
polypeptide encoded by a human gene whose coding portion
includes DNA which is at least 90~ identical to (preferably
at least 95% identical to and more preferably at least 97~ or
100% identical to) one of the polynucleotides included in
ATCC Deposit No. 97175 deposited June 2, 1995.
In accordance with an aspect of the present invention,
there are provided isolated nucleic acids (polynucleotides)
which encode for the mature polypeptides having the ~llc~
amino acid sequence of Figure 1 (SEQ ID NO:1) or fra~ment~,
analogues or derivatives thereof.
The polynucleotides of the present invention may be in
the form of RNA or in the form of DNA, which DNA includes
cDNA, genomic DNA, and synthetic DNA. The DNA may be double-
stranded or single-stranded, and if single stranded may be
the coding strand or non-coding (anti-sense) strand. The
coding sequence which encodes the mature polypeptide may
include DNA identical to Figures 1-20 (SEQ ID NO:1-20) or
that of the deposited clone or may be a different coding
sequence which coding sequence, as a result of the r~lln~ncy
or degeneracy of the genetic code, encodes the same mature
polypeptide as the coding sequence of a gene which coding
sequence includes the DNA of Figures 1-20 (SEQ ID NO:1-20) or
the deposited cDNA.
The polynucleotide which encodes a mature polypeptide of
the present invention may include, but is not limited to:
only the coding sequence for the mature polypeptide; the
coding sequence for the mature polypeptide and additional
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coding sequence such as a leader or secretory sequence or a
proprotein sequence; the coding sequence for the mature
polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequence
5' and/or 3' of the coA~ ng sèquence for the mature
polypeptide.
Thus, the term ~polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding
sequence for the polypeptide as well as a polynucleotide
which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the
hereinabove described polynucleotides which encode fragments,
analogs and derivatives o~ a mature polypeptide of the
present invention. The variant of the polynucleotide may be
a naturally occurring allelic variant of the polynucleotide
or a non-naturally occurring variant o~ the polynucleotide.
Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as hereinabove described
as well as variants of such polynucleotides which variants
encode a fragment, derivative or analog of a polypeptide of
the invention. Such nucleotide variants include deletion
variants, substitution variants and addition or insertion
variants.
The polynucleotides of the invention may have a coding
sequence which is a naturally occurring allelic variant of
the human gene whose coding sequence includes DNA as shown in
Figures 1-20 (SEQ ID NO:1-20) or of the coding sequence of
the DNA in the deposited clone. As known in the art, an
allelic variant is an alternate ~orm of a polynucleotide
sequence which may have a substitution, deletion or addition
o~ one or more nucleotides, which does not substantially
alter the function of the encoded polypeptide.
The present invention also includes pol~nucleotides,
wherein the coding sequence for the mature polypeptide may be
fused in the same reading frame to a polynucleotide sequence
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which aids in expression and secretion o$ a polypeptide from
a host cell, for example, a leader sequence which functions
as a secretory sequence for controlling transport of a
polypeptide from the cell. The polypeptide having a leader
sequence is a preprotein and may have the leader sequence
cleaved by the host cell to form the mature form of the
~ polypeptide. The polynucleotides may also encode a
otein which is the mature protein plus additional 5'
amino acid residues. A mature protein having a prosequence
is a proprotein and is an inactive form of the protein. Once
the prosequence is cleaved an active mature protein rPm~; n~,
Thus, for example, the polynucleotide o~ the present
invention may encode a mature protein, or a protein having a
prosequence or a protein having both a presequence and a
presequence (leader sequence).
The polynucleotides of the present invention may also
have the coding sequence fused in frame to a marker sequence
which allows for purification of the polypeptide of the
present invention. The marker sequence may be a hexa-
histidine tag supplied by a pQE-9 vector to provide ~or
purification of the mature polypeptide ~used to the marker in
the case o~ a bacterial host, or, ~or example, the marker
sequence may be a hemayglutinin (HA) tag when a ~ lian
host, e.g. COS-7 cells, is used. The HA tag corresponds to
an epitope derived ~rom the influenza hemagglutinin protein
(Wilson, I., et al., Cell, 37:767 (198~)).
The present invention ~urther relates to
polynucleotides which hybridize to the hereinabove-described
polynucleotides if there is at least 70%, pre~erably at least
90%, and more preferably at least 95% identity between the
sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to
the hereinabove-described polynucleotides. As herein used,
the term "stringent conditions" means hybridization will
occur only if there is at least 95% and preferably at least
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97% identity between the sequences. The polynucleotides
which hybridize to the her~n~hove described polynucleotides
in a preferred emboA~m~nt encode polypeptides which retain
substantially the same biological function or activity as the
mature polypeptide of the present invention encoded by a
coding sequence which includes the DNA of Figures 1-20 (SEQ
ID NO:1-20) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 10
or 20 bases, preferably at least 30 bases, and more
preferably at least 50 bases which hybridize to a
polynucleotide of the present invention and which has an
identity thereto, as hereinabove described, and which may or
may not retain activity. For example, such polynucleotides
may be employed as probes for polynucleotides, for example,
for recovery o~ the polynucleotide or as a diagnostic probe
or as a PCR primer.
Thus, the present invention is directed to
polynucleotides having at least a 70~ identity, preferably at
least 90~ and more preferably at least 95~ identity to a
polynucleotide which encodes the mature polypeptide encoded
by a human gene which includes the DNA of one of Figures 1-20
(SEQ ID NO:1-20) as well as ~ragments thereof, which
fragments have at least 30 bases and preferably at least 50
bases and to polypeptides encoded by such polynucleotides.
The partial sequences are specific tags for messenger
RNA molecules. The complete sequence of that messenger RNA,
in the form of cDNA, can be determined using the partial
sequence as a probe to identify a cDNA clone corresponding to
a full-length transcript, followed by sequencing of that
clone. The partial cDNA clone can also be used as a probe to
identify a genomic clone or clones that contA~ n the complete
gene including regulatory and promoter regions, exons, and
introns.
The partial sequences of Figures 2-20 (SEQ ID NO:2-20)
may be used to identify the corresponding full length gene
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~rom which they were derived. The partial sequence~ can be
nick-translated or end-labelled with 32p using polynucleotide
kinase using labelling methods known to those with skill in
the art (Basic Methods in Molecular Biology, L.G. Davis, M.D.
Dibner, and ~.F. Battey, ed., Elsevier Press, NY, 19~6). A
lambda library prepared ~rom human breast tissue can be
directly screened with the labelled sequences of interest or
the library can be converted en masse to pBluescript
(Stratagene Cloning Systems, La Jolla, CA 92037) to
~acilitate bacterial breasty screening. Regarding
pBluescript, see Sambrook et al., Molecular Cloning-A
Laboratory ~AnnAl, Cold Spring Harbor Laboratory Press
(1989), pg. 1.20. Both methods are well known in the art.
Brie~ly, ~ilters with bacterial colonies cont~ n~ ng the
library in pBluescript or bacterial lawns cOntA~n~ng l~m~
plaques are denatured and the DNA is ~ixed to the filters.
The filters are hybridized with the labelled probe using
hybridization conditions described by Davis et al., su~ra.
The partial sequences, cloned into lAmh~ or pBluescript, can
be used as positive controls to assess background binding and
to adjust the hybridization and washing stringencies
necessary ~or accurate clone identi~ication. The resulting
autoradiograms are compared to duplicate plates o~ colonies
or plaques; each exposed spot corresponds to a positive
breasty or plaque. The colonies or plaques are selected,
~xrAn~d and the DNA is isolated ~rom the colonies ~or
~urther analysis and sequencing.
Positive cDNA clones are analyzed to determine the
amount o~ additional sequence they contA~n using PCR with one
primer ~rom the partial sequence and the other primer ~rom
the vector. Clones with a larger vector-insert PCR product
than the original partial sequence are analyzed by
restriction digestion and DNA sequencing to determine whether
they contain an insert o~ the same size or similar as the
mRNA size determined ~rom Northern blot Analysis.
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Once one or more overlapping cDNA clones are identified,
the complete sequence of the clones can be determined. The
preferred method is to use ~on~lclease III digestion
(McCombie, W.R, Kirkness, E., Fleming, J.T., Kerlavage, A.R.,
Iovannisci, D.M., and Martin~ ~do, R., Methods, 3:33-40,
1991). A series of deletion clones are generated, each of
which is sequenced. The resulting overlapping sequences are
assembled into a single contiguous sequence of high
r~lln~ncy (usually three to five overlapping sequences at
each nucleotide position), resulting in a highly accurate
~inal sequence.
The DNA sequences (as well as the corresponrl;ng RNA
sequences) also include sequences which are or contain a DNA
sequence identical to one cont~;ne~ in and isolatable from
ATCC Deposit No. 97175, deposited June 2, l99S, and ~ras~-nts
or portions of the isolated DNA sequences (and corresponding
RNA sequences), as well as DNA (RNA) sequences encoding the
same polypeptide.
The deposit(s) referred to herein will be maintained
under the terms o~ the Budapest Treaty on the International
Recognition o~ the Deposit o~ Micro-org~n;Fm~ for purposes o~
Patent Procedure. These deposits are provided merely as
convenience to those of skill in the art and are not an
admission that a deposit is required under 35 U.S.C. 112.
The sequence o~ the polynucleotides cont~;ned in the
deposited materials, as well as the amino acid sequence of
the polypeptides encoded thereby, are incorporated herein by
reference and are controlling in the event of any conflict
with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and
no such license is hereby granted.
The present invention further relates to polynucleotides
which have at least 10 bases, pre~erably at least 20 bases,
and may have 30 or more bases, which polynucleotides are
hybridizable to and have at least a 70~ identity to RNA (and
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DNA which corresponds to such RNA) transcribed from a hllm~n
gene whose coding portion includes DNA as here;n~bove
described.
Thus, the polynucleotide sequences which hybridize as
described above may be used to hybridize to and detect the
expression of the human genes to which they correspond for
use in diagnostic assays as hereinafter described.
In accordance with still another aspect of the present
invention there are provided diagnostic assays for detecting
micrometastases of breast ~nc~ in a host. While applicant
does not wish to limit the reasoning of the present invention
to any specific scientific theory, it is believed that the
presence of active transcription of a breast specific gene of
the present invention in cells o~ the host, other than those
derived from the breast, is indicative of breast cancer
metastases. This is true because, while the breast specific
genes are found in all cells of the body, their transcription
to mRNA, cDNA and expression products is primarily limited to
the breast in non-diseased individuals. However, if breast
cancer is present, breast cancer cells migrate from the
cancer to other cells, such that these other cells are now
actively transcribing and expressing a breast specific gene
at a greater level than is normally found in non-diseased
individuals, i.e., transcription is higher than found in non-
breast tissues in healthy individuals. It is the detection
of this ~nh~nced transcription or ~nh~nced protein expression
in cells, other than those derived from the breast, which is
indicative of metastases of breast cancer.
In one example of such a ~;~gnostic assay, an RNA
sequence in a sample derived from a tissue other than the
breast is detected by hybridization to a probe. The sample
contains a nucleic acid or a mixture of nucleic acids, at
least one of which is suspected of cont~; n; ng a h~ n breast
specific gene or ~ragment thereof of the present invention
which is transcribed and expressed in such tissue. Thus, for
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example, in a form of an assay for determining the presence
of a specific RNA in cells, initially RNA is isolated from
the cells.
A sample may be obt~;neA from cells derived from tissue
other than from the breast including but not limited to
blood, urine, saliva, tissue biopsy and autopsy material.
The use of such methods for detecting ~nh~n~ed transcription
to mRNA from a human breast specific gene of the present
invention or fragment thereof in a sample obt~;ne~ from cells
derived from other than the breast is well within the scope
of those skilled in the art from the teachings herein.
The isolation of mRN~ comprises isolating total cellular
RNA by disrupting a cell and performing differential
centrifugation. Once the total RNA is isolated, mRNA is
isolated by making use of the ~ n; ne nucleotide residues
known to those skilled in the art as a poly(A) tail found on
virtually every eukaryotic mRNA molecule at the 3' end
thereof. Oligonucleotides composed of only deoxythymidine
toligo(dT)] are linked to cellulose and the oligo(dT)-
cellulose packed into small columns. When a preparation of
total cellular RNA is passed through such a column, the mR~A
molecules bind to the oligo(dT) by the poly(A)tails while the
rest of the RNA flows through the column. The bound mRNAs
are then eluted from the column and collected.
One example of detecting-isolated mRNA transcribed from
a breast specific gene of the present invention comprises
screening the collected mRNAs with the gene specific
oligonucleotide probes, as hereinabove described.
It is also appreciated that such probes can be and are
preferably labeled with an analytically detectable reagent to
facilitate identification of the probe. Useful reagents
include but are not limited to radioactivity, fluorescent
dyes or enzymes capable of catalyzing the fq~mation of a
detectable product.
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An example o~ detecting a polynucleotide complementary
to the mRNA sequence (cDNA) utilizes the polymerase chain
reaction (PCR) in conjunction with reverse transcriptase.
PCR is a very powerful method ~or the specific amplification
of DNA or RNA stretches (Saiki et al., Nature, 234:163-166
(1986)). One application of this technology is in nucleic
acid probe technology to bring up nucleic acid sequences
present in low copy numbers to a detectable level. Numerous
diagnostic and scientific applications of thi~ method have
been described by E.A. Erlich (ed.) in PCR Technology-
Principles and Applications for DNA Amplification, Stockton
Press, USA, 1989, and by M.A. Inis (ed.) in PCR Protocols,
Ac~em~c Press, San Diego, USA, 1990.
RT-PCR is a combination o~ PCR with the reverse
transcriptase enzyme. Reverse transcriptase is an enzyme
which produces cDNA molecules from corresponding mRNA
molecules. This is important since PCR amplifies nucleic
acid molecules, particularly DNA, and this DNA may be
produced ~rom the mRNA isolated ~rom a sample derived ~rom
the host.
A speci~ic example o~ an RT-PCR diagnostic assay
involves removing a sample ~rom a tissue o~ a host. Such a
sample will be ~rom a tissue, other than the breast, ~or
example, blood. There~ore, an example o~ such a diagnostic
assay comprises whole blood gradient isolation o~ nucleated
cells, total RNA extraction, RT-PCR o~ total RNA and agarose
gel electrophoresis of PCR products. The PCR products
comprise cDNA complement~y to RNA transcribed from one or
more breast speci~ic genes o~ the present invention or
~ragments thereo~. More particularly, a blood sample is
obtained and the whole blood is combined with an equal volume
of phosphate bu~ered saline, centri~uged and the lymphocyte
and granulocyte layer is care~ully aspirated and rediluted in
phosphate buffered saline and centrifuged again. The
supernate is discarded and the pellet cont~; n; ng nucleated
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cells is used ~or RNA extraction using the RNazole B method
as described by the manu~acturer (Tel-Test Inc., Friendswood,
TX).
Oligonucleotide primers and probes are prepared with
high speci~icity to the DNA sequences o~ the present
invention. The probes are at least 10 base pairs in length,
pre~erably at least 30 base pairs in length and most
pre~erably at least 50 base pairs in length or more. The
reverse transcriptase reaction and PCR ampli~ication are
per~ormed se~uentially without interruption. Taq polymerase
is used during PCR and the PCR products are concentrated and
the entire sample is run on a Tris-borate-EDTA agarose gel
contA;ning ethidium bromide.
In accordance with another aspect o~ the present
invention, there is provided a method o~ diagnosing a
disorder o~ the breast, ~or example breast cancer, by
determining altered levels of the breast speci~ic
polypeptides o~ the present invention in a biological sample,
derived ~rom tissue other than ~rom the breast. Elevated
levels o~ the breast speci~ic polypeptides o~ the present
invention, indicates active transcription and expression o~
the corresponding breast speci~ic gene product. Assays used
to detect levels o~ a breast speci~ic gene polypeptide in a
sample derived ~rom a host are well-known to those skilled in
the art and include radioimmllnoassays, co7npetitive-~;n~.7ing
assays, Western blot analysis, ELISA assays and "sandwich"
assays. A biological sample may include, but is not limited
to, tissue extracts, cell samples or biological ~luids,
however, in accordance with the present invention, a
biological sample speci~ically does not include tissue or
cells o~ the breast.
An ELISA assay (Coligan, et al., Current Protocols in
T7nml7noloqy, 1(2), Chapter 6, 1991) initially comprises
preparing an antibody speci~ic to a breast speci~ic
polypeptide o~ the present invention, pre~erably a monoclonal
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antibody. In addition, a reporter antibody is prepared
against the monoclonal ~nt;hody. To the reporter antibody is
attached a detectable reagent such as radioactivity,
i~luorescence or, in this example, a horser;lci; h peroxidase
enzyme. A sample is removed ~rom a host and incubated on a
solid support, e.g., a polystyrene dish, that binds the
proteins in the sample. Any ~ree protein h;n~;ng sites on
the dish are then covered by incubating with a non-speci~ic
protein, such as BSA. Next, the monoclonal ~n~;hody is
incubated in the dish during which time the monoclonal
antibodies attach to the breast speci~ic polypeptide attached
to the polystyrene dish. All unbound monoclonal antibody is
washed out with bu~er. The reporter ~nt;hody linked to
horseradish peroxidase is now placed in the dish resulting in
h;n~,ng o~ the reporter antibody to any monorlon~l ~nt;hody
bound to the breast specific gene polypeptide. Unattached
reporter antibody is then washed out. Peroxidase substrates
are then added to the dish and the amount o~ color developed
in a given time period is a measurement o~ the amount o~ the
breast speci~ic polypeptide present in a given volume o~
patient sample when ~o~r~ed against a st~n~d curve.
A competition assay may be employed where antibodies
speci~ic to a breast speci~ic polypeptide are attached to a
solid support. The breast speci~ic polypeptide is then
labeled and the labeled polypeptide a sample derived ~rom the
host are passed over the solid support and the amount o~
label detected, ~or example, by liquid scintillation
chromatography, can be correlated to a quantity o~ the breast
speci~ic polypeptide in the sample.
A "sandwich" assay is similar to an ELISA assay. In a
"sandwich~ assay, breast speci~ic polypeptides are passed
over a solid support and bind to antibody attached to the
solid support. A second antibody is then bound to the breast
speci~ic polypeptide. A third antibody which is labeled and
is speci~ic to the second antibody, is then passed over the
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solid support and binds to the second antibody and an amount
can then be quantified.
In alternative methods, labeled antibodies to a breast
speci~ic polypeptide are used. In a one-step assay, the
target molecule, if it is present, is ~ h; lized and
incllh~ted with a labeled antibody. The labeled ~nt;ho~y
binds to the immobilized target molecule. After washing to
remove the unbound molecules, the sample is assayed for the
presence of the label. In a two-step assay, ~ h;l ~zed
target molecule is incubated with an unlabeled antibody. The
target molecule-labeled antibody complex, if present, is then
bound to a second, labeled antibody that is specific for the
unlabeled Ant;hody The sample is washed and assayed for the
presence of the label.
Such antibodies specific to breast speci~ic gene
proteins, for example, anti-idiotypic Ant;hodies, can be used
to detect breast cancer cells by being labeled and described
above and h;n~;ng tightly to the breast cancer cells, and,
therefore, detect their presence.
The Ant;hodies may also be used to target breast cancer
cells, for example, in a method of homing interaction agents
which, when contacting breast cancer cells, destroy them.
This is true since the antibodies are specific for breast
specific genes which are primarily expressed in breast
cancer, and a linking of the interaction agent to the
antibody would cause the interaction agent to be carried
directly to the breast.
Antibodies of this ~ype may also be used to do in vivo
imaging, for example, by labeling the Ant;hodies to
facilitate scAnn;ng of the breast. One method for imaging
comprises contacting any cancer cells of the breast to be
imaged with an anti-breast specific gene protein antibody
labeled with a detectable marker. The method is performed
under conditions such that the labeled antibody binds to the
~'~'.'',',~"~.~;~!~ ~:.~ ~:..'.'.~'','.. '~ .r.~L:~n~ . In a specific example, the
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antibodies interact with the breast, for example, breast
cancer cells, and fluoresce upon such contact such that
imaging and visibility o~ the breast is ~nh~nced to allow a
determination of the diseased or non-diseased state of the
breast.
The choice of marker used to label the antibodies will
vary depending upon the application. However, the choice of
marker is readily determ~ n~hl e to one skilled in the art.
These labeled antibodies may be used in ;~mllno~ssays as well
as in histological applications to detect the presence of the
proteins. The labeled antibodies may be polyclonal or
monoclonal.
The presence of active transcription, which is greater
than that normally ~ound, of the breast specific genes in
cells other than from the breast, by the presence of an
altered level of m-RNA~ CDNA or expression products is an
important indication o~ the presence of a breast cancer which
has metastasized, since breast cancer cells are migrating
from the breast into the general circulation. Accordingly,
this ph~nom~non may have important clinical implications
since the method of treating a localized, as opposed to a
metastasized, tumor is entirely different.
Of the 20 breast speci~ic genes disclosed, only breast
speci~ic gene 1 is a ~ull-length gene. Breast speci~ic gene
1 is 79% identical and 83~ similar to human Al~he~m~ disease
amyloid gene. Breast speci~ic gene 2 is 30% identical and
48% 5~m;1~ to hnm~n hydroxyindole-o-methyltrans~erase gene.
Breast speci~ic gene 3 is 58% identical and 62~ similar to
human 06-methylgll~n~nF~-DNA methyltransferase gene. Breast
speci~ic gene 4 is 34~ identical and 65~ similar to the mouse
pl20 gene. Breast speci~ic gene 5 is 78~ identical and 89
similar to human p70 ribosomal S6 kinase alpha-II gene.
Breast speci~ic gene 6 is 77% identical and 79% similar to
the human transcription factor NFATp gene.
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As stated previously, the breast speci~ic genes o~ the
present inv~nt; ~n are putative molecular markers in the
diagnosis of breast c~nç~ ~ormation, and breast cancer
metastases. As shown in the ~ollowing Table 1, the presence
o~ the breast speci~ic genes when tested in normal breast,
breast cancer, embryo and other ç~nc~ libraries, the breast
speci~ic genes of the present invention were ~ound to be most
prevalent in the breast cancer library, indicating that the
genes o~ the present invention may be employed ~or detecting
breast cancer, as discussed previously. The table also
indicates a putative identi~ication, based on homology, o~
BSG1 through BSG6 to known genes.
Table 1
Genes Homolog Gene Norm Br Ca Embryo Other Others
Name (Class) Br Can-
cers
BSGl AD Amyloid (3) 1 6
BSG2 Hyd~yindole- 3
o-methytrans-
ferase (2)
BSG3 0-6- 3
methylguanine-
DNA
methyltrans-
ferase (1)
BSG4 P120 (3) 3
BSGs p70 ribosomal - 3
S6 kinase
alpha-II (2)
BSG6 Transcription 2
factor NFATp(3)
BSG7 2
BSG8 4 3
BSG8 2
BSG9 3
BSG10 3
BSGll 3
BSG12 3 3
BSG13 3
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BSG14 . 2
BSG15 3
BSG16
BSG17 2
BSG18 2
BSGl9
BSG20 2
The assays described above may also be used to test
whether bone marrow preserved before chemotherapy is
cont~m;n~ted with micrometastases of a breast cancer cell.
In the assay, blood cells from the bone marrow are isolated
and treated as described above, this method allows one to
determine whether preserved bone marrow is still suitable for
transplantation a$ter chemotherapy.
The present invention $urther relates to mature
polypeptides, for example the BSG1 polypeptide, as well as
~ragments, analogs and derivatives of such polypeptide.
The terms "$ragment,~ derivative~ and ~analog" when
referring to the polypeptides Pnco~p~ by the genes of the
invention means a polypeptide which retains essentially the
same biological $unction or activity as surh polypeptide.
Thus, an analog includes a proprotein which can be activated
by cleavage of the proprotein portion to produce an active
mature polypeptide.
The polypeptides of the present invention may be
recombinant polypeptides, natural polypeptides or synthetic
polypeptides, preferably recombinant polypeptides.
The $ragment, derivative or analog of the polypeptides
encoded by the genes of the invention may be (i) one in which
one or more of the amino acid residues are substituted with
a conserved or non-conserved amino acid residue (preferably
a conserved amino acid residue) and such substituted amino
acid residue may or may not be one encoded by the genetic
code, or (ii) one in which one or more of the amino acid
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residues includes a substituent group, or (iii) one in which
the polypeptide is $used with another compound, such as a
compound to increase the hal$-li$e o$ the polypeptide ($or
example, polyethylene glycol), or (iv) one in which the
additional amino acids are $used to the polypeptide, such as
a leader or secretory seguence or a sequence which is
employed for purification of the mature polypeptide or a
proprotein sequence. Such fra~m~nt~, derivatives and analogs
are deemed to be within the scope o$ those skilled in the art
$rom the teachings herein.
The polypeptides and polynucleotides o$ the present
invention are preferably provided in an isolated $orm, and
preferably are puri$ied to homogeneity.
The term "isolated~ means that the material is removed
$rom its original envilv~ t (e.g., the natural envi~v-~ t
if it is naturally occurring). For example, a naturally-
occurring polynucleotide or polypeptide present in a living
~n;m~l is not isolated, but the same polynucleotide or
polypeptide, separated $rom some or all of the coexisting
materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural envi~ ellt.
The polypeptides of the-present invention include the
polypeptides of Figure 1 (SEQ ID NO:1) (in particular the
mature polypeptides) as well as polypeptides which have at
least 70~ similarity (preferably at least a 70% identity) to
the polypeptides o$ Figure 1 (SBQ ID NO:1) and more
preferably at least a 90~ sim; 1 ~rity (more preferably at
least a 90% identity) to the polypeptides of Figures 8 and 9
(SEQ ID NO:8 and 9) and still more preferably at least a 95%
similarity (still more preferably at least 95% identity) to
the polypeptides of Figure 1 (SEQ ID NO:1) and also include
portions of such polypeptides with such portion of the
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polypeptide generally cont~;n;ng at least 30 amino acids and
more preferably at least 50 amino acids.
As known in the art "s;m;l~ityn between two
polypeptides is determ;n~n by comparing the amino acid
sequence and its conserved amino acid substitutes of one
polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present
invention may be employed for pro~lc;ng the correspQnn;ng
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for pron~lc;ng the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to
synthesize full-length polynucleotides of the present
invention.
The present invention also relates to vectors which
include polynucleotides of the present invention, host cells
which are genetically engineered with vectors of the
invention and the production of polypeptides of the invention
by recombinant techniques.
Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this
invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the
form of a plasmid, a viral particle, a phage, etc. The
engineered host cells can -be cultured in conventional
nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the breast
specific genes. The culture conditions, such as temperature,
pH and the like, are those previously used with the host cell
selected for expression, and will be apparent to those of
ordinarily skill in the art.
The polynucleotides of the present invention may be
employed for producing polypeptides by recombinant
techniques. Thus, for example, the polynucleotide may be
included in any one of a variety of expression vectors for
CA 0222~824 1997-12-24
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expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA se~l~ncP~, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast pl ~m; ~; vectors derived from
combinations of pl ~Fm; ~ and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is
replicable and viable in the host.
The ~L~. iate DNA sequence may be inserted into the
vector by a variety of procedures. In general, the DNA
sequence is inserted into an ~l~riate restriction
~n~Qnllclease site~s) by procedures known in the art. Such
procedures and others are deemed to be within the scope o$
those skilled in the art.
The DNA sequence in the expression vector is operatively
linked to an d~' ~riate expression control sequence(s)
(promoter) to direct mRNA synthesis. As representative
examples of such promoters, there may be mentioned: LTR or
SV40 promoter, the E. coli. lac or tr~, the phage lambda PL
promoter and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome binding site
for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for
amplifying expression.
In addition, the expression vectors preferably contain
one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic
cell culture, or such as tetracycline or ampicillin
resistance in E. coli.
The vector cont~;n;ng the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate
host to permit the host to express the protein.
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As representative examples of a~.o~riate hosts, there
may be mentioned: bacterial cells, such as E. coli,
StrePtOmYCeS, SA1~ne1 lA ty~himll~ium; fungal cells, such as
yeast; insect cells such as Drosophila S2 and SpodoPtera S~9;
Ani 1 cells such as CHO, COS or Bowes m~l An~mA;
adenovirusesi plant cells, etc. The selection of an
~ r iate host i~ deemed to be within the scope o~ those
skilled in the art ~rom the teachings herein.
More particularly, the present invention also includes
recombinant constructs comprising one or more o~ the
sequences as broadly described above. The constructs
co~ ~ise a vector, such as a plasmid or viral vector, into
which a sequence of the invention has been inserted, in a
~orward or reverse orientation. In a pre~erred aspect o~
this embodiment, the construct ~urther comprises regulatory
sequences, including, ~or example, a promoter, operably
linked to the sequence. Large nl hers o$ suitable vectors
and promoters are known to those o~ skill in the art, and are
commercially available. The following vectors are provided
by way o~ example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),
pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS,
pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-
3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO,
pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL (Pharmacia). However, any other plasmid or vector may
be used as long as they are replicable and viable in the
host.
Promoter regions can be selected ~rom any desired gene
using CAT (chloramphenicol trans~erase) vectors or other
vectors with selectable markers. Two a~ro~riate vectors are
pKK232-8 and pCM7. Particular ~ bacterial promoters
include lacI, lacZ, T3, T7, gpt, 1Am1r1A PR~ PL and trp.
Eukaryotic promoters include CMV imm~i Ate early, HSV
thymidine kinase, early and late SV40, LTRs ~rom retrovirus,
and mouse metallothionein-I. Selection o~ the appropriate
CA 0222~824 1997-12-24
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vector and promoter is well within the level o~ ordinary
skill in the art.
In a ~urther embo~;m~nt, the present invention relates
to host cells contA;n;ng the above-described constructs. The
host cell can be a higher eukaryotic cell, such as a
m~mm~ lian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host
cell can be e~ected by calcium phosphate trans~ection, D~AE-
Dextran mediated trans~ection, or electroporation (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology,
(1986)).
The constructs in host cells can be used in a
conventional mAnne~ to produce the gene product ~nco~ by
the recombinant sequence. Alternatively, the polypeptides o~
the invention can be synthetically produced by conventional
peptide synthesizers.
Proteins can be expressed in mam~Al;An cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-~ree translation systems can also be
employed to produce such proteins using RNAs derived ~rom the
DNA constructs of the present invention. Appropriate cloning
and expression vectors ~or use with prokaryotic and
eukaryotic hosts are described by Sambrook, et al., Molecular
Cloning: A Laboratory ~nllAl-, Second Bdition, Cold Spring
Harbor, N.Y., (1989), the disclosure o~ which is hereby
incorporated by re~erence.
Transcription o~ the DNA Pnco~;ng the polypeptides o~
the present invention by higher eukaryotes is increased by
inserting an ~nhAncer sequence into the vector. RnhAnrers
are cis-acting elements of DNA, usually about ~rom 10 to 300
bp that act on a promoter to increase its transcription.
Examples including the SV40 enhAncer on the la~e side o~ the
replication origin bp 100 to 270, a cytomegalovirus early
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promoter ~nh~ncer, the polyoma enhAncer on the late side of
the replication origin, and adenovirus PnhAn~5.
Generally, recombinant expression vectors will include
origins of replication and selectable markers penmitting
trans_ormation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 gene, and
-
a promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such
promoters can be derived from operons ~nCoA~ng glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor,
acid phosphatase, or heat shock proteins, among others. The
heterologous structural sequence is assembled in ~l~riate
phase with translation initiation and termination sequences.
Optionally, the heterologous sequence can PnCo~e a _usion
protein including an N-terminal identification peptide
imparting desired characteristics, e.g., stAh;l~zation or
simpli~ied puri$ication of expres~ed recombinant product.
Useful expression vectors for bacterial use are
constructed by inserting a structural DNA sequence encoding
a desired protein together with suitable translation
initiation and termination signals in operable r~;ng ~rame
with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of
replication to ensure maintenance o~ the vector and to, i~
desirable, provide ampli~ication within the host. Suitable
prokaryotic hosts ~or transformation include E. coli,
Bacillus subtilis, S~lmon~lla tYph~mll~ium and various species
within the genera Psell~mnn~ Streptomyces, and
Staphylococcus, although others may also be employed as a
matter o~ choice.
As a representative but nonlimiting example, use~ul
expression vectors for bacterial use can comprise a
selectable marker and bacterial origin o~ replication derived
~rom commercially available plasmids comprising genetic
elements of the well known cloning vector pBR322 (ATCC
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37017). Such commercial vectors include, ~or example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and OE M1
(P~ eyd Biotec, Madison, WI, USA). These pBR322 nh~ckhone"
sections are comh~n~ with an d~Lo~riate promoter and the
structural sequence to be expressed.
Following trans~ormation of a suitable host strain and
growth of the host strain to an ~~ iate cell density, the
selected promoter is ;n~llc~ by a~ iate means (e.g.,
temperature shi~t or chemical induction) and cells are
cultured for an additional period.
Cells are typically harvested by centri~ugation,
disrupted by physical or chemical means, and the resulting
crude extract retAin~ ~or ~urther puri~ication.
Microbial cells employed in expression of proteins can
be disrupted by any convenient method, including ~reeze-thaw
cycling, sonication, mechanical disruption, or use o~ cell
lysing agents, such methods are well know to those skilled in
the art.
Various m~mm~ n cell culture systems can also be
employed to express recombinant protein. Examples of
m~rm-lian expression systems include the COS-7 lines o~
monkey kidney ~ibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines capable o~ expressing a
compatible vector, ~or example, the C127, 3T3, CHO, HeLa and
BHK cell lines. ~Amm~lian expression vectors will comprise
an origin of replication, a suitable promoter and ~nh~ncer,
and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' ~lanking
nontranscribed sequences. DNA se~l~nces derived from the
SV40 splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
The breast speci~ic gene polypeptides can be recovered
and puri~ied ~rom recombinant cell cultures by methods
including ~m~on;um sul~ate or ethanol precipitation, acid
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extraction, anion or cation ~xchAnge chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding
steps can be used, as necessary, in completing configuration
of the mature protein. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification
steps.
The polynucleotides of the present invention may have
the coding se~uence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. An example of a marker sequence is a
hPx~h;stidine tag which may be supplied by a vector,
preferably a pQE-9 vector, which provides for purification of
the polypeptide fused to the marker in the case of a
bacterial host, or, for example, the marker sequence may be
a hemagglutinin (HA) tag when a m~m~ n host, e.g. COS-7
cells, is used. The HA tag corresponds to an epitope derived
from the influenza hemagglutinin protein (Wilson, I., et al.,
Cell, 37:767 (1984)).
The polypeptides of the present invention may be a
naturally purified product, or a product of chemical
synthetic procedures, or produced by recombinant techniques
from a prokaryotic or eukaryotic host ($or example, by
bacterial, yeast, higher plant, insect and, -l;an cells in
culture). Depending upon the host employed in a recomh;n~nt
production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
BSG1, and other breast specific genes, and the protein
product thereof may be employed for early detection of breast
cancer since they are over-expressed in the breast cancer
state.
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In accordance with another aspect of the present
invention there are provided assays which may be used to
screen for therapeutics to ;nh; hi t the action of the breast
specific genes or breast specific proteins of the present
invention. The present invention discloses methods ~or
selecting a therapeutic which forms a complex with breast
specific gene proteins with su~ficient affinity to ~leve-lt
their biological action. The methods include various assays,
including competitive assays where the proteins are
;m~nh; lized to a support, and are contacted with a natural
substrate and a labeled therapeutic either simultaneously or
in either consecutive order, and determining whether the
therapeutic ef~ectively competes with the natural substrate
in a m~nne~ sufficient to prevent h;n~;ng of the protein to
its substrate.
In another embodiment, the substrate is ; -h;l; zed to
a support, and is ~ont~cted with both a labeled breast
specific polypeptide and a therapeutic (or unlabeled proteins
and a labeled therapeutic), and it is determined whether the
amount of the breast specific polypeptide bound to the
substrate is reduced in comparison to the assay without the
therapeutic added. The breast specific polypeptide may be
labeled with antibodies.
Potential therapeutic compounds include antibodies and
anti-idiotypic ~nt;hodies as- described above, or in some
cases, an oligonucleotide, which binds to the polypeptide.
Another example is an antisense construct prepared using
antisense technology, which is directed to a breast specific
polynucleotide to prevent transcription. Antisense
technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or
RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is used to design an
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antisense RNA oligonucleotide o~ ~rom about 10 to 40 base
pairs in length. A DNA oligonucleotide is designed to be
complementary to a region o~ the gene involved in
transcription (triple helix -see Lee et al., Nucl. Acids
Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby
evel-ting transcription and the production of a breast
specific polynucleotide. The antisense RNA oligonucleotide
hybridizes to the mRNA in vi~o and blocks translation of the
mRNA molecule into the breast speci~ic genes polypeptide
(antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1988)). The
oligonucleotides described above can also be delivered to
cells such that the antisense RNA or DNA may be expressed in
vivo to ; nh~h; t production of the breast specific
polypeptides.
Another example is a small molecule which binds to and
occupies the active site of the breast speci~ic polypeptide
thereby making the active site inaccessible to substrate such
that normal biological activity is prevented. ~xamples o~
small molecules include but are not limited to small peptides
or peptide-like molecules.
These compounds may be employed to treat breast cancer,
since they interact with the function o~ breast specific
polypeptides in a m~n~er sufficient to inhibit natural
~unction which is necessary ~or the v;~h;l;ty o~ breast
cancer cells. This is true since the BSGs and their protein
products are primarily expressed in breast cancer tissues and
are, therefore, suspected o~ being critical to the ~ormation
of this state.
The compounds may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereina~ter
described.
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The compounds of the present invention may be employed
in combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of
the polypeptide, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to
saline, buffered ~1 ;n~, dextrose, water, glycerol, ethanol,
and r~mh~ n~tions thereof. The formulation should suit the
mode of ~m; n; stration.
The invention also provides a pharmaceutical pack or kit
comprising one or more cont~;ne~s filled with one or more of
the ingredients of the pharmaceutical romrositions of the
invention. Associated with such cont~;nen(s) can be a notice
in the ~orm prescribed by a governm~nt~l agency regulating
the manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale ~or human ~m;n; stration. In
addition, the pharmaceutical compositions may be employed in
conjunction with other therapeutic compounds.
The pharmaceutical compositions may be ~m;n; stered in
a convenient m~nnen such as by the oral, topical,
intravenous, intraperitoneal, intramuscular, subclltAn~ous,
intranasal, intra-anal or intradermal routes. The
pharmaceutical compositions are ~m; n; ~tered in an amount
which is effective for treating and/or prophylaxis of the
speci~ic indication. In general, they are ~Am; n; ~tered in an
amount of at least about 10 ~g/kg body weight and in most
cases they will be ~m;n; stered in an amount not in excess o~
about 8 mg/Kg body weight per day. In most cases, the dosage
is from about 10 ~g/kg to about 1 mg/kg body weight daily,
taking into account the routes of ~m; n; stration, symptoms,
etc.
The breast specific genes and compounds which are
polypeptides may also be employed in accorda~ce with the
present invention by expression of such polypeptides in vivo,
which is often referred to as "gene therapy."
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Thus, for example, cells from a patient may be
engineered with a polynucleotide ~DNA or RNA) encoding a
polypeptide ex vivo, with the engineered cells then being
provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells
may be engineered by procedures known in the art by use of a
retroviral particle cont~;n;ng RNA ~nco~;ng a polypeptide of
the present invention.
Similarly, cells ,m,~y be engineered in vivo for
expression of a polypeptide in vivo by, for example,
procedures known in the art. As known in the art, a producer
cell for pro~nc~ng a retroviral particle cont~; n~ ng RNA
encoding a polypeptide of the present invention may be
~m; n;stered to a patient for engineering cells in vivo and
expression of the polypeptide in vivo. These and other
methods for ~mi n~Qtering a polypeptide of the present
invention by such method should be apparent to those skilled
in the art from the teachings of the present invention. For
example, the expression vehicle ~or engineering cells may be
other than a retrovirus, for example, an adenovirus which may
be used to engineer cells in vivo after combination with a
suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not
limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis virus, gibbon ape leukemia
virus, hllm~n ~ lnodeficiency virus, adenovirus,
Myeloproliferative Sarcoma virus, and m~m~y tumor virus.
In one embodiment, the retroviral plasmid vector is derived
~rom Moloney Murine Leukemia virus.
The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited
to, the retroviral LTR; the SV40 promoter; and the hnm~n
cytomegalovirus (CMV) promoter described in Miller, et al.,
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Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other
promoter (e.g., cellular promoters such as eukaryotic
cellular promoters including, but not limited to, the
histone, pol III, and ~-actin promoters). Other viral
promoters which may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters,
and B19 parvovirus promoters. The selection o~ a suitable
promoter will be apparent to those skilled in the art ~rom
the teachings contA; n~ herein.
The nucleic acid sequence encoding the polypeptide o~
the present invention is under the control o~ a suitable
promoter. Suitable promoters which may be employed include,
but are not limited to, adenoviral promoters, such as the
adenoviral major late promoter; or heterologous promoters,
such as the cyt~m~g~lovirus (CMV) promoter; the respiratory
syncytial virus (RSV) promoter; i n~llc; hle promoters, such as
the MMT promoter, the metallothionein ~l. Ler; heat shock
promoters; the albumin ~",oLer; the ApoAI promoter; human
globin promoters; viral thymidine kinase promoters, such as
the Herpes Simplex thymidine kinase promoter; retroviral LTRs
(including the modi~ied retroviral LTRs hereinabove
described); the ~-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter
which controls the genes encoding the polypeptides.
The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples
o~ packaging cells which may be transfected include, but are
not limited to, the pEsol~ PA317, ~-2, ~-AM, PA12, T19-14X,
VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell
lines as described in Miller, Human Gene Thera~Y, Vol. 1,
pgs. 5-14 (1990), which is incorporated herein by re~erence
in its entirety. The vector may transduce the packaging
cells through any means known in the art. Such means
include, but are not limited to, electroporation, the use o~
liposomes, and CaPO4 precipitation. In one alternative, the
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retroviral plasmid vector may be encapsulated into a
liposome, or coupled to a lipid, and then ~mi n~stered to a
host.
The producer cell line generates in~ectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles
then may be employed, to transduce eukaryotic cells, either
in vitro or in vivo. The transduced eukaryotic cells will
express the nucleic acid sequence(s) encoding the
polypeptide. Euk~ryotic cells which may be transduced
include, but are not limited to, embryonic stem cells,
embryonic carcino~ cells, as well as hematopoietic stem
cells, hepatocytes, ~ibroblasts, myoblasts, keratinocytes,
endothel~l cells, and bro~rh;~l epithelial cells.
This invention is also related to the use o~ a breast
speci~ic genes of the present invention as a diagnostic. For
example, some diseases result ~rom inherited de~ective genes.
The breast speci~ic genes, CSG7 and CSG10, ~or example, have
been ~ound to have a reduced expression in breast cancer
cells as compared to that in normal cells. Further, the
r~m~n;ng breast speci~ic genes of the present invention are
overexpressed in breast cancer. Accordingly, a mutation in
these genes allows a detection of breast disorders, ~or
example, breast cancer. A mutation in a breast speci~ic gene
o~ the present invention at the DNA level may be detected by
a variety o~ techniques. Nucleic acids used ~or diagnosis
(genomic DNA, mRNA, etc.) may be obtained ~rom a patient~s
cells, other than ~rom the breast, such as ~rom blood, urine,
saliva, tissue biopsy and autopsy material. The genomic DNA
may be used directly ~or detection or may be ampli~ied
enzymatically by using PCR (Saiki, et al., Nature, 324:163-
166 (1986)) prior to analysis. RNA or cDNA may also be used
~or the same purpose. As an example, PCR primers
complementary to the nucleic acid o~ the instant invention
can be used to identi~y and analyze mutations in a breast
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speci~ic polynucleotide of the present invention. For
example, deletions and insertions can be detected by a change
in size of the amplified product in romr~ison to the normal
genotype. Point mutations can be identified by hybridizing
ampli~ied DNA to radiolabelled breast speci~ic RNA or,
alternatively, radiolabelled antisense DNA sequences.
Another well-establ;!~h~l method ~or screening for
mutations in particular se~~nt~ of DNA a~ter PCR
amplification is single-strand conformation polymorphism
(SSCP) analysis. PCR products are ~repared for SSCP by ten
cycles of reamplification to incorporate 32P-dCTP, digested
with an appropriate restriction enzyme to generate 200-300 bp
fragments, and denatured by heating to 85~C for 5 min. and
then plunged into ice. ~lectrophoresis is then carried out
in a non~n~turing gel (5% glycerol, 5~ acrylamide) (Glavac,
D. and Dean, M., ~l n Mutation, 2:404-414 (1993)).
Sequence di~ferences between the reference gene and
"mutants" may be revealed by the direct DNA sequencing
method. In addition, cloned DNA se~nts may be used as
probes to detect specific DNA se~m~nts. The sensitivity of
this method is greatly ~nh~nced when comh;n~ with PCR. For
example, a sequencing primer is used with double-stranded PCR
product or a single-stranded template molecule generated by
a modified PCR. The sequence determination is performed by
conventional procedures with-radiolabeled nucleotides or by
automatic sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic
mobility of DNA fragments and gels with or without denaturing
agents. Small sequence deletions and insertions can be
visualized by high-resolution gel electrophoresis. DNA
fragments of different sequences may be distinguished on
denaturing formamide gradient gels in which the mobilities of
di~ferent DNA fragments are retarded in the gel at different
positions according to their specific melting or partial
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melting temperatures (see, e.g., Myers, et al., Science,
230:1242 (1985)). In addition, sequence alterations, in
particular small deletions, may be detected as changes in the
migration pattern of DNA.
Sequence changes at specific locations may also be
revealed by nuclease protection assays, such as Rnase and S1
protection or the chemical cleavage method (e.g., Cotton, et
al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of the specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing, or the use of
restriction enzymes (e.g., Restriction ~ragment Length
Polymorphisms (RFLP)) and Southern blotting.
The sequences of the present invention are also valuable
for chromosome identification. The sequence is specifically
targeted to and can hybridize with a particular location on
an individual hnm~n chromosome. Moreover, there is a current
need for identifying particular sites on the chromosome. Few
chromosome marking reagents based on actual sequence data
(repeat polymorph~sms) are presently available for marking
chromosomal location. The mapping o~ DNAs to chromosomes
according to the present invention is an important first step
in correlating those sequences with genes associated with
disease.
Briefly, sequences can-be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) ~rom the cDNA.
Computer analysis o~ the 3~ untranslated region is used to
rapidly select primers that do not span more than one exon in
the genomic DNA, thus complicating the amplification process.
These primers are then used for PCR screening of somatic cell
hybrids cont~;n;ng individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer
will yield an amplified ~ragment.
PCR mapping of somatic cell hybrids is a rapid procedure
for assigning a particular DNA to a particular chromosome.
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U~ing the present invention with the same oligonucleotide
primers, sublocalization can be achieved with p~n~ls o~
~ragments from specific chromosomes or pools o~ large genomic
clones in an analogous ~nn~ Other mapping strategies that
can s~m~l~ly be used to map to its chromosome include in
situ hybridization, prescr~n;n~ with labeled ~low-sorted
chromosomes and preselection by hybridization to construct
chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA
clone to a met~ph~e chromosomal spread can be used to
provide a precise chromosomal location in one step. This
technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see Verma et al., ~nm~n
Cl,lo...~somes: a M~nll~l of Basic Techniques, Pe ydu~vll Press,
New York (1988).
Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such
data are found, for example, in V. McKusick, M~n~lian
Inheritance in Man (available on line through Johns Hopkins
University Welch Medical Library). The relationship between
genes and diseases that have been mapped to the same
chromosomal region are then identi$ied through linkage
analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in
the cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then
the mutation is likely to be the causative agent of the
disease.
With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a
chromosomal region associated with the disease ~ould be one
of between 50 and 500 potential causative genes. (This
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assumes 1 megabase mapping resolution and one gene per 20
kb).
The polypeptides, their fragments or other derivatives,
or analogs thereof, or cells expressing them can be used as
an ~ -nogen to produce Ant~ho~es thereto. These antibodies
can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes ch;m~ic~ single chain,
and hl~m~n;zed Ant;hodies, as well as Fab fragments, or the
product of an Fab expression library. Various procedures
known in the art may be used for the production of such
antibodies and fra_ - ~s.
Antibodies generated against the polypeptides
corresponding to a sequence of the present invention can be
obtA;n~ by direct injection of the polypeptides into an
An;m~- or by A~m;n;~tering the polypeptides to an ~n~m~l,
preferably a nnnhnmAn The Ant;ho~y so obtA;n~ will then
bind the polypeptides itself. In this ~-nn~, even a
sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native
polypeptides. Such ~nt;hodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For pr~paration of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line
cultures can be used. Examples include the hybridoma
technique (Kohler and Milstei-n, 1975, Nature, 256:495-497),
the trioma technique, the human B-cell hybridoma technique
(Kozbor et al., 1983, Tmmllnology Today 4:72), and the EBV-
hybridoma technique to produce human monoclonal antibodies
(Cole, et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain
antibodies (U.S. Patent 4,946,778) can be adapted to produce
single chain antibodies to ;m~-lnogenic polypeptide products
of this invention. Transgenic mice may also be used to
generate antibodies.
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The antibodies may also be employed to target breast
cancer cells, ~or example, in a method o~ homi ng interaction
agents which, when c~nt~cting breast cancer cells, destroy
them. This is true since the ~nt~h~dies are speci~ic ~or the
breast speci~ic polypeptides oi~ the present invention. A
linking o~ the interaction agent to the antibody would cause
the interaction agent to be carried directly to the breast.
~ nt;hoA;es o~ this type may also be used to do in vivo
imaging, for example, by labeling the antibodies to
facilitate sc~nn; ng o~ the pelvic area and the breast. One
method for imaging comprises cont~cting any cAn~r cells o~
the breast to be imaged with an anti-breast speci~ic protein-
antibody labeled with a detectable m~rk~r The method is
per~ormed under conditions such that the labeled ~nt; hody
binds to the breast speci~ic polypeptides. In a specific
example, the ~nt;hoA;es interact with the breast, ~or
example, breast cancer cells, and fluoresce upon contact such
that imaging and visibility o~ the breast are ~nh~nced to
allow a determination o~ the diseased or non-diseased state
o~ the breast.
The present invention will be ~urther described with
re~erence to the ~ollowing examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified,
are by weight.
In order to ~acilitate underst~n~; ng o~ the ~ollowing
examples certain ~requently occurring methods and/or terms
will be described.
"Plasmids~ are designated by a lower case p preceded
and/or ~ollowed by capital letters and/or numbers. The
starting plasmids herein are either ~omm~rcially available,
publicly available on an unrestricted basis, or can be
constructed ~rom available plasmids in accord with published
procedures. In addition, equivalent plasmids to those
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described are known in the art and will be apparent to the
ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the
DNA with a restriction enzyme that acts only at certain
sequences in the DNA. The various restriction enzymes used
herein are commercially avA;lAhle and their reaction
conditions, cofactors and other requir~m~nt~ were u~ed as
would be known to the ordinarily skilled artisan. For
analytical purposes, typically 1 ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~1
of buffer solution. For the purpose of isolating DNA
fragments ~or plasmid construction, typically 5 to 50 ~g of
DNA are digested with 20 to 250 units of enzyme in a larger
volume. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the
manu~acturer. Incubation times of about 1 hour at 37 C are
ordinarily used, but may vary in accordance with the
supplier's instructions. After digestion the reaction is
electrophoresed directly on a polyacrylamide gel to isolate
the desired fragment.
Size separation of the cleaved fragments is performed
using 1 percent TAE agarose gel described by Sambrook, et
al., ~Molecular Cloning: A Laboratory ~AnllAl" Cold Spring
Laboratory Press,(1989).
~ 'Oligonucleotides~ refers to either a single stranded
polydeoxynucleotide or two compl~m~ntAry polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not
ligate to another oligonucleotide without adding a phosphate
with an ATP in the presence of a kinase. A synthetic
oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
"Ligation" refers to the process of forming
phosphodiester bonds between two double stranded nucleic acid
fragments (Maniatis, T., et al., Id., p. 146). Unless
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otherwise provided, ligation may be accompl~h~ using known
buffers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 ~Lg of d~ ~o~imately equimolar amounts of
the DNA fra~m~nt~ to be ligated.
Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A.,
Virology, 52:456-457 (1973).
Example 1
Determination of Transcription of a breast sPecific qene
To assess the presence or absence of active
transcription of a breast specific gene RNA, a~,~ximately 6
ml o~ venous blood is obt~;ne~ with a st~n~d venipuncture
technique using heparinized tubes. Whole blood is mixed with
an equal volume of phosphate buffered ~Al in~, which is then
layered over 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in
a 15-ml polystyrene tube. The gradient is centrifuged at
1800 X g for 20 min at 5~C. The lymphocyte and granulocyte
layer (a~L~imately 5 ml) is carefully aspirated and
rediluted up to 50 ml with phosphate-buffered ~line in a 50-
ml tube, which is centrifuged again at 1800 X g for 20 min.
at 5~C. The supernatant is discarded and the pellet
cont~ining nucleated cells is used for RNA extraction using
the RNazole B method as described by the manufacturer (Tel-
Test Inc., Friendswood, TX).
To determine the quantity of mRNA from the gene of
interest, a probe is designed with an identity to at least a
portion of the mRNA sequence transcribed from a human gene
whose coding portion includes a DNA sequence of Figures 1-20
(SEQ ID NO:1-20). This probe is mixed with the extracted RNA
and the m; xe~ DNA and RNA are precipitated with ethanol -70~C
for 15 minutes). The pellet is resuspended in hybridization
buffer and dissolved. The tubes cont~;n;ng the mixture are
incubated in a 72~C water bath ~or 10-15 mins. to denature
the DNA. The tubes are rapidly transferred to a water bath
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at the desired hybridization temperature. Hybridization
temperature depend~ on the G + C content of the DNA.
Hybridization is done for 3 hrs. 0.3 ml of nuclease-S1
buffer is ~ and m; ~ well. 50 ~l of 4.0 M ~m~n;um
acetate and 0.1 M EDTA is added to stop the reaction. The
mixture is extracted with phenol/chloro~orm and 20 ~g of
carrier tRNA is ~ and precipitation is done with an equal
volume of isopropanol. The precipitate is dissolved in 40 ~l
of TE (pH 7.4) and run on an alk~l tn~ agarose gel. Following
electrophoresis, the RNA is microsequenced to con~irm the
nucleotide sequence. (See Favaloro, J. et al., Methods
Enzymol., 65:718 (1980) for a more detailed review).
Two oligonucleotide primers are employed to amplify the
sequence isolated by the above methods. The 5' primer is 20
nucleotides long and the 3' primer is a complimentary
sequence ~or the 3' end of the isolated mRNA. The primers
are custom designed according to the isolated mRNA. The
reverse transcriptase reaction and PCR amplification are
per~ormed sequentially without interruption in a Perkin Elmer
9600 PCR machine (Emeryville, CA). Four hundred ng total RNA
in 20 ~l diethylpyrocarbonate-treated water are placed in a
65~C water ~ath for 5 min. and then quickly chilled on ice
immediately prior to the addition o~ PCR reagents. The 50-~l
total PCR volume consisted o~ 2.5 units Taq polymerase
(Perkin-Elmer). 2 units avian myeloblastosis virus reverse
transcriptase (Boehringer M~nnh~;m, Tn~;~n~rolis~ IN); 200 ~M
each of dCTP, d~TP, dGTP and dTTP (Perkin Elmer); 18 pM each
primer, 10 mM Tris-HCl; 50 mM KCl; and 2 mM MgCl2 (Perkin
Elmer). PCR conditions are as follows: cycle 1 is 42~C for
15 min then 97~C for 15 s (1 cycle); cycle 2 is 95~C for 1
min. 60~C for 1 min, and 72~C for 30 s (15 cycles); cycle 3
is 95~C ~or 1 min. 60~C ~or 1 min., and 72~C ~or 1 min. (10
cycles); cycle 4 is 95~C ~or 1 min., 60~C for 1 min., and
72~C for 2 min. (8 cycles); cycle 5 is 72~C ~or 15 min. (1
cycle); and the final cycle is a 4~C hold until sample is
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taken out of the machine. The 50-~l PCR products are
concentrated down to 10 ~1 with vacuum centrifugation, and a
sample is then run on a thin 1.2 % Tris-borate-EDTA agarose
gel contA; n; ng ethidium bromide. A band of expected size
would indicate that this gene is present in the tissue
assayed. The amount of RNA in the pellet may be ~l~nt;fied
in numerous ways, for example, it may be weighed.
Verification of the nucleotide sequence of the PCR
products is done by microse~nc;ng. The PCR product is
purified with a Qiagen PCR Product Puri~ication Kit (Qiagen,
Chatsworth, CA) as described by the manufacturer. One ~g of
the PCR product undergoes PCR se~lenc;ng by using the Taq
DyeDeoxy Terminator Cycle se~ncing kit in a Perkin-Elmer
9600 PCR machine as described by Applied Biosystems (Foster,
CA). The sequenced product is purified using Centri-Sep
columns (Princeton Separations, Adelphia, NJ) as described by
the company. This product is then analyzed with an ABI model
373A DNA se~l~nc;ng system (Applied Biosystems) integrated
with a Macintosh IIci computer.
Example 2
Bacterial Ex~ression and Purification of the BSG Proteins and
Use For Preparinq a Monoclonal ~nt; hoAy
The DNA sequence encoding a polypeptide of the present
invention, for this example BSG1, ATCC # 97175, is initially
amplified using PCR oligonucleotide primers correspon~;ng to
the 5' sequences of the protein and the vector sequences 3'
to the protein. Additional nucleotides corresponA;ng to the
DNA sequence are added to the 5' and 3' sequences
respectively. The 5' oligonucleotide primer has the sequence
5' GCCACCATGGAl~rl~l~l-~AAG 3~ (SEQ ID NO:21) and cont~;n~ an
NcoI restriction enzyme site followed by 15 nucleotides of
coding sequence starting from the initial amin~ acid of the
processed protein. The 3' sequence 5' GCGCAGATCTGTCT
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CCCCCA~-l--l~C 3' (SEQ ID NO:22) and contains a compl~m~nt~ry
sequence to a BglII restriction enzyme site and is followed
by 18 nucleotides of the nucleic acid sequence ~nCo~;ng the
protein. The restriction enzyme sites correspond to the
restriction enzyme sites on a bacterial expression vector,
pQE-60 (Qiagen, Inc. Chatsworth, CA). pQE-60 ~nro~s
antibiotic resistance (Ampr), a bacterial origin of
replication (ori), an IPTG-regulatable ~---~Ler operator
(P/O), a ribosome b~n~ng site (RBS), a 6-His tag and
restriction enzyme sites. pQE-60 is then digested with NcoI
and BglII. The ampli~ied se~l~nc~s are ligated into pQE-60
and inserted in frame with the sequence ~nro~ng for the
histidine tag and the RBS. The ligation mixture is then used
to trans~orm an E. coli strain M15/rep 4 (Qiagen) by the
procedure described in Sambrook, ~. et al., Molecular
Cloning: A Laboratory ~nll~l, Cold Spring Laboratory Press,
(1989). M15/rep4 cont~nc multiple copies of the plasmid
pREP4, which expresses the lacI repressor and also confers
kanamycin resistance (Kanr). Transformants are identi~ied by
their ability to grow on LB plates and ampicillin/kanamycin
resistant colonies are selected. Plasmid DNA is isolated and
confirmed by restriction analysis.
Clones cont~;n~ng the desired constructs are grown
overnight (O/N) in liquid culture in LB media supplemented
with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of
1:100 to 1:250. The cells are grown to an optical density
600 (O.D.~') o~ between 0.4 and 0.6. IPTG ("Isopropyl-B-D-
thiogalacto pyranoside") i8 then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene
expression. Cells are grown an extra 3 to 4 hours. Cells
are then harvested by centri$ugation. The cell pellet is
solubilized in the chaotropic agent 6 Molar Guanidine HCl.
A~ter clarification, solubilized protein i8 purified ~rom
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this solution by chromatography on a Nickel-Chelate column
under conditions that allow for tight h; nA; n~ by proteins
r~nt~;n~n~ the 6-His tag (Hochuli, E. et al., J.
C~ tography 411:177-184 (1984)). BSG1 protein (,90~ pure)
is eluted ~rom the column in 6 molar guanidine HCl pH 5.0 and
~or the purpose o~ renaturation adjusted to 3 molar guanidine
HCl, lOOmM sodium phosphate, 10 mmolar gl~ltAth;one (reduced)
and 2 mmolar glutathione (oxidized). After incubation in
this solution ~or 12 hours the protein is dialyzed to 10
mmolar sodium phosphate.
The protein puri~ied in this m~nner may be used as an
epitope to raise monoclonal antibodies speci~ic to such
protein. The monoclonal antibodies generated against the
polypeptide the isolated protein can be obtA; n~A by direct
injection o~ the polypeptides into an An;m~l or by
AAm;n~stering the polypeptides to an An;m-l. The antibodies
so obtained will then bind to the protein itsel~. Such
antibodies can then be used to isolate the protein ~rom
tissue expressing that polypeptide by the use o~ an, for
example, ELISA assay.
Exam~le 3
Preparation o~ cDNA Libraries ~rom Breast Tissue
Total cellular RNA is prepared ~rom tissues by the
guanidinium-phenol method as previously described (P.
Chomczynski and N Sacchi, Anal. Biochem., 162: 156-159
(1987)) using RNAzol (Cinna-Biotecx). An additional ethanol
precipitation o~ the RNA is included. Poly A mRNA is
isolated ~rom the total RNA using oligo dT-coated latex beads
(Qiagen). Two rounds of poly A selection are per~ormed to
ensure better separation ~rom non-polyadenylated material
when suf~icient ~uantities o~ total RNA are available.
The mRNA selected on the oligo dT is used ~or the
synthesis o~ cDNA by a modi~ication of the method o~ Gob~ler
and Ho~man (Gobbler, U. and B.J. Ho~man, 1983, Gene,
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25:263). The first strand synthesis is performed using
either Moloney murine sarcoma virus reverse transcriptase
(Stratagene) or Superscript II (RNase H minus Moloney murine
reverse transcriptase, Gibco-BRL). First strand synthesis is
primed u~ing a primer/linker co~t~; n; ng an Xho I restriction
site. The nucleotide mix used in the synthesis c~nt~; n~
methylated dCTP to prevent restriction within the cDNA
sequence. ~or second-strand synthesis E. coli polymerase
Klenow fragment is used and [32p~ -d~TP is incorporated as a
tracer of nucleotide incorporation.
Following 2nd strand synthesis, the cDNA is made blunt
ended using either T4 DNA polymerase or Klenow fragment. Eco
RI adapters are A~e~ to the cDNA and the cDNA is restricted
with Xho I. The cDNA is size fractionated over a Sephacryl
S-500 column (Pharmacia) to remove excess linkers and cDNAs
under approximately 500 base pairs.
The cDNA is cloned unidirectionally into the Eco RI-Xho
I ~ites of either pBluescript II phagemid or lAmh~A Uni-zap
XR (Stratagene). In the case of cloning into pBluescript II,
the plasmids are electroporated into E.coli SURE competent
cells (Stratagene). When the cDNA is cloned into Uni-Zap XR
it is packaged using the Gigipack II packaging extract
(Stratagene). The packaged phage is used to infect SURE
cells and amplified. The pBluescript phagemid contA;n;ng the
cDNA inserts are excised from-the lAmh~A Zap phage using the
helper phage ~xAssist (Stratagene). The rescued phagemid is
plated on SOLR ~.coli cells (Stratagene).
Pre~aration of Seauencing TemPlates
Template DNA for se~lenc;~g is prepared by 1) a boiling
method or 2) PCR amplification.
The boiling method is a modification of the method of
Holmes and Quigley (Holmes, D.S. and M. Quigley, 1981, Anal.
Biochem., 114:193). Colonies from either cDNA cloned into
Bluescript II or rescued Bluescript phagemid are grown in an
enriched bacterial media overnight. 400 ~1 of cells are
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centri~uged and resuspended in STET (O.lM NaCl, lOmM TRIS Ph
8.0, 1.0 mM EDTA and 5~ Triton X-100) including lysozyme (80
~g/ml) and RNase A (4 ~g/ml). Cells are boiled for 40
seconds and centrifuged for 10 minutes. The supernatant is
removed and the DNA is precipitated with PBG/NaCl and ~ che~
with 70~ ethanol (2x). Templates are resuspended in water at
d~~ tely 250 ng/~
Preparation of templates by PCR is a modification of the
method of Ros~nth~l et al. (Rospnth~l~ et al., Nucleic Acids
Res., 1993, 21:173-174). Colonies cont~n~ng cDNA cloned
into pBluescript II or rescued pBluescript phagemid are grown
overnight in LB cont~;n;ng ampicillin in a 96 well tissue
culture plate. Two ~1 of the cultures are used as template
in a PCR reaction (Saiki, RK, et al., Science, 239:487-493,
1988; and Saiki, RR, et al., Science, 230:1350-1354, 1985)
using a tricine buffer system (Ponce and Micol., Nucleic
Acids Res., 1992, 20:1992.) and 200 ~M dNTPs. The primer set
chosen for amplification of the templates is outside of
primer sites chosen ~or sequencing of the templates. The
primers used are 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO:23)
which is 5' of the M13 reverse sequence in pBluescript and
5'-GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:24) which is 3' o~ the
M13 forward primer in pBluescript. Any primers which
correspond to the sequence flanking the M13 forward and
reverse sequences can be- used. Perkin-Elmer 9600
thermocyclers are used ~or amplification of the templates
with the ~ollowing cycler conditions: 5 min at 94~C (1
cycle); (20 sec at 94~C); 20 sec at 55~C (1 min at 72~C) (30
cycles); 7 min at 72~C (1 cycle). Following amplification
the PCR templates are precipitated using PEG/NaC1 and washed
three times with 70~ ethanol. The templates are resuspended
in water.
Example 4
Isolation of a Selected Clone From Breast Tissue
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Two approaches are used to isolate a particular clone
from a cDNA library prepared from human breast ti~sue.
In the first, a clone is isolated directly by screening
the library using an oligonucleotide probe. To isolate a
particular clone, a specific oligonucleotide with 30-40
nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to one of the partial sequences
described in this application. The oligonucleotide is
labeled with 32p_ -ATP using T4 polynucleotide kinase and
purified according to the st~n~d protocol (~n~t;S et al.,
Molecular Cloning: A Laboratory ~n~ , Cold Spring ~A~h
Press, Cold Spring, NY, 1982). The T-~mh~ cDNA library is
plated on 1.5% agar plate to a density of 20,000-50,000
pfu/150 mm plate. These plates are screened using Nylon
".~"~Lanes according to the st~n~d phage screening protocol
(Stratagene, 1993). Specifically, the Nylon membrane with
denatured and fixed phage DNA is prehybridized in 6 x SSC, 20
mM NaH2PO4, 0.4% SDS, 5 x Denhardt~s 500 ~g/ml denatured,
sonicated s~lm~n sperm DNA; and 6 x SSC, 0.1% SDS. After one
hour of prehybridization, the membrane is hybridized with
hybridization buffer 6 x SSC, 20 mM NaH2PO4, 0.4% SDS, 500
~g/ml denatured, sonicated salmon sperm DNA with 1 x 1o6
cpm/ml 32P-probe overnight at 42~C. The ",~"~ldne is w~he~ at
45-50~C with washing buffer 6 x SSC, 0.1% SDS for 20-30
minutes dried and exposed to Kodak X-ray film overnight.
Positive clones are isolated and puri~ied by secondary and
tertiary screening. The purified clone sequenced to verify
its identity to the partial sequence described in this
app~ication.
An alternative approach to screen the cDNA library
prepared from ~n~n breast tissue is to prepare a DNA probe
corresponding to the entire partial sequence. To prepare a
probe, two oligonucleotide primers of 17-20 nucleotides
derived from bo~h ends of the partial sequence reported are
synthesized and purified. These two oligonucleotides are
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used to ampli~y the probe using the cDNA library template.
The DNA template is prepared ~rom the phage lysate o~ the
cDNA library according to the stAn~d phage DNA preparation
protocol tManiatis et al.). The polymerase chain reaction is
carried out in 25 ~l reaction mixture with 0.5 ~g of the
above cDNA template. The reaction mixture is 1.5-5 mM MgCl2,
O.01~ (w/v) gelatin, 20 ~M each of dATP, dCTP, dGTP, dTTP, 25
pmol of each primer and 0.25 Unit o~ Taq polymerase. Thirty
~ive cycles o~ PCR (denaturation at 94~C ~or 1 min; ~nne~l;ng
at 55~C ~or 1 min; elongation at 72~C ~or 1 min) are
performed with the Perkin-Blmer Cetus automated thermal
cycler. The ampli~ied product is analyzed by agarose gel
electrophoresis and the DNA band with expected molecular
weight is excised and puri~ied. The PCR product is veri~ied
to be the probe by subcloning and se~l~nc~ng the DNA product.
The probe is labeled with the Multiprime DNA Labelling System
(Amersham) at a speci~ic activity c 1 x 109 dmp/~g. This
probe is used to screen the l~mh~A cDNA library according to
Stratagene's protocol. Hybridization is carried out with 5X
TEN 920XTEN:0.3M Tris-HCl pH 8.0, 0.02M EDTA and 3MNaCl), 5X
Denhardt's, 0.5% sodium pyrophosphate, 0.1% SDS, 0.2 mg/ml
heat denatured salmon sperm DNA and 1 x 1o6 cpm/ml o~ t32P]-
labeled probe at 55~C ~or 12 hours. The ~ilters are w~:hF~
in 0.5X TEN at room temperature ~or 20-30 min., then at 55~C
~or 15 min. The ~ilters are dried and autoradiographed at -
70~C using Kodak XAR-5 ~ilm. The positive clones are
puri~ied by secon~ry and tertiary screening. The sequence
o~ the isolated clone are veri~ied by DNA se~l~n~ng
General procedures ~or obt~;n;ng complete sequences ~rom
partial sequences described herein are summarized as ~ollows;
Procedure 1
Selected human DNA ~rom the partial sequence clone (the
cDNA clone that was sequenced to give the partial sequence)
is puri~ied e.g., by endonuclease digestion using ~co-R1, gel
electrophoresis, and isolation o~ the clone by removal ~rom
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low melting agarose gel. The isolated insert DNA, is
radiolabeled e.g., with 32p labels, preferably by nick
translation or r~n~m primer labeling. The labeled in~ert is
used as a probe to screen a lAmb~ phage cDNA library or a
plasmid cDNA library. Colonies con~A;n;ng clones related to
the probe cDNA are identi~ied and puri~ied by known
purification methods. The ends of the newly puri~ied clones
are nucleotide sequenced to irl~nt; fy full length sequences.
Complete sequencing of full length clones is then performed
by R~o~llclease III digestion or primer walking. Northern
blots of the mRNA ~rom various tissues using at least part of
the deposited clone from which the partial sequence is
obtained as a probe can optionally be performed to check the
size of the mRNA against that of the purported full length
cDNA.
The ~ollowing procedures 2 and 3 can be used to obtain
full length genes or full length coding portions of genes
where a clone isolated from the deposited clone mixture does
not contain a full length sequence. A library derived from
human breast tissue or from the deposited clone mixture is
also applicable to obtAin;ng full length sequences from
clones obtained from sources other than the deposited mixture
by use of the partial sequences of the present invention.
Pr~ e 2
RACE Protocol For Recovery of Full-Length Genes
Partial cDNA clones can be made full-length by utilizing
the rapid amplification of cDNA ends (RACE) procedure
described in Frohman, M.A., Dush, M.K. and Martin, G.R.
(1988) Proc. Nat'l. Acad. Sci. USA, 85:8998-9002. A cDNA
clone missing either the 5~ or 3~ end can be reconstructed to
include the absent base pairs extending to the translational
start or stop codon, respectively. In most cases, cDNAs are
missing the start of translation therefor. The following
brie~ly describes a modification of this original 5' RACE
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procedure. Poly A+ or total RNA is reverse transcribed with
Superscript II (Gibco/BRL) and an antisense or complementary
primer speci~ic to the cDNA sequence. The primer is removed
~rom the reaction with a Microcon Concentrator (Amicon). The
first-strand cDNA is then tailed with dATP and terminal
deoxynucleotide trans~erase (Gibco/BRL). Thus, an anchor
sequence is produced which is ne~e~ ~or PCR ampli~ication.
The second strand is synthesized ~rom the d~-tail in PCR
bu~er, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT
primer cont~;n;ng three adjacent restriction sites (XhoI,
SalI and ClaI) at the 5' end and a primer contA;n;ng just
these restriction sites. This double-stranded cDNA is PCR
ampli~ied ~or 40 cycles with the same primers as well as a
nested cDNA-speci~ic antisense primer. The PCR products are
size-separated on an ethidium bromide-agarose gel and the
region o~ gel c~nt~; n; ng cDNA products the predicted size of
missing protein-coding DNA is removed. cDNA is puri~ied ~rom
the agarose with the Magic PCR Prep kit (Promega),
restriction digested with XhoI or SalI, and ligated to a
plasmid such as pBluescript SRII (Stratagene) at ShoI and
EcoRV sites. This DNA is trans~ormed into bacteria and the
plasmid clones sequenced to identi~y the correct protein-
coding inserts. Correct 5' ends are con~irmed by comparing
this sequence with the putatively identi~ied homologue and
overlap with the partial cDNA clone.
Several quality-controlled kits are available ~or
purchase. S;m; 1 Ar reagents and methods to those above are
supplied in kit ~orm $rom Gibco/BRL. A second kit is
available ~rom Clontech which is a modi~ication o~ a related
technique, SLIC (single-stranded ligation to single-stranded
cDNA) developed by Dumas et al. (Dumas, J.B., Edwards, M.,
Delort, J. and Mallet, Jr., 1991, Nucleic Acids Res.,
19:5227-5232). The major di~erences in procedure are that
the RNA is alkaline hydrolyzed a~ter reverse transcription
and RNA ligase is used to join a restriction site-cont~;n;ng
CA 0222~824 1997-12-24
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~nchor primer to the first-strand cDNA. This obviates the
necessity for the dA-~ ng reaction which results in a
polyT stretch that is difficult to sequence past.
An alternative to generating 5' cDNA from RNA is to use
cDNA library double-stranded DNA. An asymmetric PCR-
amplified antisense cDNA strand is synthesized with an
antisense cDNA-specific primer and a plasmid-anchored primer.
These primers are le~ ~d and a symmetric PCR reaction is
performed with a nested cDNA-specific antisense primer and
the plasmid-~ncho~ed primer.
Pror~ ~e 3
RNA Ligase Protocol For Generating The 5' End Se~n~es To
Obtain Full ~ength Gene~
Once a gene of interest is identified, several methods
are available for the identi~ication o~ the 5~ or 3' portions
of the gene which may not be present in the original
deposited clone. These methods include but are not limited
to filter probing, clone enrirhm~nt using specific probes and
protocols similar and identical to 5' and 3' RACE. While the
full length gene may be present in a library and can be
identi~ied by probing, a useful method for generating the 5~
end is to use the existing sequence information from the
original partial sequence to generate the missing
information. A method similar to 5~ RACE is available for
generating the misQing 5~ end of a desired full-length gene.
(This method was pnhl~sh~tl by Fr~ t-Racine et al, Nucleic
Acids Res., 21(7):1683-1684 (1993). Briefly, a specific RNA
oligonucleotide is ligated to the 5~ ends of a population of
RNA presumably cont~n;ng full-length gene RNA transcript and
a primer set cont~ n~ ng a primer specific to the ligated RNA
oligonucleotide. A primer specific to a known sequence (EST)
of the gene of interest is used to PCR amplify the 5' portion
of the desired full length gene which may then be sequenced
and used to generate the full length gene. This method
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starts with total RNA isolated from the desired source, poly
A RNA may be used but is not a prerequisite for this
procedure. The RNA preparation may then be treated with
phosphatase if necessary to ~1 ;m~ n~te 5' phosphate groups on
degraded or damaged RNA which may inter~ere with the later
RNA ligase step. The phosphatase if used is then inactivated
and the RNA is treated with tobacco acid pyrophosphatase in
order to remove the cap structure present at the 5' ends of
messenger RNAs. This reaction leaves a 5~ phosphate group at
the 5' end of the cap-cleaved RNA which can then be ligated
to an RNA oligonucleotide using T4 RNA ligase. This modi~ied
RNA preparation can then be used as a template for first
strand cDNA synthesis using a gene-speci~ic oligonucleotide.
The first stand synthesis reaction can then be used as a
template for PCR amplification of the desired 5' end using a
primer specific to the ligated RNA oligonucleotide and a
primer specific to the known sequence (EST) of the gene of
interest. The resultant product is then sequenced and
analyzed to confirm that the 5' end sequence belongs to the
partial sequence.
ExamPle 5
Cloninq and exPression o~ BSG1 usin~ the baculovirus
expression system
The DNA sequence encoding the ~ull length BSG1 protein,
ATCC # 97175, was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' AAAGGA~I~CCATCATGG
Al~rl-l-lCAAGAAG 3~ (SEQ ID NO:25) and contains a BamHI
restriction enzyme site (in bold) followed by 8 nucleotides
resembling an efficient signal for the initiation of
translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,
196:947-950 (1987) of the BSG1 gene (the initiation codon for
translation ~ATG~ is underlined).
The 3~ primer has the sequence 5~ A~ATCTAGACTAGTCTCCCCC
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ACTCTG 3' (S~Q ID NO:26) and contains the cleavage site for
the restriction ~n~onllclease XbaI and 21 nucleotides
complementary to the 3' sequence of the BSG1 gene. The
amplified sequences were isolated from a 1~ agarose gel using
a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Ca.). The fragment was then digested with the
A , ~n~nll~leases BamHI and XbaI and then purified again on a 1
agarose gel. This fragment is designated F2.
The vector pA2 (modification of pVL941 vector, discussed
below) is used for the expression of the BSG1 protein using
the baculovirus expression system (for review see: Summers,
M.D. and Smith, G.E. 1987, A ~-nll~l of methods for
baculovirus vectors and insect cell culture procedures, Texas
Agricultural Bxper~m~nt~l Station Bulletin No. 1555). This
expression vector ront~nfi the strong polyhedrin promoter of
the Autographa cali$ornica nuclear polyhedrosis virus
(AcMNPV) followed by the recognition sites for the
restriction ~nnllcleases BamHI and XbaI. The
polyadenylation site of the simian virus (SV)40 is used for
efficient polyadenylation. For an easy selection of
recombinant virus the beta-galactosidase gene from E.coli is
inserted in the same orientation as the polyhedrin promoter
followed by the polyadenylation signal of the polyhedrin
gene. The polyhedrin sequences are $1anked at both sides by
viral sequences for the cell-mediated homologous
recombination of co-transfected wild-type viral DNA. Many
other baculovirus vectors could be used in place of pA2 such
as pRG1, pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers,
M.D.~ Virology, 170:31-39).
The plasmid was digested with the restriction enzymes
BamHI and XbaI and dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The DNA was then
isolated $rom a 1~ agarose gel using the commercially
available kit ("Geneclean" BI0 101 Inc., La Jolla, Ca.).
This vector DNA is designated V2.
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Fragment F2 and the dephosphorylated plasmid pA2 were
ligated with T4 DNA ligase. E.coli HB101 cells were then
transformed and bacteria ;~nt;fied that ront~;ne~ the
plasmid (pBacBSG1) with the BSG1 gene using the enzymes BamHI
and XbaI. The sequence of the cloned fragment was confirmed
by DNA seqllencing.
5 ~g of the plasmid pR~cR-SG1 was co-transfected with 1.0
~g of a commercially av;~ hl e l;n~ized baculovirus
("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.)
using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the p~asmid
pBacBSG1 were m;xr~A in a sterile well o~ a microtiter plate
cont~;n;ng 50 ~l of serum free Grace's medium (Life
Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l
Lipofectin plus 90 ~l Grace's medium were added, mixed and
incubated for 15 minutes at room temperature. Then the
transfection mixture was added drop-wise to the Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate
with 1 ml Grace's medium without serum. The plate was rocked
back and forth to mix the newly added solution. The plate
was then incubated ~or 5 hours at 27~C. A~ter 5 hours the
transfection solution was removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum
was ~ ~. The plate was put back into an incubator and
cultivation cont;nlleA at 27~C ~or ~our days.
After four days the supernatant was collected and a
plague assay performed similar as described by Summers and
Smith (supra). As a modification an agarose gel with "Blue
Gal" ~Li~e Technologies Inc., Gaithersburg) was used which
allows an easy isolation of blue st~; n~ plaques. (A
detailed description of a "plaque assay" can also be found in
the user's guide for insect cell culture and b~culovirology
distributed by Life ~echnologies Inc., Gaithersburg, page 9-
10) .
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Four days after the serial dilution, the virus was added
to the cells and blue st~n~ plaques were picked with the
tip of an Eppendor~ pipette. The agar ront~;ning the
recombinant viruses was then resuspended in an Eppendorf tube
cont~;n;ng 200 ~l of Grace's medium. The agar was removed by
a brief centrifugation and the supernatant cont~; n; ng the
recomh~n~nt baculovirus was used to infect Sf9 cells seeded
in 35 mm dishes. Four days later the supernatants of these
culture ~;Ch~s were harvested and then stored at 4~C.
S~9 cells were grown in Grace~s medium supplemented with
10% heat-inactivated FBS. The cells were infected with the
recombinant baculovirus V-BSG1 at a multiplicity of infection
(MOI) of 2. Six hours later the medium was removed and
replaced with SF900 II medium minus methionine and cysteine
(Li~e Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci
o~ 35S-methionine and 5 ~Ci 35S cysteine (Amersham) were added.
The cells were further incubated for 16 hours before they
were harvested by centrifugation and the labelled proteins
visualized by SDS-PAGE and autoradiography.
BxamPle 6
Expression o~ Recombinant BSG1 in COS cells
The expression of plasmid, BSG1 HA is derived ~rom a
vector pcDNAI/Amp (Invitrogen) cont~;n;ng: 1) SV40 origin of
replication, 2) ampicillin- resistance gene, 3) E.coli
replication origin, 4) CMV promoter followed by a polylinker
region, an SV40 intron and polyadenylation site. A DNA
~ragment encoding the entire precursor and a HA tag ~used in
frame to its 3~ end was cloned into the polylinker region o~
the vector, there~ore, the recombinant protein expression is
directed under the CMV promoter. The HA tag corresponds to
an epitope derived from the influenza hemagglutinin protein
as previously described (I. Wilson, H. Niman, R. Heighten, A
Cherenson, M. Connolly, and R. T.~rn~r, 1984, Cell 37:767,
(1984)). The in~usion of HA tag to the target protein allows
CA 02225824 1997-12-24
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easy detection o~ the re~omh~nAnt protein with an antibody
that recognizes the HA epitope.
The plasmid construction strategy is described as
~ollows:
The DNA sequence enco~; ng BSGl, ATCC # 97175, was
constructed by PCR using two primers: the 5' primer AAAGGA
T~CCCC~CCATCATGGA~ lCAAGA~G 3' (SEQ ID N0:27) rontA~n~ a
BamHI site ~ollowed by 18 nucleotides o~ BSGl coding sequence
starting ~rom the initiation codon; the 3' sequence AAATC
TAGAcTA~AGcGTA~ Alw~lA~ L~ 'A~-J'~
3' (SEQ ID NO:28) contA;ns complementary se~-Pnc~s to an XbaI
site, translation stop codon, HA tag and the last 18
nucleotides o~ the BamHI coding sequence (not including the
stop codon). There~ore, the PCR product cont~;nC an BamXI
site, BSGl coding sequence ~ollowed by HA tag ~used in ~rame,
a translation termination stop codon next to the HA tag, and
an XbaI site. The PCR ampli~ied DNA ~ragment and the vector,
pcDNAI/Amp, were digested with BamHI and XbaI restriction
enzyme and ligated. The ligation mixture was trans~ormed into
E. coli strain SURE (available ~rom Stratagene Cloning
Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037)
the trans~ormed culture was plated on ampicillin media plates
and resistant colonies were selected. Plasmid DNA was
isolated ~rom trans~ormants and ~xAm;ne~ by restriction
analysis ~or the presence o~ the correct ~ragment. For
expression o~ the rec~mh;nAnt BSG protein, COS cells were
trans~ected with the expression vector by DEAE-DEXTRAN method
(~. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory MAnll~Al~ Cold Spring Laboratory Pre~s, (1989)).
The expression o~ the BSG HA protein was detected by
radiolab~ll~ng and immunoprecipitation method (E. Harlow, D.
Lane, Antibodies: A Laboratory MAnllAl, Cold Spring Harbor
Laboratory Press, (1988)). Cells were lA~lled ~or 8 hours
with 35S-cysteine two days post trans~ection. Culture media
was then collected and cells were lysed with detergent (RIPA
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buffer (150 mM NaCl, 1~ NP-40, 0.1~ SDS, 1~ NP-40, 0.5~ DOC,
50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).
Both cell lysate and culture media were precipitated with an
HA specific monoclonal ~nt; ho~y ~ Proteins precipitated were
analyzed on 15% SDS-PAGE gels.
Numerous modi~ications and variations o~ the present
invention are possible in light of the above teachings and,
there~ore, within the scope of the appended claims, the
invention may be practiced otherwise than as particularly
described.
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