The present invention relates to a novel BCSG1 protein. In particular,
isolated nucleic acid molecules are provided encoding the human
BCSG1 protein. BCSG1 polypeptides are also provided, as are vectors,
host cells and recombinant methods for producing the same. Also
provided are diagnostic methods for detecting breast cancer.
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about 1 to about
127 in SEQ ID NO:2; (b) a nucleotide sequence encoding a polypeptide
comprising amino acids from about 2 to about 127 in SEQ ID NO:2;
(c) a nucleotide sequence encoding a polypeptide having the amino
acid sequence encoded by the cDNA clones contained in ATCC Deposit
No. 97175 or 97856; (d) and a nucleotide sequence of a fragment
of the sequence shown in SEQ ID NO:1, wherein said fragment comprises
at least 50 contiguous nucleotides of SEQ ID NO:1 (e) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c) or (d).
2. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of an BCSG1 polypeptide having an amino acid sequence in (a), (b),
(c) or (d) of claim 1.
3. The isolated nucleic acid molecule of claim 2, which encodes
an epitope-bearing portion of a BCSG1 polypeptide selected from
the group consisting of: a polypeptide comprising amino acid residues
from about 94 to about 107 in FIG. 1 (SEQ ID NO:2); and a polypeptide
comprising amino acid residues from about 120 to about 127 in FIG.
1 (SEQ ID NO:2).
4. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
5. A recombinant vector produced by the method of claim 4.
6. A method of making a recombinant host cell comprising introducing
the recombinant vector of claim 5 into a host cell.
7. A recombinant host cell produced by the method of claim 6.
8. A recombinant method for producing any of the BCSG1 polypeptides,
comprising culturing the recombinant host cell of claim 7 under
conditions such that said polypeptide is expressed and recovering
9. An isolated BCSG1 polypeptide having an amino acid sequence
at least 95% identical to a sequence selected from the group consisting
of: (a) amino acids from about 1 to about 127 in SEQ ID NO:2; (b)
amino acids from about 2 to about 127 in SEQ ID NO:2; (c) the amino
acid sequence of the BCSG1 polypeptide having the amino acid sequence
encoded by the cDNA clones contained in ATCC Deposit No. 97856 or
97175; and (d) the amino acid sequence of an epitope-bearing portion
of any one of the polypeptides of (a), (b) or (c).
10. The isolated polypeptide of claim 1 comprising an epitope-bearing
portion of the BCSG1 protein, wherein said portion is selected from
the group consisting of: a polypeptide comprising amino acid residues
from about 94 to about 107 in FIG. 1 (SEQ ID NO:2); and a polypeptide
comprising amino acid residues from about 120 to about 127 in FIG.
1 (SEQ ID NO:2).
11. An isolated antibody that binds specifically to a BCSG1 polypeptide
of claim 9.
12. A method for breast tumor diagnosis in an individual comprising
assaying the expression level of the gene encoding the BCSG1 protein
in cells or body fluid of the individual and comparing the gene
expression level with a standard BCSG1 gene expression level, whereby
an increase in the gene expression level over the standard is indicative
of malignant breast cancer.
CROSS-REFERENCE TO RELATED APPLICATIONS
 This application is a divisional of U.S. Application Ser.
No. 09/017,715, filed Feb. 3, 1998, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No. 60/037,080
filed Feb. 3, 1997. Each of these applications is herein incorporated
by reference in their entirety.
FIELD OF THE INVENTION
 The present invention relates to a novel breast cancer specific
marker. More specifically, isolated nucleic acid molecules are provided
encoding a human breast cancer specific gene 1 (BCSG1). BCSG1 polypeptides
are also provided, as are vectors, host cells and recombinant methods
for producing the same. Also provided are diagnostic methods for
detecting breast cancer. The invention further provides an isolated
BCSG1 polypeptide having an amino acid sequence encoded by a polynucleotide
BACKGROUND OF THE INVENTION
 More than 190,000 new cases of breast cancer are diagnosed
in the United States every year, with incidence increasing by approximately
1% annually (Goldhirsch, A., JNCI 97:1141 -1145 (1995); Emster,
V. L., et al., JAMA 275:913-918 (1996)). Studies linked to the discovery
of new genetic markers and additional risk factors could provide
new information that fits into the complex patient management issues
surrounding breast cancer. Many new prognostic and predictive factors
have been proposed and studied for breast cancer. HER 2/neu positive
tumors respond poorly to endocrine treatment (Allred D. C., et al.,
J. Clin Oncol. 10:599-605 (1992); Gusterson B. A., et al., J. Clin
Oncol. 10:1049-56 (1992)). p53 alteration has an indication of poorer
prognosis and poor response to tamoxifen (Bergh J., et al., Nature
Medicine 10: 1029-34 (1995); Elledge R. M., et al., Breast Cancer
Res Treat 27:95-102 (1993)). The lack of Nm23 expression has an
indicative value of metastatic potential and poor prognosis in invasive
ductal carcinoma (Steeg P. S., et al., Breast Cancer Res Treat 25:175-87
(1993)). Cathepsin D, a protease suggested to have a role in breast
cancer, appears to affect the potential for invasive growth (Velculescu,
V. E., et al., Science 270:484-7 (1995); Schena, M., et al., Science
270:467-70 (1995); M. L. Angerer & R. C. Angerer, In: In situ
hybridization, D. Rickwood and B. D. Hames (ed.). London: LRL Press.,
(1992), pp.15-32; Femo M., et al., Eur J. Cancer 30A:2042-8 (1994)).
Positive immunostaining of tumor sections with Factor VIII antibodies
seems to be a marker for angiogenesis (Klijn J. G. M., et al., Breast
Cancer 18:165-98 (1993); Harris A. L., et al., Eur J. Cancer 31A:831-2
(1995); Gasparini G., et al., JNCI 85:1206-19 (1993) (errata JNCI
85:1605 (1993))). It has been postulated that these tumors are targets
for anti-angiogenesis drug treatment. Expression of the mdr-1 gene
is proposed to be an indicator of multidrug resistance (Harris A.
L., et al., Eur J. Cancer 31A:831-2 (1995); Gasparini G., et al.,
JNCI 85:1206-19 (1993) (errata JNCI 85:1605 (1993))). Poor response
to endocrine therapy has been indicated for uPA/PAI-1, a plasminogen
activator/inhibitor (Foekens J. A., et al., JNCI 87:751-6(1995)).
Also receiving major attention are the familial breast cancerrelated
genes, BRCA1 and BRCA2 (Miki, Y., et al., Science 266:66-71 (1994);
Wooster, R., et al., Science 265:2088-2090 (1994); Futreal, P. A.,
et al., Science 266:120-122 (1994)).
 Thus, the onset and progression of breast cancer is accompanied
by multiple genetic changes that result in qualitative and quantitative
alterations in individual gene expression (Porter-Jordan, K. &
Lippman, M. E., Hematol. Oncol. Clin. N. Am. 8:73-100 (1994)). Many
of these quantitative genetic changes may manifest themselves as
alterations in the cellular complement of novel transcribed mRNAs.
Identification of these mRNAs could provide clinically useful information
for patient management and prognosis while enhancing our understanding
of breast cancer pathogenesis.
 Identification of quantitative changes in gene expression
that occur in the malignant mammary gland may yield novel molecular
markers which may be useful in the diagnosis and treatment of human
breast cancer. Several differential cloning methods, such as differential
display polymerase chain reaction and subtractive hybridization,
have been used to identify the genes differentially expressed in
breast cancer biopsies, as compared to normal breast tissue controls
(Watson, M. A. & Fleming, T. P., Cancer Res. 54:4598-4602 (1994);
Sager, R., et al., FASEB J. 7:964-970 (1993); Chen, Z. & Sager,
R., Mol. Med. 1:153-160 (1995); Zhang, M., et al., Cancer Res. 55:2537-2541
(1995); Zou, Z., et al., Science 263:526-529)). However, these investigations
have involved the relatively time- and labor-intensive steps of
subcloning, library screening, and cDNA sequencing of individual
genes (Sager, R., et al., FASEB J 7:964-970 (1993); Liang, P., et
al., Cancer Res. 52:6966-6968 (1992)).
 Although pathological endpoints such as tumor size, lymph
node status and status of estrogen receptor and progesterone receptor
remain the most useful guides in prognosis and selecting treatment
strategies for breast cancer (Manning, D. L., et al., Acta Oncol.
34:641-646 (1995)), there is still a need to further investigate
the molecular mechanisms that determine the properties of an individual
tumor e.g., probability of metastasis. While numerous prognostic
factors have been identified, few have contributed to defining clinical
response to therapy.
SUMMARY OF THE INVENTION
 The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding the BCSG1 polypeptide having
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino
acid sequence encoded by the cDNA clones deposited in a bacterial
host as ATCC Deposit Number 97175 on Jun. 2, 1995 or as ATCC Deposit
Number 97856 on Jan. 23, 1997.
 The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors,
as well as to methods of making such vectors and host cells and
for using them for production of BCSG1 polypeptides or peptides
by recombinant techniques.
 In accordance with another aspect of the present invention,
there is provided a method of and products for diagnosing breast
cancer metastases by detecting an altered level of a polypeptide
corresponding 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.
 The present invention further relates to antibodies specific
to the polypeptides of the present invention, which may be employed
to detect breast cancer cells or breast cancer metastasis.
 The polynucleotides and polypeptides described herein are
useful as markers for breast cancer.
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino
acid (SEQ ID NO:2) sequences of BCSG1. The protein has a deduced
molecular weight of about 14.2 kDa. The predicted amino acid sequence
of the BCSG1 protein is also shown.
 FIG. 2 shows the differential cDNA sequencing approach.
Messenger RNAs from normal and diseased tissues were extracted and
used for making the cDNA libraries. These libraries are searched
by EST method involving automated DNA sequence analysis of randomly
selected cDNA clones. The ESTs with overlapping sequences were grouped
into unique EST groups. Each unique EST group, which does not overlap
to each other in sequence, was analyzed for its relative expression
by examining the number of expressed individual EST in the libraries
of normal vs diseased tissues. Three EST groups are listed. Blue
EST group represents gene that is equally expressed in both libraries.
Green EST group represents gene that is more expressed in normal
library compared to diseased library. Red EST group represent gene
that is more expressed in diseased library compared to normal library.
 FIG. 3 shows a schematic representation of the pHE4-5 expression
vector (SEQ ID NO:10) and the subcloned BSCG-1 cDNA coding sequence.
The locations of the kanamycin resistance marker gene, the BSCG-1
coding sequence, the oriC sequence, and the lacIq coding sequence
 FIG. 4 shows the nucleotide sequence of the regulatory elements
of the pHE promoter (SEQ ID NO:11). The two lac operator sequences,
the Shine-Delgamo sequence (S/D), and the terminal HindiI and NdeI
restriction sites (italicized) are indicated.
 The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding a BCSG1 polypeptide having
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2), which was
determined by sequencing a cloned cDNA. The BCSG1 protein of the
present invention shares sequence homology with human AD amyloid.
The nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) was obtained
by sequencing the 184,497 clone, which was deposited on Jan. 23,
1997 at the American Type Culture CollectionPatent Depository, 10801
University Boulevard, Manassas, Va. 20110-2209, and given accession
number 97856. The deposited clone is contained in the pBluescript
SK(-) plasmid (Stratagene, La Jolla, Calif.). The BSCG-1 gene was
also deposited on Jun. 2, 1995 at the American Type Culture CollectionPatent
Depository, 10801 University Boulevard, Manassas, Va. 20110-2209,
and given accession number 97175.
 Nucleic Acid Molecules
 Unless otherwise indicated, all nucleotide sequences determined
by sequencing a DNA molecule herein were determined using an automated
DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.),
and all amino acid sequences of polypeptides encoded by DNA molecules
determined herein were predicted by translation of a DNA sequence
determined as above. Therefore, as is known in the art for any DNA
sequence determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90% identical,
more typically at least about 95% to at least about 99.9% identical
to the actual nucleotide sequence of the sequenced DNA molecule.
The actual sequence can be more precisely determined by other approaches
including manual DNA sequencing methods well known in the art. As
is also known in the art, a single insertion or deletion in a determined
nucleotide sequence compared to the actual sequence will cause a
frame shift in translation of the nucleotide sequence such that
the predicted amino acid sequence encoded by a determined nucleotide
sequence will be completely different from the amino acid sequence
actually encoded by the sequenced DNA molecule, beginning at the
point of such an insertion or deletion.
 Using the information provided herein, such as the nucleotide
sequence in FIG. 1, a nucleic acid molecule of the present invention
encoding a BCSG1 polypeptide may be obtained using standard cloning
and screening procedures, such as those for cloning cDNAs using
mRNA as starting material. Illustrative of the invention, the nucleic
acid molecule described in FIG. 1 (SEQ ID NO:1) was discovered in
a cDNA library derived from breast cancer. The gene was also identified
in cDNA libraries from brain tissue. The determined nucleotide sequence
of the BCSG1 cDNA of FIG. 1 (SEQ ID NO:1) contains an open reading
frame encoding a protein of 127 amino acid residues, with an initiation
codon at positions 12-14 of the nucleotide sequence in FIG. 1 (SEQ
ID NO:1), and a deduced molecular weight of about 14.2 kDa. The
BCSG1 protein shown in FIG. 1 (SEQ ID NO:2) is about 54% identical
to non-A.beta. fragment of human Alzheimer's disease (AD) amyloid
 As one of ordinary skill would appreciate, due to the possibilities
of sequencing errors, the predicted BCSG1 polypeptide encoded by
the deposited cDNA comprises about 127 amino acids, but may be anywhere
in the range of 110-140 amino acids.
 As indicated, nucleic acid molecules of the present invention
may be in the form of RNA, such as mRNA, or in the form of DNA,
including, for instance, cDNA and genomic DNA obtained by cloning
or produced synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known
as the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
 By "isolated" nucleic acid molecule(s) is intended
a nucleic acid molecule, DNA or RNA, which has been removed from
its native environment. For example, recombinant DNA molecules contained
in a vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include recombinant
DNA molecules maintained in heterologous host cells or purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
DNA molecules of the present invention. Isolated nucleic acid molecules
according to the present invention further include such molecules
 Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) shown
in FIG. 1 (SEQ ID NO:1) and DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode the BCSG1 protein.
Of course, the genetic code is well known in the art. Thus, it would
be routine for one skilled in the art to generate such degenerate
 In another aspect, the invention provides isolated nucleic
acid molecules encoding the BCSG1 polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid deposited
as ATCC Deposit No. 97856 on Jan. 23, 1997 or contained in the plasmid
deposited as ATCC Deposit No. 97175 on Jun. 2, 1995. The invention
further provides an isolated nucleic acid molecule having the nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1) or the nucleotide sequence
of the BCSG1 cDNA contained in the above-described deposited clone,
the full-length BCSG1 polypeptide lacking the N-terminal methionine
or a nucleic acid molecule having a sequence complementary to one
of the above sequences. Such isolated molecules, particularly DNA
molecules, are useful as probes for gene mapping, by in situ hybridization
with chromosomes, and for detecting expression of the BCSG1 gene
in human tissue, for instance, by Northern blot analysis.
 The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in FIG. 1
(SEQ ID NO:1) is intended fragments at least about 15 nt, and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably, at least about 40 nt in length
which are useful as diagnostic probes and primers as discussed herein.
Of course, larger fragments 50, 75, 100, 125, 150, 175, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550
nt in length are also useful according to the present invention
as are fragments corresponding to most, if not all, of the nucleotide
sequence of the deposited cDNA or as shown in FIG. 1 (SEQ ID NO:1).
By a fragment at least 20 nt in length, for example, is intended
fragments which include 20 or more contiguous bases from the nucleotide
sequence of the deposited cDNA or the nucleotide sequence as shown
in FIG. 1 (SEQ ID NO:1). SEQ ID NO:12 is full length cDNA sequence
of breast specific gene 1 of the present invention.
 Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions
of the BCSG1 protein. In particular, such nucleic acid fragments
of the present invention include nucleic acid molecules encoding:
a polypeptide comprising amino acid residues from about 94 to about
107 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid
residues from about 120 to about 127 in FIG. 1 (SEQ ID NO:2). The
inventors have determined that the above polypeptide fragments are
antigenic regions of the BCSG1 protein. Methods for determining
other such epitope-bearing portions of the BCSG1 protein are described
in detail below.
 In another aspect, the invention provides an isolated nucleic
acid molecule comprising a polynucleotide which hybridizes under
stringent hybridization conditions to a portion of the polynucleotide
in a nucleic acid molecule of the invention described above, for
instance, the cDNA clones contained in ATCC Deposits 97856 or 97175.
By "stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50% formamide,
5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt.times.s solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA, followed
by washing the filters in 0.1.times.SSC at about 65.degree. C.
 By a polynucleotide which hybridizes to a "portion"
of a polynucleotide is intended a polynucleotide (either DNA or
RNA) hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide. These are useful as diagnostic probes and primers
as discussed above and in more detail below.
 By a portion of a polynucleotide of "at least 20 nt
in length," for example, is intended 20 or more contiguous
nucleotides from the nucleotide sequence of the reference polynucleotide
(e.g., the deposited cDNA or the nucleotide sequence as shown in
FIG. 1 (SEQ ID NO: 1)). Of course, a polynucleotide which hybridizes
only to a poly A sequence (such as the 3' terminal poly(A) tract
of the BCSG1 cDNA shown in FIG. 1 (SEQ ID NO:1)), or to a complementary
stretch of T (or U) resides, would not be included in a polynucleotide
of the invention used to hybridize to a portion of a nucleic acid
of the invention, since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the complement
thereof (e.g., practically any double-stranded cDNA clone).
 As indicated, nucleic acid molecules of the present invention
which encode a BCSG1 polypeptide may include those encoding the
amino acid sequence of the polypeptide, by itself; the coding sequence
for the polypeptide and additional sequences, such as those encoding
an amino acid leader or secretory sequence, such as a pre-, or pro-
or prepro- protein sequence; the coding sequence of the polypeptide,
with or without the aforementioned additional coding sequences,
together with additional, non-coding sequences, including for example,
but not limited to introns and non-coding 5' and 3' sequences, such
as the transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and polyadenylation
signals, for example--ribosome binding and stability of mRNA; an
additional coding sequence which codes for additional amino acids,
such as those which provide additional functionalities. Thus, the
sequence encoding the polypeptide may be fused to a marker sequence,
such as a sequence encoding a peptide which facilitates purification
of the fused polypeptide. In certain preferred embodiments of this
aspect of the invention, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (Qiagen, Inc.),
among others, many of which are commercially available. As described
in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for
instance, hexa-histidine provides for convenient purification of
the fusion protein. The "HA" tag is another peptide useful
for purification which corresponds to an epitope derived from the
influenza hemagglutinin protein, which has been described by Wilson
et al., Cell 37: 767 (1984). As discussed below, other such fusion
proteins include the BCSG1 fused to Fc at the N- or C-terminus.
 The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode portions,
analogs or derivatives of the BCSG1 protein. Variants may occur
naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally
occurring variants may be produced using art-known mutagenesis techniques.
 Such variants include those produced by nucleotide substitutions,
deletions or additions, which may involve one or more nucleotides.
The variants may be altered in coding regions, non-coding regions,
or both. Alterations in the coding regions may produce conservative
or non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions, additions
and deletions, which do not alter the properties and activities
of the BCSG1 protein or portions thereof. Also especially preferred
in this regard are conservative substitutions.
 Further embodiments of the invention include isolated nucleic
acid molecules comprising a polynucleotide having a nucleotide sequence
at least 90% identical, and more preferably at least 95%, 96%, 97%,
98% or 99% identical to (a) a nucleotide sequence encoding the BCSG1
polypeptide having the amino acid sequence in FIG. 1 (SEQ ID NO:2);
(b) a nucleotide sequence encoding the polypeptide having the amino
acid sequence in SEQ ID NO:2, but lacking the N-terminal methionine;
(c) a nucleotide sequence encoding the BCSG1 polypeptide having
the amino acid sequence encoded by the cDNA clones contained in
ATCC Deposit Nos. 97856 or 97175; or (d) a nucleotide sequence complementary
to any of the nucleotide sequences in (a), (b) or (c).
 By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide
sequence encoding a BCSG1 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference sequence
except that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide sequence
encoding the BCSG1 polypeptide. In other words, to obtain a polynucleotide
having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide,
or a number of nucleotides up to 5% of the total nucleotides in
the reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or
3' terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
 As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%,96%, 97%,98% or 99% identical to,
for instance, the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1)
or to the nucleotides sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as
the Bestfit program (Wisconsin Sequence Analysis Package, Version
8 for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711. Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics
2: 482-489 (1981), to find the best segment of homology between
two sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for instance,
95% identical to a reference sequence according to the present invention,
the parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference nucleotide sequence
and that gaps in homology of up to 5% of the total number of nucleotides
in the reference sequence are allowed.
 The present application is directed to nucleic acid molecules
at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid
sequence of the deposited cDNA, irrespective of whether they encode
a polypeptide having BCSG1 activity. This is because even where
a particular nucleic acid molecule does not encode a polypeptide
having BCSG1 activity, one of skill in the art would still know
how to use the nucleic acid molecule, for instance, as a hybridization
probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic
acid molecules of the present invention that do not encode a polypeptide
having BCSG1 activity include, inter alia, (1) isolating the BCSG1
gene or allelic variants thereof in a cDNA library; (2) in situ
hybridization (e.g., "FISH") to metaphase chromosomal
spreads to provide precise chromosomal location of the BCSG1 gene,
as described in Verma et al., Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York (1988); and Northern Blot analysis
for detecting BCSG1 mRNA expression in specific tissues.
 Preferred, however, are nucleic acid molecules having sequences
at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid
sequence of the deposited cDNA which do, in fact, encode a polypeptide
having BCSG1 protein activity. By "a polypeptide having BCSG1
activity" is intended polypeptides exhibiting activity similar,
but not necessarily identical, to an activity of the BCSG1 protein
of the invention, as measured in a particular biological assay.
BCSG1 protein is believed to be involved with apoptosis. BCSG1 protein
activity can be measured using assays that measure apoptosis. For
example, human breast cancer cells cultured on Lab-Tek chamber slides
(Nunc, Inc.) are treated with or without recombinant BCSG1 protein
or a candidate BCSG1 protein. The cells are then treated with several
concentrations of an apoptotic inducer, such as adriamycin. Apoptosis
is compared between the treated and control cells where DNA fragmentation
is the criteria for apoptotic death using the following assay. At
various time points after the adriamycin treatment, adherent cells
are stained with DNA-specific fluorochrome diamino-2 phenylindole
(Boehringer Mannheim) in a 1 .mu.g/ml methanol solution. Cells are
counted within 20 minutes of staining on a Zeiss Axiophot epiflouresence
microscope. Experiments are performed in triplicate with at least
150 cells scored at each point. Fragmented or condensed nuclei are
scored as apoptotic. Intact or mitotic nuclei are scored as normal.
 Of course, due to the degeneracy of the genetic code, one
of ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence
of the deposited cDNA or the nucleic acid sequence shown in FIG.
1 (SEQ ID NO:1) will encode a polypeptide "having BCSG1 protein
activity." In fact, since degenerate variants of these nucleotide
sequences all encode the same polypeptide, this will be clear to
the skilled artisan even without performing the above described
comparison assay. It will be further recognized in the art that,
for such nucleic acid molecules that are not degenerate variants,
a reasonable number will also encode a polypeptide having BCSG1
protein activity. This is because the skilled artisan is fully aware
of amino acid substitutions that are either less likely or not likely
to significantly effect protein function (e.g., replacing one aliphatic
amino acid with a second aliphatic amino acid).
 For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to
Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein
the authors indicate that proteins are surprisingly tolerant of
amino acid substitutions.
 Vectors and Host Cells
 The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of BCSG1 polypeptides or fragments thereof by recombinant
 The polynucleotides may be joined to a vector containing
a selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector
is a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
 The DNA insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac,
trp and tac promoters, the SV40 early and late promoters and promoters
of retroviral LTRs, to name a few. Other suitable promoters will
be known to the skilled artisan. The expression constructs will
further contain sites for transcription initiation, termination
and, in the transcribed region, a ribosome binding site for translation.
The coding portion of the mature transcripts expressed by the constructs
will preferably include a translation initiating at the beginning
and a termination codon (UAA, UGA or UAG) appropriately positioned
at the end of the polypeptide to be translated.
 As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as
E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and SpodopteraSf9
cells; animal cells such as CHO, COS and Bowes melanoma cells; and
plant cells. Appropriate culture mediums and conditions for the
above-described host cells are known in the art.
 In addition to the use of expression vectors in the practice
of the present invention, the present invention further includes
novel expression vectors comprising operator and promoter elements
operatively linked to nucleotide sequences encoding a protein of
interest. One example of such a vector is pHE4-5 which is described
in detail below.
 As summarized in FIGS. 3 and 4, components of the pHE4-5
vector (SEQ ID NO:10) include: 1) a neomycinphosphotransferase gene
as a selection marker, 2) an E. coli origin of replication, 3) a
T5 phage promoter sequence, 4) two lac operator sequences, 5) a
Shine-Delgamo sequence, 6) the lactose operon repressor gene (lacIq).
The origin ofreplication (oriC) is derived from pUC19 (LTI, Gaithersburg,
Md.). The promoter sequence and operator sequences were made synthetically.
Synthetic production of nucleic acid sequences is well known in
the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East
Meadow Circle, Palo Alto, Calif. 94303. A nucleotide sequence encoding
BSCG-1 (SEQ ID NO:1), is operatively linked to the promoter and
operator by inserting the nucleotide sequence between the NdeI and
Asp718 sites of the pHE4-5 vector.
 As noted above, the pHE4-5 vector contains a lacIq gene.
LacIq is an allele of the lacI gene which confers tight regulation
of the lac operator. Amann, E. et al., Gene 69:301-315 (1988); Stark,
M., Gene 51:255-267 (1987). The lacIq gene encodes a repressor protein
which binds to lac operator sequences and blocks transcription of
down-stream (i.e., 3') sequences. However, the lacIq gene product
dissociates from the lac operator in the presence of either lactose
or certain lactose analogs, e.g., isopropyl B-D-thiogalactopyranoside
(IPTG). BSCG-1 thus is not produced in appreciable quantities in
uninduced host cells containing the pHE4-5 vector. Induction of
these host cells by the addition of an agent such as IPTG, however,
results in the expression of the BSCG-1 coding sequence.
 The promoter/operator sequences of the pHE4-5 vector (SEQ
ID NO:11) comprise a T5 phage promoter and two lac operator sequences.
One operator is located 5' to the transcriptional start site and
the other is located 3' to the same site. These operators, when
present in combination with the lacIq gene product, confer tight
repression of down-stream sequences in the absence of a lac operon
inducer, e.g., IPTG. Expression of operatively linked sequences
located down-stream from the lac operators may be induced by the
addition of a lac operon inducer, such as IPTG. Binding of a lac
inducer to the lacIq proteins results in their release from the
lac operator sequences and the initiation of transcription of operatively
linked sequences. Lac operon regulation of gene expression is reviewed
in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS,
4th Edition (1997), pages 802-807.
 The pHE4 series of vectors contain all of the components
of the pHE4-5 vector except for the BSCG-1 coding sequence. Features
of the pHE4 vectors include optimized synthetic T5 phage promoter,
lac operator, and Shine-Delagarno sequences. Further, these sequences
are also optimally spaced so that expression of an inserted gene
maybe tightly regulated and high level of expression occurs upon
 Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic
promoters include the CMV immediate early promoter, the HSV thymidine
kinase promoter, the early and late SV40 promoters, the promoters
of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV),
and metallothionein promoters, such as the mouse metallothionein-I
 The pHE4-5 vector also contains a Shine-Delgamo sequence
5' to the AUG initiation codon. Shine-Delgamo sequences are short
sequences generally located about 10 nucleotides up-stream (i.e.,
5') from the AUG initiation codon. These sequences essentially direct
prokaryotic ribosomes to the AUG initiation codon.
 Thus, the present invention is also directed to expression
vector useful for the production of the proteins of the present
invention. This aspect of the invention is exemplified by the pHE4-5
vector (SEQ ID NO:10).
 Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available
from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable
vectors will be readily apparent to the skilled artisan.
 Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection or other methods. Such methods are described
in many standard laboratory manuals, such as Davis et al., Basic
Methods In Molecular Biology (1986).
 The polypeptide may be expressed in a modified form, such
as a fusion protein, and may include not only secretion signals,
but also additional heterologous functional regions. For instance,
a region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve stability
and persistence in the host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties may be added
to the polypeptide to facilitate purification. Such regions may
be removed prior to final preparation of the polypeptide. The addition
of peptide moieties to polypeptides to engender secretion or excretion,
to improve stability and to facilitate purification, among others,
are familiar and routine techniques in the art. A preferred fusion
protein comprises a heterologous region from immunoglobulin that
is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved pharmacokinetic
properties (EP-A 0232 262). On the other hand, for some uses it
would be desirable to be able to delete the Fc part after the fusion
protein has been expressed, detected and purified in the advantageous
manner described. This is the case when Fc portion proves to be
a hindrance to use in therapy and diagnosis, for example when the
fusion protein is to be used as antigen for immunizations. In drug
discovery, for example, human proteins, such as, hIL5-receptor has
been fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. See, D. Bennett et al.,
Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson
et al., The Journal of Biological Chemistry, Vol. 270, No. 16:9459-9471
 The BCSG1 protein can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography
and lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention include naturally purified
products, products of chemical synthetic procedures, and products
produced by recombinant techniques from a prokaryotic or eukaryotic
host, including, for example, bacterial, yeast, higher plant, insect
and mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention
may be glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
 BCSG1 Polyypeptides and Fragments
 The invention further provides an isolated BCSG1 polypeptide
having the amino acid sequence encoded by the deposited cDNA clones,
or the amino acid sequence in FIG. 1 (SEQ ID NO:2), or a peptide
or polypeptide comprising a portion of the above polypeptides.
 It will be recognized in the art that some amino acid sequences
of the BCSG1 polypeptide can be varied without significant effect
of the structure or function of the protein. If such differences
in sequence are contemplated, it should be remembered that there
will be critical areas on the protein which determine activity.
 Thus, the invention further includes variations of the BCSG1
polypeptide which show substantial BCSG1 polypeptide activity or
which include regions of BCSG1 protein such as the protein portions
discussed below. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions. As indicated above, guidance concerning
which amino acid changes are likely to be phenotypically silent
can be found in Bowie, J. U., et al., "Deciphering the Message
in Protein Sequences: Tolerance to Amino Acid Substitutions,"
Science 247:1306-1310 (1990).
 Thus, the fragment, derivative or analog of the polypeptide
of FIG. 1 (SEQ ID NO:2), or that encoded by the deposited cDNA,
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 residues
includes a substituent group, or (iii) one in which the polypeptide
is fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol),
or (iv) one in which the additional amino acids are fused to the
polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory sequence or a sequence which is employed for purification
of the polypeptide or a proprotein sequence. Such fragments, derivatives
and analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
 Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or negatively
charged amino acids. The latter results in proteins with reduced
positive charge to improve the characteristics of the BCSG1 protein.
The prevention of aggregation is highly desirable. Aggregation of
proteins not only results in a loss of activity but can also be
problematic when preparing pharmaceutical formulations, because
they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340
(1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.
Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
 As indicated, changes are preferably of a minor nature,
such as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic Phenylalanine
Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar
Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic
Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine
 Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those described
above. Generally speaking, the number of substitutions for any given
AIM-II polypeptide will not be more than 50, 40, 30, 25, 20, 15,
10, 5 or 3.
 Amino acids in the BCSG1 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis
(Cunningham and Wells, Science 244:1081-1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity such as receptor binding or in vitro, or in
vitro proliferative activity. Sites that are critical for ligand-receptor
binding can also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312
 The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide"
is intended a polypeptide removed from its native environment. Thus,
a polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides
that have been purified, partially or substantially, from a recombinant
host cell or from a native source. For example, a recombinantly
produced version of the BCSG1 polypeptide can be substantially purified
by the one-step method described in Smith and Johnson, Gene 67:31-40
 The polypeptides of the present invention include the polypeptide
encoded by the deposited cDNA; a polypeptide comprising amino acids
about 1 to about 127 in SEQ ID NO:2 (FIG. 1); a polypeptide comprising
amino acids about 2 to about 127 in SEQ ID NO:2; as well as polypeptides
which are at least 80% identical, more preferably at least 90% or
95% identical, still more preferably at least 96%, 97%, 98% or 99%
identical to the polypeptide encoded by the deposited cDNA, to the
polypeptide of FIG. 1 (SEQ ID NO:2), and also include portions of
such polypeptides with at least 30 amino acids and more preferably
at least 50 amino acids.
 By a polypeptide having an amino acid sequence at least,
for example, 95% "identical" to a reference amino acid
sequence of a BCSG1 polypeptide is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of the BCSG1 polypeptide. In other words, to obtain a polypeptide
having an amino acid sequence at least 95% identical to a reference
amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino
acid, or a number of amino acids up to 5% of the total amino acid
residues in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur
at the amino or carboxy terminal positions of the reference amino
acid sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or
in one or more contiguous groups within the reference sequence.
 As a practical matter, whether any particular polypeptide
is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or to the
amino acid sequence encoded by deposited cDNA clone can be determined
conventionally using known computer programs such the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive, Madison,
Wis. 53711. When using Bestfit or any other sequence alignment program
to determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present invention,
the parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference amino acid sequence
and that gaps in homology of up to 5% of the total number of amino
acid residues in the reference sequence are allowed.
 The polypeptide of the present invention could be used as
a molecular weight marker on SDS-PAGE gels or on molecular sieve
gel filtration columns using methods well known to those of skill
in the art.
 In another aspect, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide of the invention.
The epitope of this polypeptide portion is an immunogenic or antigenic
epitope of a polypeptide described herein. An "immunogenic
epitope" is defined as a part of a protein that elicits an
antibody response when the whole protein is the immunogen. On the
other hand, a region of a protein molecule to which an antibody
can bind is defined as an "antigenic epitope." The number
of immunogenic epitopes of a protein generally is less than the
number of antigenic epitopes. See, for instance, Geysen et al.,
Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).
 As to the selection of peptides or polypeptides bearing
an antigenic epitope (i.e., that contain a region of a protein molecule
to which an antibody can bind), it is well known in that art that
relatively short synthetic peptides that mimic part of a protein
sequence are routinely capable of eliciting an antiserum that reacts
with the partially mimicked protein. See, for instance, Sutcliffe,
J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies
that react with predetermined sites on proteins. Science 219:660-666.
Peptides capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be characterized
by a set of simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals.
 Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including monoclonal
antibodies, that bind specifically to a polypeptide of the invention.
See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
 Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about at least
about 15 to about 30 amino acids contained within the amino acid
sequence of a polypeptide of the invention. Non-limiting examples
of antigenic polypeptides or peptides that can be used to generate
BCSG1-specific antibodies include: apolypeptide comprising amino
acid residues from about 94 to about 107 in FIG. 1 (SEQ ID NO:2);
a polypeptide comprising amino acid residues from about 120 to about
127 in FIG. 1 (SEQ ID NO:2). As indicated above, the inventors have
determined that the above polypeptide fragments are antigenic regions
of the BCSG1 protein.
 The epitope-bearing peptides and polypeptides of the invention
may be produced by any conventional means. Houghten, R. A. (1985).
General method for the rapid solid-phase synthesis of large numbers
of peptides: specificity of antigen-antibody interaction at the
level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135.
This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Pat. No. 4,631,211 to Houghten
et al. (1986).
 As one of skill in the art will appreciate, BCSG1 polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased half-life
in vivo. This has been shown, e.g., for chimeric proteins consisting
of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of
mammalian immunoglobulins (EPA 394,827; Trauneckeretal., Nature
331:84- 86 (1988)). Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG part can also be more efficient
in binding and neutralizing other molecules than the monomeric BCSG1
protein or protein fragment alone (Fountoulakis et al., J. Biochem
 Cancer Diagnosis and Prognosis
 There are two classes of genes affecting tumor development.
Genes influencing the cancer phenotype that act directly as a result
of changes (e.g., mutation) at the DNA level, such as BRCA1, BRCA2,
and p53, are called Class I genes. The Class II genes affect the
phenotype by modulation at the expression level. Development of
breast cancer and subsequent malignant progression is associated
with alterations of a variety of genes of both classes. Identification
of quantitative changes in gene expression that occur in the malignant
mammary gland, if sufficiently characterized, may yield novel molecular
markers which may be useful in the diagnosis and treatment of human
 The present inventors have identified a new breast cancer
marker that is overexpressed in advanced infiltrating breast cancer
cells. The lack of expression of BCSG1 in normal or benign breast
epithelial cells and a weak expression in low grade in situ carcinomas
suggest that overexpression of BCSG1 indicates breast cancer malignant
progression. (See, Examples 6 and 7). It is unlikely that BCSG1
is overexpressed as a secondary effect of cellular proliferation
because no detectable BCSG1 expression is evident in rapidly proliferating
nonmalignant breast lesions. (See, Example 7).
 BCSG1 maybe useful in clinical management and treatment
of breast cancer. In this regard, the expression of BCSG1 transcripts
was observed in the neoplastic epithelial cells of infiltrating
breast carcinoma but not in epithelial cells of normal and benign
breast tissue. (See, Example 7). The overexpression of BCSG1 in
malignant infiltrating breast epithelial cells compared to the low
level expression in the low grade in situ carcinoma suggests that
up-regulation of BCSG1 expression is associated with breast malignant
progression and may signal the more advanced invasive/metastatic
phenotype of human breast cancer. This implication is further supported
by detection of BCSG1 expression in 4/4 breast cancer cell lines
derived from ductal infiltrating carcinomas but not (0/3) in breast
cancer cell lines derived from primary solid carcinoma (See, Example
6). BCSG1 overexpression in ductal carcinoma in situ (DCIS) may
indicate a malignant progression leading to metastasis. There was
a marked increase in DCIS incidence beginning in the early 1980s
(Emster, V. L., et al., JAMA 275:913-918 (1996)). The total estimated
number of DCIS cases in the United States in 1992 was 200% higher
than expected based on 1983 rates and trends between 1973 and 1983
(Emster, V. L., et al., JAMA 275:913-918 (1996)). While early detection
of invasive breast cancer is beneficial, the value of DCIS detection
is currently unknown. There is cause for concern about the large
number of DCIS cases that are being diagnosed as a consequence of
screening mammography, most of which are treated by some form of
surgery. In addition, the proportion of cases treated by mastectomy
may be inappropriately high (Emster, V. L., et al., JAMA 275:913-918
(1996)). BCSG1 expression may provide some prognostic information
on distinguishing the DCIS which is not likely to become invasive
from the DCIS which is most likely to become invasive, which will
help to reduce some inappropriate or unnecessary mastectomies. In
addition, the use of BCSG1 gene could be of great importance in
differentiating atypical proliferative breast lesions from cancer
and may be useful in screening of breast biopsies for potential
 It is interesting to note that the predicted amino acid
sequence of BCSG1 gene shares high sequence homology with the recently
cloned non-A.beta. component of Alzheimer's disease (AD) amyloid
precursor protein (Ueda, K., et al., Proc. Natl. Acad. Sci. USA.
90(23):11282-6 (1993)). A neuropathological hallmark of AD is a
widespread amyloid deposition resulting from beta-amyloid precursor
proteins (beta APPS). Beta APPs are large membrane-spanning proteins
that either give rise to the beta A4 peptide (AB fragment) (Masters,
C. L., et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985)) or
a non-A.beta. component of AD amyloid (Ueda, K., et al., Proc. Natl.
Acad. Sci. USA. 90(23):11282-6 (1993)) that is either deposited
in AD amyloid plaques or yielding soluble forms. While the insoluble
membrane-bound AD amyloid destabilizes calcium homeostasis and thus
renders cell vulnerable to excitotoxic conditions of calcium influx
resulting from energy deprivation or overexcitation (Mattson, M.
P., et al., Ann. N.Y. Acad. Sci. 679:121 (1993)), the soluble AD
amyloid proteins are neuroprotective against glucose deprivation
and glutamate toxicity, perhaps through their ability to lower the
intraneuronal calcium concentration (Barger, S. W., J. Neurochem.
64:2087-96 (1995)). It is possible that BCSG1, like soluble AD amyloid,
may be potentially involved in tissue damage resulting from tissue
remodeling due to the local cancer invasion. Nevertheless, Examples
6 and 7 demonstrate a stage-specific BCSG1 expression and an association
of BCSG1 overexpression with clinical aggressiveness of breast cancers.
BCSG1 overexpression may indicate breast cancer malignant progression
from benign breast or low grade in situ carcinoma to the highly
 The Examples demonstrate that certain tissues in mammals
with cancer express significantly enhanced levels of the BCSG1 protein
and mRNA encoding the BCSG1 protein when compared to a corresponding
"standard" mammal, i.e., a mammal of the same species
not having the cancer. Further, it is believed that enhanced levels
of the BCSG1 protein can be detected in certain body fluids (e.g.,
sera, plasma, urine, and spinal fluid) from mammals with cancer
when compared to sera from mammals of the same species not having
the cancer. Thus, the invention provides a diagnostic method useful
during tumor diagnosis, which involves assaying the expression level
of the gene encoding the BCSG1 protein in mammalian cells or body
fluid and comparing the gene expression level with a standard BCSG1
gene expression level, whereby an increase in the gene expression
level over the standard is indicative of certain tumors.
 Where a tumor diagnosis has already been made according
to conventional methods, the present invention is useful as a prognostic
indicator, whereby patients exhibiting enhanced BCSG1 gene expression
will experience a worse clinical outcome relative to patients expressing
the gene at a lower level.
 By "assaying the expression level of the gene encoding
the BCSG1 protein" is intended qualitatively or quantitatively
measuring or estimating the level of the BCSG1 protein or the level
of the mRNA encoding the BCSG1 protein in a first biological sample
either directly (e.g., by determining or estimating absolute protein
level or mRNA level) or relatively (e.g., by comparing to the BCSG1
protein level or mRNA level in a second biological sample).
 Preferably, the BCSG1 protein level or mRNA level in the
first biological sample is measured or estimated and compared to
a standard BCSG1 protein level or mRNA level, the standard being
taken from a second biological sample obtained from an individual
not having the cancer. As will be appreciated in the art, once a
standard BCSG1 protein level or mRNA level is known, it can be used
repeatedly as a standard for comparison.
 By "biological sample" is intended any biological
sample obtained from an individual, cell line, tissue culture, or
other source which contains BCSG1 protein or mRNA. Biological samples
include mammalian body fluids (such as sera, plasma, urine, synovial
fluid and spinal fluid) which contain secreted mature BCSG1 protein,
and ovarian, prostate, heart, placenta, pancreas liver, spleen,
lung, breast and umbilical tissue.
 The present invention is useful for detecting cancer in
mammals. In particular the invention is useful during diagnosis
of the following types of cancers in mammals: breast, ovarian, prostate,
bone, liver, lung, pancreatic, and spleenic. Preferred mammals include
monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.
Particularly preferred are humans.
 Total cellular RNA can be isolated from a biological sample
using the single-step guanidinium-thiocyanate-phenol-chloroform
method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159
(1987). Levels of mRNA encoding the BCSG1 protein are then assayed
using any appropriate method. These include Northern blot analysis
(Haada et al., Cell 63:303-312 (1990)), S1 nuclease mapping (Fujita
et al., Cell 49:357-367 (1987)), the polymerase chain reaction (PCR),
reverse transcription in combination with the polymerase chain reaction
(RT-PCR) (Makino et al., Technique 2:295-301 (1990)), and reverse
transcription in combination with the ligase chain reaction (RT-LCR).
 Assaying BCSG1 protein levels in abiological sample can
occur using antibody-based techniques. For example, BCSG1 protein
expression in tissues can be studied with classical immunohistological
methods (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)).
 Other antibody-based methods useful for detecting BCSG1
protein gene expression include immunoassays, such as the enzyme
linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
 Suitable labels are known in the art and include enzyme
labels, such as, Glucose oxidase, and radioisotopes, such as iodine
(.sup.125I, .sup.121I, carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
 Chromosome Assays
 The nucleic acid molecules 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
human chromosome. The mapping of DNAs to chromosomes according to
the present invention is an important first step in correlating
those sequences with genes associated with disease.
 In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a BCSG 1 protein
gene. This can be accomplished using a variety of well known techniques
and libraries, which generally are available commercially. The genomic
DNA then is used for in situ chromosome mapping using well known
techniques for this purpose.
 In addition, in some cases, sequences can be mapped to chromosomes
by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer
analysis of the 3' untranslated region of the gene 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 containing
individual human chromosomes.
 Fluorescence in situ hybridization ("FISH") of
a cDNA clone to a metaphase chromosomal spread can be used to provide
a precise chromosomal location in one step. This technique can be
used with probes from the cDNA as short as 50 or 60 bp. For a review
of this technique, see Verma et al., Human Chromosomes: A Manual
Of Basic Techniques, Pergamon 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, Mendelian 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 identified 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.
 Having generally described the invention, the same will
be more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
Expression and Purification of BCSG1 in E. coli
 The bacterial expression vector pQE9 (pD10) is used for
bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). pQE9 encodes ampicillin antibiotic resistance
("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding
site ("RBS"), six codons encoding histidine residues that
allow affinity purification using nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra,
and suitable single restriction enzyme cleavage sites. These elements
are arranged such that an inserted DNA fragment encoding a polypeptide
expresses that polypeptide with the six His residues (i.e., a "6.times.His
tag")) covalently linked to the amino terminus of that polypeptide.
 The DNA sequence encoding the desired portion BCSG1 protein
sequence is amplified from the deposited cDNA clone using PCR oligonucleotide
primers which anneal to the amino terminal sequences of the desired
portion of the BCSG1 protein and to sequences in the deposited construct
3' to the cDNA coding sequence. Additional nucleotides containing
restriction sites to facilitate cloning in the pQE9 vector are added
to the 5' and 3' primer sequences, respectively.
 For cloning the mature protein, the 5' primer has the sequence
5' GGGGATCCATGTTTTCAAGAAGG 3' (SEQ ID NO:3) containing the underlined
BamHI restriction site followed by 16 nucleotides complementary
to the amino terminal coding sequence of the BCSG1 sequence in FIG.
1. One of ordinary skill in the art would appreciate, of course,
that the point in the protein coding sequence where the 5' primer
begins may be varied to amplify a DNA segment encoding any desired
portion of the complete BCSG1 protein shorter or longer than the
protein. The 3' primer has the sequence 5'GGAAGCTTCTAGTCTCCCCCACTCTGG
3' (SEQ ID NO:4) containing the underlined HindIII restriction site
followed by 19 nucleotides complementary to the non-coding sequence
of the BCSG1 DNA sequence in FIG. 1.
 The amplified BCSG1 DNA fragment and the vector pQE9 are
digested with BamHI/HindIII and the digested DNAs are then ligated
together. Insertion of the BCSG1 DNA into the restricted pQE9 vector
places the BCSG1 protein coding region downstream from the IPTG-inducible
promoter and in-frame with an initiating AUG and the six histidine
 The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY (1989). E. coli
strain M15/rep4, containing multiple copies of the plasmid pREP4,
which expresses the lac repressor and confers kanamycin resistance
("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing BCSG1 protein, is available commercially
from QIAGEN, Inc., supra. Transformants are identified by their
ability to grow on LB plates in the presence of ampicillin and kanamycin.
Plasmid DNA is isolated from resistant colonies and the identity
of the cloned DNA confirmed by restriction analysis, PCR and DNA
 Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with
both ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The
O/N culture is used to inoculate a large culture, at a dilution
of approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside
("IPTG") is then added to a final concentration of 1 mM
to induce transcription from the lac repressor sensitive promoter,
by inactivating the lacI repressor. Cells subsequently are incubated
further for 3 to 4 hours. Cells then are harvested by centrifugation.
 The cells are then stirred for 3-4 hours at 4.degree. C.
in 6 M guanidine-HCI, pH 8. The cell debris is removed by centrifugation,
and the supernatant containing the BCSG1 is loaded onto a nickel-nitrilo-tri-acetic
acid ("NiNTA") affinity resin column (available from QIAGEN,
Inc., supra). Proteins with a 6.times.His tag bind to the NI-NTA
resin with high affinity and can be purified in a simple one-step
procedure (for details see: The QIAexpressionist, 1995, QIAGEN,
Inc., supra). Briefly the supernatant is loaded onto the column
in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes
of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl
pH 6, and finally the BCSG1 is eluted with 6 M guanidine-HCl, pH
 The purified protein is then renatured by dialyzing it against
phosphatebuffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer
plus 200 mM NaCl. Alternatively, the protein can be successfully
refolded while immobilized on the Ni-NTA column. The recommended
conditions are as follows: renature using a linear 6M-1M urea gradient
in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing
protease inhibitors. The renaturation should be performed over a
period of 1.5 hours or more. After renaturation the proteins can
be eluted by the addition of 250 mM immidazole. Immidazole is removed
by a final dialyzing step against PBS or 50 mM sodium acetate pH
6 buffer plus 200 mM NaCl. The purified protein is stored at 4.degree.
C. or frozen at -80.degree. C.
Cloning and Expression of BCSG1 protein in a Baculovirus Expression
 In this illustrative example, the plasmid shuttle vector
pA2 GP is used to insert the cloned DNA encoding the protein into
a baculovirus to express the BCSG1 protein, using a baculovirus
leader and standard methods as described in Summers et al., A Manual
of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,
Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
This expression vector contains the strong polyhedrin promoter of
the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed
by the secretory signal peptide (leader) of the baculovirus gp67
protein and convenient restriction sites such as BamHI, XbaI and
Asp7l8. The polyadenylation site of the simian virus 40 ("SV40")
is used for efficient polyadenylation. For easy selection of recombinant
virus, the plasmid contains the beta-galactosidase gene from E.
coli under control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA
to generate viable virus that expresses the cloned polynucleotide.
 Many other baculovirus vectors could be used in place of
the vector above, such as pAc373, pVL941 and pAcIM1, as one skilled
in the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation, secretion
and the like, including a signal peptide and an in-frame AUG as
required. Such vectors are described, for instance, in Luckow et
al., Virology 170:31-39.
 The cDNA sequence encoding the BCSG1 protein in the deposited
clone shown in FIG. 1 (SEQ ID NO:2), is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene.
 The 5' primer has the sequence 5' GGGGATCCcGATGTTTTCAAGAAGG
3' (SEQ ID NO:5) (the lowercase "c" is a nucleotide included
to preserve the coding frame) containing the underlined BamHI restriction
enzyme site followed by 16 bases of the sequence of the BCSG1 protein
shown in FIG. 1, beginning with the N-terminus of the protein. The
3' primer has the sequence 5'GGGGTACCCTAGTCTCCCCCACTCTGG 3' (SEQ
ID NO:6) containing the underlined Asp718 restriction site followed
by 18 nucleotides complementary to the 3' noncoding sequence in
 The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101
Inc., La Jolla, Calif.). The fragment then is digested with BamHI/Asp718
and again is purified on a 1% agarose gel. This fragment is designated
 The plasmid is digested with the restriction enzymes BamHI/Asp718
and optionally, can be dephosphorylated using calf intestinal phosphatase,
using routine procedures known in the art. The DNA is then isolated
from a 1% agarose gel using a commercially available kit ("Geneclean"
BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein
 Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human BCSG1 gene using the PCR method, in which one of
the primers that is used to amplify the gene and the second primer
is from well within the vector so that only those bacterial colonies
containing the BCSG1 gene fragment will show amplification of the
DNA. The sequence of the cloned fragment is confirmed by DNA sequencing.
This plasmid is designated herein pBac BCSG1.
 Five .mu.g of the plasmid pBacBCSG1 is co-transfected with
1.0 .mu.g of a commercially available linearized baculovirus DNA
("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego,
Calif.), using the lipofection method described by Felgner et al.,
Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 .mu.g of BaculoGold.TM.
virus DNA and 5 .mu.g of the plasmid pBac BCSG1 are mixed in a sterile
well of a microtiter plate containing 50 .mu.l of serum-free Grace's
medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards,
10 .mu.l Lipofectin plus 90 .mu.l Grace's medium are added, mixed
and incubated for 15 minutes at room temperature. Then the transfection
mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded
in a 35 mm tissue culture plate with 1 ml Grace's medium without
serum. The plate is rocked back and forth to mix the newly added
solution. The plate is then incubated for 5 hours at 27.degree.
C. After 5 hours the transfection solution is removed from the plate
and 1 ml of Grace's insect medium supplemented with 10% fetal calf
serum is added. The plate is put back into an incubator and cultivation
is continued at 27.degree. C. for four days.
 After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of gal-expressing
clones, which produce blue-stained plaques. (A detailed description
of a "plaque assay" of this type can also be found in
the user's guide for insect cell culture and baculovirology distributed
by Life Technologies Inc., Gaithersburg, page 9-10). After appropriate
incubation, blue stained plaques are picked with the tip of a micropipettor
(e.g., Eppendorf). The agar containing the recombinant viruses is
then resuspended in a microcentrifuge tube containing 200 .mu.l
of Grace's medium and the suspension containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are harvested
and then they are stored at 4.degree. C. The recombinant virus is
 To verify the expression of the BCSG1 gene, Sf9 cells are
grown in Grace's medium supplemented with 10% heat inactivated FBS.
The cells are infected with the recombinant baculovirus V-BCSG1
at a multiplicity of infection ("MOI") of about 2. Six
hours later the medium is removed and is replaced with SF900 II
medium minus methionine and cysteine (available from Life Technologies
Inc., Rockville, Md.). If radiolabeled proteins are desired, 42
hours later, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci .sup.35S-cysteine
(available from Amersham) are added. The cells are further incubated
for 16 hours and then they are harvested by centrifugation. The
proteins in the supernatant as well as the intracellular proteins
are analyzed by SDS-PAGE followed by autoradiography (ifradiolabeled).
Microsequencing of the amino acid sequence of the amino terminus
of purified protein may be used to determine the amino terminal
sequence of the mature protein and thus the cleavage point and length
of the secretory signal peptide.
Cloning and Expression of BCSG1 in Mammalian Cells
 A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the termination
of transcription and polyadenylation of the transcript. Additional
elements include enhancers, Kozak sequences and intervening sequences
flanked by donor and acceptor sites for RNA splicing. Highly efficient
transcription can be achieved with the early and late promoters
from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,
RSV, HTLV I, HIV I and the early promoter of the cytomegalovirus
(CMV). However, cellular elements can also be used (e.g., the human
actin promoter). Suitable expression vectors for use in practicing
the present invention include, for example, vectors such as PSVL
and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr
(ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that
could be used include, human Hela 293, H9 and Jurkat cells, mouse
NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells,
mouse L cells and Chinese hamster ovary (CHO) cells.
 Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The co-transfection
with a selectable marker such as dhfr, gpt, neomycin, or hygromycin
allows the identification and isolation of the transfected cells.
 The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS) (Murphy
et al., Biochem J. 22 7:277-279 (1991); Bebbington et al., Bio/Technology
10:169-175 (1992)). Using these markers, the mammalian cells are
grown in selective medium and the cells with the highest resistance
are selected. These cell lines contain the amplified gene(s) integrated
into a chromosome. Chinese hamster ovary (CHO) and NSO cells are
often used for the production of proteins.
 The expression vectors pC1 and pC4 contain the strong promoter
(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
contain in addition the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene.
Cloning and Expression in COS Cells
 The expression plasmid, pBCSG1 HA, is made by cloning a
cDNA encoding BCSG1 into the expression vector pcDNAI/Amp or pcDNAIII
(which can be obtained from Invitrogen, Inc.).
 The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and other
prokaryotic cells; (2) an ampicillin resistance gene for selection
of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication
for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,
an SV40 intron; (5) several codons encoding a hemagglutinin fragment
(i.e., an "HA" tag to facilitate purification) followed
by a termination codon and polyadenylation signal arranged so that
a cDNA can be conveniently placed under expression control of the
CMV promoter and operably linked to the SV40 intron and the polyadenylation
signal by means of restriction sites in the polylinker. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein described by Wilson et al., Cell 37:767 (1984). The fusion
of the HA tag to the target protein allows easy detection and recovery
of the recombinant protein with an antibody that recognizes the
HA epitope. pcDNAIII contains, in addition, the selectable neomycin
 A DNA fragment encoding the BCSG1 is cloned into the polylinker
region of the vector so that recombinant protein expression is directed
by the CMV promoter. The plasmid construction strategy is as follows.
The BCSG1 cDNA of the deposited clone is amplified using primers
that contain convenient restriction sites, much as described above
for construction of vectors for expression of BCSG1 in E. coli.
Suitable primers include the following, which are used in this example.
The 5' primer, containing the underlined BaniHI site, a Kozak sequence,
an AUG start codon and 4 codons of the 5' coding region of the complete
BCSG1 has the following sequence: 5' GGGGATccgccaccATGTTTTCAAGAAGG
3' (SEQ ID NO:7) (Kozak sequence is represented by the lowercase
letters). The 3' primer, containing the underlined BaniHI site,
a stop codon, and 19 bp of 3' coding sequence has the following
sequence (at the 3' end): 5' GGGGATCCTCAgaaagcgtagtctgggacg- tcgtatgggtaCTAGTCTCCCCCACTCTGG
3' (SEQ ID NO:8) (the HA tag is represented by the lowercase letters).
 The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with BamHI and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037), and the transformed culture is plated on ampicillin media
plates which then are incubated to allow growth of ampicillin resistant
colonies. Plasmid DNA is isolated from resistant colonies and examined
by restriction analysis or other means for the presence of the BCSG1-encoding
 For expression of recombinant BCSG1, COS cells are transfected
with an expression vector, as described above, using DEAE-DEXTRAN,
as described, for instance, in Sambrook et al., Molecular Cloning:
a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,
New York (1989). Cells are incubated under conditions for expression
of BCSG1 by the vector.
 Expression of the BCSG1-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow et al., Antibodies: A Laboratory Manual, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York (1988). To this end, two days after transfection, the cells
are labeled by incubation in media containing .sup.35S-cysteine
for 8 hours. The cells and the media are collected, and the cells
are washed and lysed with detergent-containing RIPA buffer: 150
mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described
by Wilson et al. cited above. Proteins are precipitated from the
cell lysate and from the culture media using an HA-specific monoclonal
antibody. The precipitated proteins then are analyzed by SDS-PAGE
and autoradiography. An expression product of the expected size
is seen in the cell lysate, which is not seen in negative controls.
Cloning and Expression in CHO Cells
 The vector pC4 is used for the expression of BCSG1 protein.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession
No. 37146). The plasmid contains the mouse DHFR gene under control
of the SV40 early promoter. Chinese hamster ovary- or other cells
lacking dihydrofolate activity that are transfected with these plasmids
can be selected by growing the cells in a selective medium (alpha
minus MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see, e.g.,
Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,
1978, J Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990,
Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham,
M. A. 1991, Biotechnology 9:64-68). Cells grown in increasing concentrations
of MTX develop resistance to the drug by overproducing the target
enzyme, DHFR, as a result of amplification of the DHFR gene. If
a second gene is linked to the DHFR gene, it is usually co-amplified
and over-expressed. It is known in the art that this approach may
be used to develop cell lines carrying more than 1,000 copies of
the amplified gene(s). Subsequently, when the methotrexate is withdrawn,
cell lines are obtained which contain the amplified gene integrated
into one or more chromosome(s) of the host cell.
 Plasmid pC4 contains for expressing the gene of interest
the strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March
1985:438-447) plus a fragment isolated from the enhancer of the
immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are BamHI,
XbaI, and Asp718 restriction enzyme cleavage sites that allow integration
of the genes. Behind these cloning sites the plasmid contains the
3' intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the expression,
e.g., the human .beta.-actin promoter, the SV40 early or late promoters
or the long terminal repeats from other retroviruses, e.g., HIV
and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems
and similar systems can be used to express the BCSG1 in a regulated
way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc.
Natl. Acad. Sci. USA 89: 5547-5551). For the polyadenylation of
the mRNA other signals, e.g., from the human growth hormone or globin
genes can be used as well. Stable cell lines carrying a gene of
interest integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418 or hygromycin.
It is advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
 The plasmid pC4 is digested with the restriction enzymes
BamHI/Asp718 and then dephosphorylated using calf intestinal phosphatase
by procedures known in the art. The vector is then isolated from
a 1% agarose gel.
 The DNA sequence encoding the BCSG1 protein sequence is
amplified using PCR oligonucleotide primers corresponding to the
5' and 3' sequences of the gene. The 5' primer has the sequence
5' GGGGATccgccaccATGTTTTCAAGAAGG 3' (SEQ ID NO:7) (Kozak sequence
is represented by the lowercase letters) containing the underlined
BamHI restriction enzyme site followed by an efficient signal for
initiation of translation in eukaryotes, as described by Kozak,
M., J. Mol. Biol. 196:947-950 (1987), and 15 bases of the coding
sequence of BCSG1 shown in FIG. 1 (SEQ ID NO:1). The 3' primer has
the sequence 5' GGGGTACCTCACTAGTCTCCCCCACTCTGG 3' (SEQ ID NO:9)
containing the underlined Asp718 restriction site followed by 22
nucleotides complementary to the non-translated region of the BCSG1
gene shown in FIG. 1 (SEQ ID NO:1).
 The amplified fragment is digested with the endonucleases
BamHI/Asp718 and then purified again on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed
and bacteria are identified that contain the fragment inserted into
plasmid pC4 using, for instance, restriction enzyme analysis.
 Chinese hamster ovary cells lacking an active DHFR gene
are used for transfection. 5 .mu.g of the expression plasmid pC4
is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using lipofectin
(Felgner et al., supra). The plasmid pSV2neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are trypsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then transferred
to new 6-well plates containing even higher concentrations of methotrexate
(1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration
of 100-200 .mu.M. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reverse phase HPLC